CN106701602A - Strain of fusarium chlamydosporum and application thereof - Google Patents
Strain of fusarium chlamydosporum and application thereof Download PDFInfo
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Abstract
The invention discloses a strain of fusarium chlamydosporum and an application thereof. The strain is currently collected in the CGMCC (China General Microbiological Culture Collection Center) on December 9th, 2016, with a collection number of CGMCC No.13198. After fermentation and extraction, the strain has relatively high inhibiting activity on plant pathogenic fungi such as cotton wilt fusarium and colletotrichum glecosporioides, and the minimum inhibiting concentration (MIC) is 4mu g/mL and 8mu g/mL respectively; moreover, the strain also has relatively good nematicidal activity against American ginseng root-knot nematodes, and the fatality rates of 50-times and 100-times diluents against American ginseng root-knot nematodes are 100% and 83% respectively; and thus, the strain has good application prospects in plant disease control.
Description
Technical field
The invention belongs to microbial pesticide field.Specifically, the present invention relates to one plant thick wall sickle-like bacteria and its application.
Background technology
The various plant diseases that plant pathogenic fungi causes seriously threaten the production estimation of China, the economy for causing every year
Loss is difficult to estimate.For example：Cotton-wilt fusarium has different degrees of generation in the main cotton growing area of China, and Seedling Stage can hair
Disease, or even can result in that whole cotton plant is withered to die of illness, have a strong impact on the yield and quality of cotton.
Plant pathogeny line insect is also one of plant disease for generally occurring, China main harm tobacco, pseudo-ginseng, cotton,
The industrial crops such as peanut, American Ginseng, it has also become one of key factor of harm yield of commercial crops.According to statistics, the whole world it is annual because
Crop damage caused by plant pathogeny line insect is up to tens billion of dollars.
Preventing and treating to plant disease at present is mainly utilized including chemical pesticide control, rotation method and resistant variety, is deposited
In certain limitation.Especially chemical pesticide control, easily makes plant pathogenic fungi be developed immunity to drugs with nematode, and residual
Phase is long, so being above restricted in application.And microbial pesticide is the drawbacks of effectively can overcome chemical synthetic pesticide, with being difficult
Develop immunity to drugs, environment compatibility is good, the advantages of be easy to scale fermenting and producing.It is nuisanceless as a kind of " environment-friendly "
Environment friendly agricultural, sustainability is strong, and the effect in control of plant disease is increasingly apparent.Therefore, found from microorganism and developed
Biological pesticide, has become the important directions of development plant disease green preventing and treating.
The content of the invention
The purpose of the present invention is offer one plant of thickness wall sickle-like bacteria QJP1 and its application.The bacterial strain is easy to scale fermentation life
Produce, there is preferable eelworm-killing activity to American Ginseng root-knot nematode after the extracted separation of its tunning, to cotton-wilt fusarium with
The plant pathogenic fungis such as citrus anthracnose bacterium have preferable inhibitory activity, therefore with good development prospect.
Bacterial strain provided by the present invention is thickness wall sickle-like bacteria (Fusarium chlamydosporum) QJP1, is located away from green grass or young crops
The alkaline land soil of the fluffy wetland collection of island city Jiaozhou Bay's alkali.The bacterial strain has been preserved in Chinese microorganism strain preservation management committee at present
Member can common micro-organisms center, preservation date：On December 09th, 2016, deposit number is CGMCC No.13198.
The store method of thick wall reaping hook bacteria strain QJP1：Preserved using PDA culture medium.PDA culture medium：Potato 200g,
Glucose 20g, agar 12g, distilled water 1000mL, pH are adjusted to 7.0.
It is a further object to provide a kind of fermentation process of thick wall reaping hook bacteria strain QJP1, it is characterized in that,
1) the thick wall reaping hook bacteria strain QJP1 for preserving is inoculated in PDA plate surface, 26-30 DEG C is cultivated 5-10 days, as
The strain of scale fermentation culture, it is stand-by；
2) and then take above-mentioned strain and add in fermentation medium at 26-30 DEG C shaking table or standing for fermentation culture 10-40 days, obtain
It is stand-by to zymotic fluid.The fermentation medium (with PDB culture mediums) is：Potato 200g, glucose 20g, distilled water 1000mL,
PH is adjusted to 7.0.
Above-mentioned tunning can be extracted further, specifically include following steps：
1) Solvent Extract methods are used, extract solution vacuum distillation obtains medicinal extract；The organic solvent be ethyl acetate, chloroform,
One or more in acetone, methyl alcohol, ethanol or n-butanol, ethyl acetate；
2) and then medicinal extract carries out column chromatography for separation, the column chromatography can use silica gel column chromatography, gel filtration chromatography, reversed-phase column
One or more in chromatography and macroporous adsorbent resin column chromatography, preferably silica gel column chromatography；
3) with petroleum ether-ethyl acetate gradient elution, petroleum ether-ethyl acetate volume ratio 10 is collected:The wash-out group of 1 gradient
Dividing I is used to prepare microorganism nematicide；The petroleum ether-ethyl acetate gradient is 100:1 to 1:1；
4) and then with chloroform-methanol gradient elution, chloroform-methanol volume ratio 20 is collected:The elution fraction II of 1 gradient is used for
Prepare microbial bactericide；The chloroform-methanol gradient is 60:1 to 1:1.
Advantage for present invention：Tunning of the invention can by the fermented cultures of thick wall reaping hook bacteria strain QJP1,
Extract separate and obtains, with production technology it is relatively easy, be easy to scale fermentation production, in the environment be easy to degrade and to people and animals
The advantages of toxicity is relatively low.There is preferably suppression to live to plant pathogenic fungis such as cotton-wilt fusarium and citrus anthracnose bacterium for it
Property, MIC (MIC) is respectively 4 and 8 μ g/mL；Additionally, the tunning has preferably to American Ginseng root-knot nematode
Eelworm-killing activity, its 50 times are respectively 100% and 83% to the fatal rate of American Ginseng root-knot nematode with 100 times of dilutions, therefore
Had a good application prospect in control of plant disease.
Specific embodiment
Further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and
It is apparent.But these embodiments are only exemplary, do not constitute any limitation to the scope of the present invention.People in the art
Member to the details of technical solution of the present invention and form it should be understood that can enter without departing from the spirit and scope of the invention
Row modification is replaced, but these modifications and replacement are each fallen within protection scope of the present invention.
Embodiment 1：Thick wall sickle-like bacteria QJP1 isolation and identification for strains
(1) strain isolation
Bacterial strain QJP1 of the present invention is separated and obtained with the method such as rule using plating dilutions coating from soil.(1)
Collecting soil sample：Collecting location is Jiaozhou Bay of the Qingdao City fluffy wetland of alkali, using 5 point samplings.(2) separating step：Weigh 1g
Salt-soda soil soil sample in 100mL sterilized waters, be placed in 30 DEG C in shaking table, 150r/min concussion 10min, 10 are diluted to successively-2、10-3、10-4Concentration；The 100 above-mentioned dilutions of μ L are drawn respectively, are spread evenly across on the dual anti-culture medium flat plates of LB containing 3.5% sea salt,
Each gradient coating three is parallel；After 30 DEG C of culture 2d, the microbe colony of picking media surface different shape, and containing
Rule on the dual anti-culture medium flat plates of LB of 3.5% sea salt, timing observation colony growth situation；Finally, using plate streaking
Method, separation purification of epiphyte bacterial strain, numbering is preserved.
(2) identification of strains
By QJP1 inoculations to PDA plate surface, observation after 28 DEG C of cultures 5-7 days.Bacterium colony center forms raised, bacterium colony
Surface is white, and close raw aerial hyphae flocculence, the bacterium colony back side is faint yellow；Microscope inspection can be observed that sickleshaped is large-scale, have every
Conidium, mycelia top or mycelia intermediate green grow Thick Spherical wall spore.
The rDNA gene sequencings result (ITS-5.8S-ITS2 areas) of the bacterial strain is as follows：
TCCGTAGGTGAACCTTGCGGTTAAACTCCCAAACCCCTGTGACATACCTATACGTTGCCTCGGCGGATC
AGCCCGCGCCCCGTAAAACGGGACGGCCCGCCCGAGGACCCCTAAACTCTGTTTTTAGTGGAACTTCTGAGTAAAAC
AAACAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAG
TAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATG
CCTGTTCGAGCGTCATTTCAACCCTCAAGCTCAGCTTGGTGTTGGGACTCGCGGTAACCCGCGTTCCCCAAATCGAT
TGGCGGTCACGTCGAGCTTCCATAGCGTAGTAATCATACACCTCGTTACTGGTAATCGTCGCGGCCACGCCGTAAAA
CCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGACCG
CAAGGTTCACCTACGGA
In sum, identification of strains is thick wall sickle-like bacteria (Fusarium chlamydosporum), and the bacterial strain has been protected at present
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address：Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, Institute of Microorganism, Academia Sinica), preservation date：On December 09th, 2016, the preservation of the bacterial strain
Numbering is CGMCC No.13198.
Embodiment 2：Thick wall reaping hook bacteria strain QJP1 fermented and cultureds
(1) fermented and cultured
According to the conventional culture methods of microorganism, picking is stored in the thick wall reaping hook bacteria strain on PDA culture medium inclined-plane on a small quantity
QJP1, is inoculated in PDA plate surface, and 28 DEG C are cultivated 5 days, stand-by as the strain of scale fermentation culture.
PDA culture medium：Potato 200g, glucose 20g, agar 12g, distilled water 1000mL, pH are adjusted to 7.0.
(2) cut the strain the 1/4 of flat board (consumption for) on above-mentioned PDA plate surface, be seeded to it is sterilized, fill PDB
In the conical flask of culture medium, 28 DEG C, 120rpm shaking table cultures 10 days are standby.
PDB culture mediums：Potato 200g, glucose 20g, distilled water 1000mL, pH are adjusted to 7.0.
(3) preparation of tunning
Above-mentioned cultured products are extracted 3 times with ethyl acetate, combined ethyl acetate extract solution, vacuum distillation obtains medicinal extract.Will
It carries out silica gel VLC (vacuum liquid chromatography) rapid column chromatography, according to eluent polarity incremental order,
Respectively with petroleum ether-ethyl acetate (volume ratio 100:1 to 1:1) with chloroform-methanol (volume ratio 60:1 to 1:1) it is gradient elution
Agent carries out silica gel column chromatography separation.Collect petroleum ether-ethyl acetate volume ratio 10:The elution fraction I of 1 gradient, it is micro- for preparing
GR；Collect chloroform-methanol volume ratio 20:The elution fraction II of 1 gradient, for preparing microbial bactericide.
Embodiment 3：Bacteriostatic activity
Using micro-dilution method, thick wall reaping hook bacteria strain QJP1 tunnings are determined to cotton-wilt fusarium and oranges and tangerines anthrax
The bacteriostatic activity of germ.
1) preparation of bacteria suspension
PDA culture medium surface will be inoculated in after 28 DEG C of culture 72h for examination fungi, draw the aseptic 0.85%NaCl of 2mL molten
Liquid (containing 0.25% Tween-20) washing culture, and gently scraped bacterium colony with glass slicker.Appropriate bacteria suspension is drawn in aseptic
In test tube, 0.5 Maxwell is adjusted to more standby than turbid (equivalent to 1.5 × 108CFU/mL)；
2) preparation of sample
A certain amount of testing sample (elution fraction II of tunning in embodiment 2) is taken, 100 μ L 50%DMSO are dissolved in
In, after fully mixing, in 50 μ L samples solution of absorption to another centrifuge tube, 50 μ L 50%DMSO are subsequently added into, obtain concentration
The sample solution for halving.In this way, the sample solution that 12 groups of concentration halve successively is obtained.
3) MIC assay methods
(1) sterile working is used, the sample solution of various concentrations after doubling dilution is added separately to 96 aseptic hole polyphenyl
In vinyl plate, the 1st to the 12nd hole respectively adds the sample solution of 5 μ L, and to be not added with sample well as blank, plus 5 μ L DMSO molten
Fluid apertures is solvent control.
(2) the instruction bacteria suspension of 0.5 maxwell reduced turbidity is will be equivalent to, after diluting 1000 times through sabouraud culture medium, 95 μ L is taken
Be added sequentially in 96 orifice plates so that the sample concentration in the 1st to the 12nd hole is followed successively by 512,256,128,64,32,16,8,4,2,
1st, 0.5 and 0.25 μ g/mL.All of above sample is in triplicate.After gently concussion is mixed, 96 pore plate by sealing are placed in 28 DEG C of lifes
72h is cultivated in change incubator.
(3) light absorption value per hole is determined using ELIASA under 600nm wavelength, is given birth to completely inhibiting indicator bacteria in aperture
Minimum sample concentration long is the MIC of the compound.(note：When the obvious growth experiment of indicator bacteria is just intentional in negative control hole
Justice；When single jump hole occurs in experiment, the highest drug concentration for suppressing strain growth should be recorded；Such as there are many places and jump hole, then
Result should not be reported, need to repeat to test.)
Result of the test is thick wall reaping hook bacteria strain QJP1 tunnings to cotton-wilt fusarium and the MIC of citrus anthracnose bacterium
Value is respectively 4 and 8 μ g/mL, with preferable bacteriostatic activity.
Above-mentioned the results show thick wall reaping hook bacteria strain QJP1 tunnings of the present invention have preferable bacteriostatic activity,
Can be used to prepare microbial bactericide.
Embodiment 4：Eelworm-killing activity
(1) experimental technique
For examination nematode and its cultural method
American Ginseng root-knot nematode is provided by Shandong Forest Science Academy.20g oatmeals plus 60mL water are weighed, after sterilizing
Add 1mL nematode suspensions, 25 DEG C of culture 7d.Aseptically, the cultured oat containing nematode is put in filter to picking in right amount
On paper, nematode is washed out with the graceful funnel method of simple shellfish, nematode is suspended from standby in sterilized water, concentration is about 2000/mL, 4 DEG C of guarantors
Deposit standby.
Nematicidal Activity
Take a certain amount of testing sample (elution fraction I of tunning in embodiment 2), diluted respectively with 50%DMSO 50 with
100 times, in absorption 1mL to sterile centrifugation tube, the μ L of nematode suspension 100 are added, control, 25 DEG C of standings are processed as with 50%DMSO
12h.After mixing, mixed liquor point is drawn to slide, be placed in counting verge of death borer population and line insect number (line under light microscope
Worm is stiff motionless to be considered as death), calculate the death rate.The nematode number of observation is no less than 30 every time.
Judge that the other standard of active level is：Death rate ＞ 90% is " +++ " level；70% ＜ death rates ＜ 90%, is " ++ "
Level；50% ＜ nemic death rates ＜ 70%, is "+" level；Death rate ＜ 50%, is "-" level.
(2) experimental result
50 times of dilutions of thick wall sickle-like bacteria strain fermentation product are 100% to the fatal rate of American Ginseng root-knot nematode, living
Property rank be " +++ " level；Each treatment polypide form is examined under a microscope after 12h, it is found that dead nematode loses activity, polypide
It is stiff, and compare living nematode motion vivaciously, polypide bending is in " S " type.
And 100 times of dilutions of thick wall sickle-like bacteria strain fermentation product are 83% to the fatal rate of American Ginseng root-knot nematode,
Active rank is " ++ " level.It can be seen that, although after tunning further dilutes, the fatal rate of nematode has declined, compared to sky
White control (5%) still shows obvious eelworm-killing activity.Therefore, the tunning that prepared by the bacterial strain, can effectively prevent and treat
Plant pathogeny line insect, especially American Ginseng root-knot nematode, can be used to prepare microorganism nematicide.
SEQUENCE LISTING
<110>Qingdao Agricultural University
<120>One plant thick wall sickle-like bacteria and its application
<130> 1
<140> 0
<141> 2017-02-16
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 548
<212> DNA
<213>Thick wall sickle-like bacteria（Fusarium chlamydosporum）The rRNA gene orders of bacterial strain QJP1（ITS1-
5.8S-ITS4 areas）
<400> 1
tccgtaggtg aaccttgcgg ttaaactccc aaacccctgt gacataccta tacgttgcct 60
cggcggatca gcccgcgccc cgtaaaacgg gacggcccgc ccgaggaccc ctaaactctg 120
tttttagtgg aacttctgag taaaacaaac aaataaatca aaactttcaa caacggatct 180
cttggttctg gcatcgatga agaacgcagc aaaatgcgat aagtaatgtg aattgcagaa 240
ttcagtgaat catcgaatct ttgaacgcac attgcgcccg ccagtattct ggcgggcatg 300
cctgttcgag cgtcatttca accctcaagc tcagcttggt gttgggactc gcggtaaccc 360
gcgttcccca aatcgattgg cggtcacgtc gagcttccat agcgtagtaa tcatacacct 420
cgttactggt aatcgtcgcg gccacgccgt aaaaccccaa cttctgaatg ttgacctcgg 480
atcaggtagg aatacccgct gaacttaagc atatcaataa gcggaggacc gcaaggttca 540
cctacgga 548
Claims (10)
1. thick wall sickle-like bacteria Fusarium chlamydosporum bacterial strains QJP1, the deposit number of the bacterial strain is CGMCC
No.13198。
2. the thick wall reaping hook bacteria strain QJP1 described in claim 1 is preparing preventing and treating plant pathogenic fungi disease or plant root knot
Purposes in the tunning of nematodiasis.
3. purposes as claimed in claim 2, it is characterized in that, the plant pathogenic fungi is cotton-wilt fusarium or oranges and tangerines charcoal
Subcutaneous ulcer germ.
4. the fermentation process of the thick wall reaping hook bacteria strain QJP1 described in claim 1, it is characterized in that,
1) the thick wall reaping hook bacteria strain QJP1 for preserving is inoculated in PDA plate surface, 26-30 DEG C is cultivated 5-10 days, used as scale
The strain of fermented and cultured, it is stand-by；
2) and then take above-mentioned strain and add in fermentation medium at 26-30 DEG C shaking table or standing for fermentation culture 10-40 days, sent out
Ferment product is stand-by；The fermentation medium is：Potato 200g, glucose 20g, distilled water 1000mL, pH are adjusted to 7.0.
5. the method for elution fraction I and elution fraction II being prepared using the tunning prepared by claim 4 simultaneously, its feature
It is,
1) tunning is first used into Solvent Extract methods, extract solution vacuum distillation obtains medicinal extract；
And then medicinal extract carries out column chromatography for separation 2)；
3) with petroleum ether-ethyl acetate gradient elution, petroleum ether-ethyl acetate volume ratio 10 is collected:The elution fraction I of 1 gradient
It is standby；
4) and then with chloroform-methanol gradient elution, chloroform-methanol volume ratio 20 is collected:The elution fraction II of 1 gradient is standby.
6. method as claimed in claim 5, it is characterized in that, the organic solvent is ethyl acetate, chloroform, acetone, methyl alcohol, second
One or more in alcohol or n-butanol.
7. method as claimed in claim 5, it is characterized in that, the column chromatography is using silica gel column chromatography, gel filtration chromatography, anti-phase
One or more in column chromatography or macroporous adsorbent resin column chromatography.
8. the elution fraction I in claim 5-7 prepared by any one is preparing micro- life of preventing and treating plant root-knot nematodes evil
Purposes in terms of thing nematicide.
9. the elution fraction II in claim 5-7 prepared by any one is preparing micro- life of preventing and treating plant pathogenic fungi disease
Purposes in terms of thing bactericide.
10. purposes as claimed in claim 9, it is characterized in that, the plant pathogenic fungi is cotton-wilt fusarium or oranges and tangerines
Anthrax bacteria.
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| CN201710117103.XA CN106701602B (en) | 2017-03-01 | 2017-03-01 | Fusarium chlamydosporia and application thereof |
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| CN201710117103.XA CN106701602B (en) | 2017-03-01 | 2017-03-01 | Fusarium chlamydosporia and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107858295A (en) * | 2017-11-27 | 2018-03-30 | 天津工业大学 | One plant is applied to high ammonia nitrogen landfill leachate processing bacterium and its application |
| CN108299277A (en) * | 2018-02-08 | 2018-07-20 | 青岛农业大学 | A kind of preparation and its application of indole derivatives |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104250620A (en) * | 2014-09-24 | 2014-12-31 | 山东省农业科学院植物保护研究所 | Chlamydospore fusarium strain and application thereof |
| CN105307492A (en) * | 2013-04-19 | 2016-02-03 | 拜耳作物科学股份公司 | Binary insecticidal or pesticidal mixture |
-
2017
- 2017-03-01 CN CN201710117103.XA patent/CN106701602B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105307492A (en) * | 2013-04-19 | 2016-02-03 | 拜耳作物科学股份公司 | Binary insecticidal or pesticidal mixture |
| CN104250620A (en) * | 2014-09-24 | 2014-12-31 | 山东省农业科学院植物保护研究所 | Chlamydospore fusarium strain and application thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107858295A (en) * | 2017-11-27 | 2018-03-30 | 天津工业大学 | One plant is applied to high ammonia nitrogen landfill leachate processing bacterium and its application |
| CN108299277A (en) * | 2018-02-08 | 2018-07-20 | 青岛农业大学 | A kind of preparation and its application of indole derivatives |
| CN108299277B (en) * | 2018-02-08 | 2020-11-10 | 青岛农业大学 | Preparation and application of indole derivative |
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| CN106701602B (en) | 2020-03-27 |
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