CN101182469A - Aspergillus niger strain having highly-resistant activity to carbendazol and uses thereof - Google Patents
Aspergillus niger strain having highly-resistant activity to carbendazol and uses thereof Download PDFInfo
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Abstract
The invention relates to an Aspergillus niger strain ANUV90 obtained through induced mutation, which has high and stable resistance for carbendazim. Compared with the resistance for carbendazim of the initial strain AN, the resistance for carbendazim of the strain is increased by1000 times. The Aspergillus niger strain ANUV90 can grow normally under the 1000 Mu g/mL treatment concentration of effective component of carbendazim. And the fermentation broth of the strain has certain nematode killing effects. No significant difference of colony morphology characteristic is detected between the strains before and after induced mutation. And the strain has a certain biological activity on the tomato root knot nematodes (Meloidogyne spp.). After analyzing the molecular mechanism of the resistance of the mutant strain, the primary research result shows that the site directed mutation of Beta- tubulin encoding gene leads to the generation of the biological characteristic resistance of ANUV90. The bacterial strain related to the invention is stored in China General Microbiological Culture Collection Center on 30 October, 2007. And the preservation number of the strain is CGMCC No: M2234. The classification and nomenclature of the strain is Aspergillus niger. Due to the resistance characteristic, the strain can be mixed and applied with most common pesticides for the control and prevention of various harmful insects. The invention provides a way for solving the unstable control effect problem in the biocontrol strain field, which has great application and development prospect.
Description
Technical field:
The present invention relates to the fungi field, be specifically related to aspergillus niger fungal bacterial strain and the correlated characteristic thereof of a strain, belong to biological technical field high density derosal performance resistance.
Background technology:
In soil, introduce the antagonistic microbe controlling plant diseases particularly the research of soil-borne disease carried out more than 60 year, become disease flocking biocontrol worker's common recognition, and obtained certain achievement.At present, utilize biological and ecological methods to prevent plant disease, pests, and erosion microorganism substituted chemistry sterilant to become a kind of direction of effort, but when applying, biocontrol strain nearly all can run into same obstacle, in shop experiment and greenhouse test pathogen is showed high preventive effect though that is exactly some biocontrol microorganisms, there is instability problem in biocontrol effect in the field.Unstable show introduce when use in the field bacterial strain in the different location, different year disease prevention growth-promoting effect all may be not quite similar, this has become a kind of maximum restraining factors that its large-scale commercial is applied to agriculture production.It is numerous to influence the biological and ecological methods to prevent plant disease, pests, and erosion effect factors of instability, and wherein reason is exactly that soil fungistasis influences biocontrol effect on the one hand.Multiple microorganism is to the breeding of fungi, growth and grow surely certain restraining effect is arranged in the soil.
Timper confirms that the existence meeting of microorganism in the soil greatly reduces the activity of biocontrol strain.Stirling thinks that bacteriostatic action makes biocontrol microorganisms deciding in soil grow probably and falls flat, and by controlling or create the living environment that is beneficial to biocontrol microorganisms artificially, reaches the purpose that reduces the line insect population size.The power of soil fungistasis is decided by the physico-chemical property of soil and the activity of soil microorganisms, the latter play a major role (can by to experiment confirms such as soil disinfection processing) wherein, the rhizosphere soil indigenous microorganism except that by to nutritive substance particularly the cut-throat competition of carbon, nitrogenous source limit the growth of biocontrol microorganisms, also may suppress the growth of biocontrol microorganisms by producing antifungal compound.Therefore, how breaking soil fungistasis grows and the pass that has played a role into problem biocontrol fungicide well surely in soil.Interpolation chemistry or organic substance can be broken soil fungistasis to a certain extent in the biocontrol fungicide.Wishing that bright 7 kinds of microbiotic, 6 kinds of chemical pesticides and rice bran, the stalk etc. of studies show that can effectively remove soil fungistasis; Though can remove the bacteriostatic action of soil by the artificial quantity that sterilant reduces original rhizospheric microorganism that applies, may influence the growth of biocontrol microorganisms equally, influence the preventive effect of biocontrol microorganisms, this point can not be ignored.
To rely on the future of Plant diseases biological and ecological methods to prevent plant disease, pests, and erosion biological prevention and control agent to carry out technological improvement to introducing in the soil.Biological prevention and control agent being introduced in the soil course, traditional biocontrol microorganisms is modified into and can and works in coordination with the biocontrol microorganisms of using with the chemical agent compatibility, perhaps is exactly one of so-called " technological improvement ".
Summary of the invention:
The present invention just is being based on above-mentioned situation and theory, tames directional induction by ultraviolet mutagenesis in conjunction with medicament, and it is the aspergillus niger resisting carbendazim strains A NUV90 of 100 μ g/mL substratum normal growths to carbendazim concentration that screening obtains a strain.This strains A NUV90 is aspergillus niger Aspergillus niger, be deposited in (address: Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on October 30th, 2007, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.2234.Find that simultaneously this resistant strain and most of chemical agent are compatible good, ANUV90 and most common pesticides can be worked in coordination with to be mixed and be used the multiple harmful organism of control, provide an approach for solving biocontrol strain field efficacy instability problem.
Its concrete grammar is as follows:
1. the aspergillus niger that mutagenesis screening is obtained is to measuring derosal susceptibility and stability.
2. the observation of aspergillus niger resistance mutant strain colonial morphology and thalline feature.
3. the compatibility research of aspergillus niger resistance mutant strain ANUV90 and common fungicide, Insecticides (tech) ﹠ Herbicides (tech), plant-growth regulator.
4. anti-medicine Aspergillus niger strain meta-bolites is measured the tomato root-knot eelworm toxic action.
5. aspergillus niger resistance mutant strain ANUV90 resistance molecular mechanism preliminary study.
The present invention relates to a strain has resistance to Multiple Pesticides Aspergillus niger strain, because this bacterial strain is after mutagenesis, obtain feature genetic stability, that multiple common pesticides had resistance, not performance decline aspect the nematicide ability simultaneously, can be used for the biological control of plant nematode, especially be mixed and prevent and treat the tomato root-knot eelworm disease with Multiple Pesticides.
Embodiment:
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1 aspergillus niger ANUV90 is to the susceptibility and the stability of derosal
Aspergillus niger ANUV90 is inoculated in the pastille PDA culture medium flat plate that contains 50 μ g/ml, 100 μ g/ml, 500 μ g/ml, 1000 μ g/ml derosal effective constituents, behind the certain hour, measures the bacterium colony size.Be inoculated on the normal PSA substratum that does not contain derosal and cultivate obtaining drug-fast mutants which had after the mutagenesis, carried out for 10 generations continuously, measure the drug-fast genetic stability of aspergillus niger after the mutagenesis.
The making of anti-medicine substratum: 50% carbendazol wettable powder is dissolved in earlier in the 0.1mol/L dilute hydrochloric acid, be made into and contain pharmaceutically active ingredient 3750 μ g/mL (0.375g medicament+100mL sterilized water promptly obtains 3750 μ g/mL) mother liquor, it is 0.375 μ g/mL that gradient dilution becomes concentration, 37.5 μ g/mL, 375 μ g/mL, 3750 μ g/mL, getting 1mL adding volume then respectively is to sterilize in the 75mLPSA substratum, makes concentration and is respectively 0,0.05 μ g/mL, 0.5 μ g/mL, 5 μ g/mL, 50 μ g/mL pastille culture medium flat plates.To going in the glass culture dish that diameter is 9cm, every ware 15mL cooling back is standby, is contrast (CK) with the PSA substratum of no medicament, every processing repetition four times.
Aspergillus niger ANUV90 up to 1000 μ g/mL derosal concentration of treatment (this concentration through convert much larger than derosal field working concentration) but under normal growth still, each carbendazim concentration is handled with the normal aspergillus niger colony growth situation of control treatment does not have significant difference, even some concentration level (is 500 μ g/mL as concentration) shows the growth that promotes ANUV90 to a certain extent, compare with former strains A N, aspergillus niger ANUV90 is very insensitive to derosal, its resistance has improved nearly more than 1000 times (table 1, tables 2).
The comparison of table 1 aspergillus niger AN colony growth radius (mm) under the different concns derosal is handled
Data are the measured value behind the AN growth 2d in the table
The comparison of table 2ANUV90 colony growth radius (mm) under the different concns derosal is handled
Data are the measured value after ANUV90 cultivates 2d in the table
Aspergillus niger ANUV90 succeeding transfer culture after 10 generations the derosal different concns is handled and contrast AN colony growth situation does not have significant difference, shows that ANUV90 can genetic stability (table 3) to the high resistance property of medicine of derosal.
Table 3 aspergillus niger ANUV90 succeeding transfer culture is handled the comparison of colony growth radius (mm) down to the different concns derosal after 10 generations
Data are the measured value of ANUV90 behind succeeding transfer culture 10 generation 2d on the no medicine substratum in the table
Embodiment 2 aspergillus niger ANUV90 thalline features
Observe mutagenesis front and back bacterial strain mycelia form down by 40 times in microscope,, and take pictures by the scanning electron microscopic observation spore shape.
Aspergillus niger ANUV90 can find out that under identical 40 times of opticmicroscopes the mycelia of resistance mutant strain is narrow thinner than former bacterial strain, be likely the result of uv-radiation variation, the AN spore shape does not have significant difference in scanning electron microscopic observation ANUV90 spore shape and the documents and materials, (accompanying drawing 1 left side figure is the mycelia form of AN only to show irregularity slightly, middle figure is the mycelia form of ANUV90, and right figure is an ANUV90 spore head morphology under the scanning electron microscope).
The compatibility of embodiment 3 aspergillus niger resistance mutant strain ANUV90 and chemical agent commonly used
Common fungicide (Ke Lu, m-tetrachlorophthalodinitrile, thiophanate methyl, zinc manganese ethylenebisdithiocarbamate, metaxanin, frost slip, the mould prestige of second), sterilant (Decamethrin), weedicide (acetochlor, atrazine), plant-growth regulator (Plant hormones regulators,gibberellins) are made and are contained the PSA substratum that pharmaceutically active ingredient concentration is respectively 0.01 μ g/mL, 0.1 μ g/mL, 1 μ g/mL, 10 μ g/mL, 100 μ g/mL, are contrast with pastille not.After making flat board, be that the aseptic punch tool acquisition Aspergillus niger strain of 5mm and the bacterium sheet of aspergillus niger resistance mutant strain are connected to it on pastille PSA substratum with diameter.25 ℃ of following constant temperature culture, record bacterium colony spread scenarios after 2 days is with the diameter of right-angled intersection method measurement bacterium colony.
PSA substratum: potato 200g, sucrose 20g, agar-agar 20g, water 1000mL.
Except that the mould prestige of second, the compatibility of AN and ANUV90 two bacterial strains and most sterilant, weedicide, other reagent agent does not have notable difference, and substantially all be faint to the bacterial strain influence under low consistency conditions, extremely indivedual agricultural chemicals even ANUV90 had certain promotion (illustrating that these trace pesticides can be the ANUV90 strain growth nutritive substance is provided), but two bacterial strain colony diameters all diminish gradually along with the increase of drug concentration generally; Wherein with sterilant zinc manganese ethylenebisdithiocarbamate, metaxanin, the weedicide atrazine, sterilant Decamethrin and gibberellin of plant growth regulator have favorable compatibility, illustrate that two bacterial strains all can mix composite use with these chemical agents.
Table 4 AN and the colony diameter of ANUV90 under different medicament different concns are handled
The mycelium of ANUV90 is inoculated on the test tube nutrient agar, and culture medium prescription is the PDA substratum, i.e. potato 200g; Glucose 20g; Agar 17g; Water 1000mL.Cultivate 10d for 25 ℃, obtain the test tube kind.
The test tube kind is inoculated in 250mL triangular flask (the every bottled 50mL) liquid nutrient medium, culture medium prescription is (weight percent): sucrose (2.83%), ammonium sulfate (1.367%), K again
2HPO
4(0.064%), MgSO
47H
2O (0.182%), KCl (0.996%), FeSO
4(0.02%), remainder is a water, pH6.5.Under 25 ℃～28 ℃, shaking speed is with 150rmin
-1～200rmin
-1, the fermentation 240h.Fermented liquid is made into different multiples carries out the test of nematicide drug effect.
In special concave surface capsule, put into 100 μ L different concns sterilizations filtrate, after put into northern root knot nematode J about 50
2, be contrast with the sterilized water, three repetitions are calculated J at 12h, 24h, 48h respectively
2Relative death rate.(formula is as follows)
In 12h, 24h, these three times of 48h, measure the lethality rate no significant difference (accompanying drawing 2 aspergillus niger ANs and ANUV900 stoste 12,24,48 hour to root knot nematode lethality rate dynamically compare) of the mutagenesis front and back strain fermentation filtrate of same concentrations to nematode.
The drug-fast molecular mechanism of embodiment 5 ANUV90
Strains tested: aspergillus niger (AN), aspergillus niger drug-fast strain (ANUV90)
1. specimen preparation
On the PSA flat board, get the bacterium cake of strains tested with the sterilization punch tool, put into PS (potato juice and sucrose) nutrient solution with tweezers gripping bacterium cake then, in 25 ℃ of constant incubators of 180r/min vibration, cultivate, the back taking-up of one week, filter mycelia with double gauze, after sterile distilled water washes 3 times repeatedly, the mycelia surface-moisture is fully blotted with filter paper, put into 50 ℃ of-55 ℃ of baking ovens oven dry to suitable state, 4 ℃ of refrigerators are preserved standby.
2. the extraction of genomic dna
Urea extraction method (Liu Shaohua etc. are adopted in this test, 2005): the ANUV90 oven dry mycelia that 0.5g is ground adds 10mL urea extracting solution (7mol/L Urea, 50mmol/L Tris-HCl pH8.0,62.5mmol/L Nacl, 1%SDS), shake up, change in the centrifuge tube, with rotating speed 12000r/min, high speed frozen centrifugation 5min gets supernatant liquid, repeats once.
Supernatant is transferred in another new pipe, add isopyknic phenol: chloroform: primary isoamyl alcohol (volume ratio=25: 24: 1) solution, shake up for several times with forced oscillation, put into refrigerated centrifuge again, with rotating speed 12000r/min, centrifugal 5min gets the NaAc (pH=5.2) that adds isopyknic Virahol and 1/10 volume 3mol/L in the new pipe of supernatant liquor to,-20 ℃ leave standstill 30min, with the centrifugal again 5min of 12000r/min, abandon supernatant liquor, inversion is flow to end tube wall liquid, use 70% ethanol, the absolute ethanol washing precipitation, room temperature is placed dry 5-10min, is dissolved in the 200 μ L distilled waters, adds RNaseA (10 μ g/ μ L) 2 μ L, 37 ℃ of water-bath 30min ,-20 ℃ of preservations are standby.
3. polymerase chain reaction (PCR)
Primer: it is synthetic to give birth to worker (Sangon) with B1 and B3 by Shanghai, and sequence is respectively:
B1?5’A?(AG)?AT?(CT)?ACCCA?(CT)?TC?(CT)?CT?(CT)?GGTGGTGG 3’
B3?5’CTCCAT?(CT)?TC?(AG)?TCCAT?(ACT)?CC?(CT)?TC?(AG)?CC?3’
Connecing for the examination strain gene group DNA is template amplification 'beta '-tubulin gene.
Contain 100ng genomic dna template in the 50 μ L reaction systems, 5L 10 * bufer (10mmoL/L Tris-HCl, 50mmoL/L KCl), MgCl2 2mmol/L, Taq archaeal dna polymerase 1U (Shanghai Promga), dNTP 0.2mmol/L, each 0.5 μ mol/L of primer.
Reaction conditions is: 94 ℃ of 5min, 1 circulation; 94 ℃ of 1min, 65 ℃ of 2min, 72 ℃ of 2min, 30 circulations; Last 72 ℃ are extended the 5min end.
The PCR product is electrophoresis detection on 1.0% sepharose, and ethidium bromide (EB) dyeing 30min puts gel observe and take pictures (accompanying drawing 3 AN and the total DNA of ANUV and 'beta '-tubulin gene amplification segment electrophoretogram) in ImageMaster ultraviolet imagery system.
4. the comparison in sequence homology analysis and mutational site
With primer B1, B3 the amplification output is directly checked order, adopt VectorNTI Suit9 AN and ANUV90 base and aminoacid sequence comparative analysis.
5.PCR amplified production sequencing and AN and ANUV90 bacterial strain 'beta '-tubulin gene fragment compare of analysis are directly handed in the order-checking of extra large Sangon company with the pcr amplification segment, by B1 and B3 primer the 'beta '-tubulin gene of aspergillus niger AN and ANUV90 bacterial strain is carried out pcr amplification, all obtain the dna fragmentation (accompanying drawing 3) of equal length, two-way order-checking is after splicing, the 'beta '-tubulin gene fragment length of determining above-mentioned bacteria strain is 854bp, and the β-microtubule protein-2 genes sequence alignment nucleotide homology by Blast and aspergillus niger generic typical module strains A spergillus nidulans (benA) is 92.3%.Therefore confirm further that it clearly is aspergillus niger AN and ANUV90 coding for beta-tubulin gene that these two fragments can be distinguished.The order-checking fragment is the result show, AN is different with the indivedual sites of ANUV90 two bacterial strain nucleotide sequences, be mutated into C at 81 A, and cause amino acid that variation has taken place, 26 L-glutamic acid (Glu) of AN replace to aspartic acid (Asp) (E → D), this site may be in the mutational site, thereby (the 'beta '-tubulin dna sequence dna of accompanying drawing 4 aspergillus niger ANUV90 and the amino acid of original AN bacterial strain comparing difference and deduction change, the total DNA of 1 AN to have caused ANUV90 drug-fast generation on biological characteristics; The total DNA of 2 ANUV90; The 'beta '-tubulin gene of 3 AN; The 'beta '-tubulin gene of 4 ANUV90; The 'beta '-tubulin gene of 5 ANUV90; M DNAMaker).
The classification called after aspergillus niger Aspergillus niger of this strains A NUV90, be deposited in (address: Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on October 30th, 2007, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.2234.
By the Aspergillus niger strain ANUV90 that the screening acquisition has higher stable resistance to derosal, this bacterial strain has certain biological activity to tomato root-knot eelworm simultaneously, and various chemical medicaments is had stable resistance; Show through sequencing, comparison, coding for beta-tubulin gene generation locating point sudden change in the gene order, thus caused ANUV90 drug-fast generation on biological characteristics.These characteristics of this bacterial strain make it can work in coordination with the multiple harmful organism of use control that is mixed with most common pesticides, provide an approach for solving biocontrol strain field efficacy instability problem, have the excellent development application prospect.
Claims (7)
1. the present invention is that a strain has drug-fast fungal bacterial strain to derosal, this strains A NUV90 is aspergillus niger Aspergillusniger, be deposited in (address: Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on October 30th, 2007, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNo.2234.
2. aspergillus niger according to claim 1, the fermented liquid that it is characterized by this bacterium has stable resistance to derosal, and (Meloidogyne spp.) has certain cytotoxicity to tomato root-knot eelworm.
3. the resistance that the aspergillus niger ANUV90 bacterial strain that the present invention relates to is had is mutated into C at 81 by A for the coding for beta-tubulin gene takes place behind ultraviolet mutagenesis, and causes changing the highly-resistant activity of generation to derosal.
4. the application in the biological control in agriculture production according to claim 1 and the described aspergillus niger ANUV90 of claim 2 bacterial strain.
According to claim 1 and the described aspergillus niger of claim 2 in the application of plant nematode especially tomato root-knot eelworm (Meloidogyne spp.) aspect preventing and treating.
6. a mematocide is characterized in that, comprises the described fungi of claim 3 itself or its metabolite as activeconstituents.
7. the product that forms in accordance with the method for claim 5 can be aqua, pulvis or suspension concentrate, and application process can adopt the whole bag of tricks such as seed dressing, seed pelleting, filling root and soil treatment.
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| US20180127707A1 (en) * | 2015-04-06 | 2018-05-10 | Industry Foundation Of Chonnam National University | Aspergillus niger f22 strain having nematicidal activity against plant-parasitic nematodes, and use thereof |
| CN109844095A (en) * | 2015-04-06 | 2019-06-04 | 全南大学校产学协力团 | There is the aspergillus niger F22 bacterial strain and application thereof of eelworm-killing activity to plant nematode |
| CN109844095B (en) * | 2015-04-06 | 2022-03-25 | 全南大学校产学协力团 | Aspergillus niger F22 strain having nematicidal activity against plant parasitic nematodes and use thereof |
| CN104928194A (en) * | 2015-06-26 | 2015-09-23 | 云南大学 | Aspergillus niger strain and application thereof |
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