CN104403960B - A kind of biocontrol bacterial strain and its using method for reducing Eupatorium cannabinum L. (Eupatorium coelestinum L.) allelopathy - Google Patents
A kind of biocontrol bacterial strain and its using method for reducing Eupatorium cannabinum L. (Eupatorium coelestinum L.) allelopathy Download PDFInfo
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Abstract
The invention discloses a kind of biocontrol bacterial strain for reducing Eupatorium cannabinum L. (Eupatorium coelestinum L.) allelopathy and its application.The biocontrol bacterial strain is arthrobacterium (Athrobacter sp.) ZS3, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number CGMCC NO.9283.Concentration can be in 5 days in minimal medium the arthrobacterium ZS3 of the present invention degraded 49.8% and 56.8% of Herba Lycopi's diketone and hydroxyl Herba Lycopi's ketone of 100ug/mL.Ratio spraying concentration with 4mL/100g soil is as 109～1010After CFU/mL ZS3 microbial inoculums, the allelopathy of Eupatorium cannabinum L. (Eupatorium coelestinum L.) reduces 46.8%.This shows that the bacterial strain effectively can be degraded allelochemical, have broad application prospects in terms of prevention and control Eupatorium cannabinum L. (Eupatorium coelestinum L.).
Description
Technical field
The invention belongs to technical field of plant protection, being specifically related to one kind can effectively reduce Alien Invasive Plants purple stem
The biocontrol bacterial strain of Herba Lycopi's allelopathy.Meanwhile, the invention further relates to using method of the biocontrol bacterial strain in Biological control.
Background technology
Eupatorium cannabinum L. (Eupatorium coelestinum L.) (Ageratina adenophorum) is the perennial pernicious exotic invasive weed of Compositae, is originated in
The Mexico and Costa Rica in Central America, is widely distributed in the world torrid zone, more than 30, subtropical zones countries and regions at present.Purple stem
Herba Lycopi's invasion causes serious impact to local environment, is mainly manifested in the growth for squeezing local other plant, is formed
Single excellent population, destroys bio-diversity.Eupatorium cannabinum L. (Eupatorium coelestinum L.) invasion farmland, forest land, behind pasture, fight for crops, herbage and forest
Crops and economic plants, meadow maintenance, the renewal of forest are had significant impact by fertilizer, water, sunlight and space.Research finds purple
The stem of stem Herba Lycopi, leaf extract and root secretion contain allelochemical, can significantly inhibit the sprouting and growth of endemic plant.Poplar
National Day etc. (2006) tracks the method for combining using chromatographic isolation and raw survey, separates and filter out Eupatorium cannabinum L. (Eupatorium coelestinum L.) leaching approach
2 kinds of main effect allelochemicals, and Structural Identification has been carried out to which using nuclear magnetic resonance, NMR and makings technology used in conjunction：
It is respectively designated as I:4,7- dimethyl -1- (propane -2- methylene) -1,4,4a, 8a- naphthanes -2,6 (1H, 7H) -
Diketone (Herba Lycopi's diketone, DTD) and II:Light base -5- isopropyls -3,8- dimethyl-the 4a of 6-, 5,6,7,8., 8a- hexahydro naphthalenes -2
(1H) -one (hydroxyl base Herba Lycopi's ketone, HHO).
For a long time, to anti-Zhiduo County of Eupatorium cannabinum L. (Eupatorium coelestinum L.) from cultural control and chemical prevention, prevention effect is not obvious, and
And cause a certain degree of environmental pollution.Therefore, Biological control starts more and more to be weighed due to the advantage of its own
Depending on.But research of the prior art in terms of Biological control is relatively fewer, and mainly carry out in terms of the natural enemy of Eupatorium cannabinum L. (Eupatorium coelestinum L.),
Such as Herba Lycopi eats fly (Procecidochares utilis Stone) and Cercospora eupatorii (Cercospora eupatorii) etc..
If will can be effective control Eupatorium cannabinum L. (Eupatorium coelestinum L.) from reducing the allelopathy of Eupatorium cannabinum L. (Eupatorium coelestinum L.), weakening the angle of its competitiveness
Invasion provides a kind of new approach, but there is not yet successfully reporting in prior art.
The content of the invention
Present invention aims to the deficiencies in the prior art, there is provided one kind can effectively reduce Eupatorium cannabinum L. (Eupatorium coelestinum L.) allelopathy
Biocontrol bacterial strain.
The present invention also aims to provide using method of the described biocontrol bacterial strain in Eupatorium cannabinum L. (Eupatorium coelestinum L.) Biological control.
The purpose of the present invention is achieved by following technical proposals.
A. the present invention has separated one plant of superior microorganism ZS3 as test strain from Eupatorium cannabinum L. (Eupatorium coelestinum L.) invasion soil, to which
Carry out 16s rRNA sequencings and NCBI is compared, qualification result shows that the 16s rDNA sequences of the bacterial strain and Arthrobacter similarity reach
To 99%, its Classification And Nomenclature is arthrobacterium (Arthrobacter sp) .ZS3, and preserving number is CGMCC No.9283.
The morphological feature of arthrobacterium ZS3 (Arthrobacter sp ZS3) bacterial strain of the present invention is：Cell is not in
Rule is shaft-like, long 1～3 μm, a width of 0.4～1 μm.Often V-shaped arrangement, end circle.It is shaft-like to fragment into bead, 0.5～1 μm of diameter,
It is single, arrange in pairs, irregular stack.There is the club cycle.Gram-positive, does not give birth to spore, aerobic, hydrolysis starch, oxidase
Feminine gender, contacts enzyme positive.
The invention provides a kind of pole bacteria preparation, said preparation is prepared according to a conventional method, will arthrobacterium ZS3
(Arthrobacter sp ZS3) makes microbial inoculum after expanding fermentation culture.
The preparation method of microbial inoculum is preferably：Arthrobacterium ZS3 (Arthrobacter sp ZS3) is carried out with activation medium
Activation；Will be the amplification culture base of sterilizing quantitatively standby；Strain after activation is seeded to the amplification culture base that sterilization treatment is crossed respectively
Middle amplification culture, shaken cultivation 1～5 day；Bacterium solution is centrifuged 10 minutes in 4000～6000r/min, precipitation is diluted with aquesterilisa
It is 1 × 10 into ZS3 viable bacteria concentrations sum9～1 × 1010The microbial inoculum of CFU/ml.
B. present invention also offers the arthrobacterium ZS3 (Arthrobacter sp ZS3) is in Eupatorium cannabinum L. (Eupatorium coelestinum L.) Biological control
In using method, its concrete practice is as follows：With the ZS3 biocontrol agents for preparing, (concentration is 109～1010CFU/ml) with 4mL/
The amount of 100g soil is sprayed application in soil.
Compared with prior art, the present invention has the advantages that following prominent：
It is demonstrated experimentally that arthrobacterium ZS3 (Arthrobacter sp ZS3) in minimal medium in 5 days can by DDT and
HHO (concentration is 10mg/L) degradeds 49.8% and 56.8%.Sprout experiment to show, ZS3 bacterial strains is sprayed and can reduce after the raw-soil
Eupatorium cannabinum L. (Eupatorium coelestinum L.) allelopathy 46.8%.The bacterial strain effectively can be degraded Eupatorium cannabinum L. (Eupatorium coelestinum L.) allelochemical DDT and HHO, reduce Eupatorium cannabinum L. (Eupatorium coelestinum L.)
Allelopathy, weaken its competitiveness to a certain extent, the growth of the eciophyte of the competition for giving provides more machines
Meeting, promotes the bio-diversity of Disturbed habit.Due to being biological preparation, do not use that herbicide brought completely a series of asks
Topic.The bacterial strain has broad application prospects in terms of preventing and treating Eupatorium cannabinum L. (Eupatorium coelestinum L.).
The explanation of preservation biomaterial
The bacterial strain of the present invention, on 06 09th, 2014, is deposited in China Committee for Culture Collection of Microorganisms general
Logical microorganism center (CGMCC)；The centre address：City of BeiJing, China Chaoyang District North Star West Road 1 institute 3, the Chinese Academy of Sciences is micro-
Biological study institute.The strain classification is named as arthrobacterium (Arthrobacter sp) .ZS3；Preserving number is CGMCC No.9283.
Specific embodiment
In order that the objects, technical solutions and advantages of the present invention become more apparent, by the following examples and test number
According to being described in further detail to the present invention.It should be appreciated that specific embodiment described herein is only to explain this
It is bright, it is not intended to limit the present invention.
Experimental technique used in following embodiments if no special instructions, is conventional method.
Embodiment I, the separation of DDT and HHO degradation bacteria ZS3 bacterial strains and identification
First, the separation of degradation bacteria ZS3 bacterial strain
Bacterial strain screening step:Respectively by 1g Eupatorium cannabinum L. (Eupatorium coelestinum L.) root soil input the 60ml containing Herba Lycopi's diketone and hydroxyl Herba Lycopi's ketone without
Machine salt culture medium, shaking table culture took 100 μ L respectively and coat containing on two allelochemical inorganic salt culture plates, be inverted after 1 day
In constant incubator, 30 DEG C of cultures.After bacterium grows, with the bacterium of inoculating loop picking different shape feature in two kinds of culture medium
Fall, then, in nutritive solid culture medium repeatedly line, separate, purification.Then the bacterial strain for filtering out is transferred to containing two kinds
Rule on allelochemical inorganic salt solid medium, it is isolated to have well tolerable property and degradation property to two kinds of allelochemicals
Bacterial strain.10 plants of bacterium are screened from Eupatorium cannabinum L. (Eupatorium coelestinum L.) root soil by said method.To guarantee stablizing for strains for degrading performance, further sieve
Choosing.By 10 plants of bacterium in Nutrient medium amplification culture 2 days, take that 1 collarium bacterium solution accesses 40mL diketone containing Herba Lycopi and hydroxyl is damp respectively
In the inorganic salt culture fluid of each 400 μ L of blue ketone, shaking table culture took 100 μ L respectively and coats corresponding allelochemical inorganic salt after 4 days
On solid medium, see whether that bacterium grows.The bacterium that selection is grown on allelochemical inorganic salt solid medium.Will domestication
The bacterium for obtaining accesses Nutrient medium amplification culture 2 days, takes l mL bacterium solutions (using colony counting method quantification) and accesses 10mL
In the inorganic salt culture fluid of diketone containing Herba Lycopi and each 100 μ L of hydroxyl Herba Lycopi's ketone, shaking table culture 5 days is measured by sampling wherein 2 allelopathics
The content of material.By these tests, it has been found that bacterial strain ZS3 has more stable degraded to make to the allelochemical of Eupatorium cannabinum L. (Eupatorium coelestinum L.)
With.Therefore select for bacterial strain ZS3 to be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is
CGMCC No.9283。
Nutrient medium used：Glucose 5g；Peptone 2g；Yeast extract 1g；Water 1L；Agar 15-20g；PH=
7.5。
Minimal medium：Ammonium nitrate 3g；Potassium dihydrogen phosphate 0.5g；Dipotassium hydrogen phosphate 0.5g；Trace element storing solution
2mL；Water 1L；15～20g of agar；PH=7.5.Wherein trace element storing solution is included：Magnesium sulfate 4g, copper sulfate 1g, manganese sulfate
1g, iron sulfate 1g, calcium chloride 1g, water 1L.Save backup during 4 DEG C are placed after sterilizing.
Allelochemical storing solution:Herba Lycopi's diketone and hydroxyl Herba Lycopi ketone 10mg are weighed, is dissolved in 10mL acetone, is obtained final product 1g/L
(1mg/mL) storing solution.
Allelochemical minimal medium:A certain amount of allelochemical storing solution is taken in above-mentioned minimal medium, end is made
Concentration is 1mg/L.
Allelochemical inorganic salt solid medium:On the inorganic salt solid medium for having prepared, 100uL allelopathics are coated with
Reserve supply liquid.
2nd, the identification of Eupatorium cannabinum L. (Eupatorium coelestinum L.) Allelochemical degradation bacterium ZS3
From the following aspects, identification isolate and purify in aforementioned manners the degraded Eupatorium cannabinum L. (Eupatorium coelestinum L.) allelochemical DDT that obtains and
The bacterial strain ZS3 of HHO.
Morphological Identification：Will be in exponential phase, and stable degradation bacteria ZS3 of bacterium colony size carries out single bacterium colony form and retouches
State, the main size for including bacterium colony, color, transparency, wettability, bacterium colony apparent condition (whether flat, projection, fold, depression
Deng), colony edge state (whether neat, irregular, radial etc.)；For degradation bacteria strains ZS3, Jing in exponential phase
The form of observation by light microscope thalline is adopted after smear staining.
Analysis of physio biochemical characteristics：With reference to《Common bacteria system identification handbook》(east show pearl, the wonderful English .2011. of Cai are common thin
Fungus strain system identification handbook. Beijing：Science Press) and《Microbiology Experiment》(Shen Ping, Fan Xiurong, the Li Guang force micro- lifes of .1999.
Thing tests (third edition). Beijing：Higher Education Publishing House) determine the physiological and biochemical property of above-mentioned ZS3 bacterial strains.
As a result：ZS3 strain cells are irregularly shaft-like, long 1～3 μm, a width of 0.4～1 μm.Often V-shaped arrangement, end circle.Bar
Shape fragments into bead, and 0.5～1 μm of diameter is single, arranges in pairs, irregular stack.There is the club cycle.Gram-positive, no
Raw spore, aerobic, hydrolysis starch, oxidase negative contact enzyme positive.It is sequenced by 16s rDNA, and NCBI is compared, it has been found that
The 16s rDNA sequences of bacterial strain ZS3 reach 99% with Arthrobacter similarity.During bacterial strain ZS3 is preserved in in June, 2014
State's Microbiological Culture Collection administration committee common micro-organisms center, preserving number are CGMCC No.9283.
Embodiment II, arthrobacterium (Arthrobacter sp.) ZS3 degraded DDT and HHO ability quantitative determinations
First, the standard curve making of allelochemical DDT and HHO
DDT the and HHO standard solution of 5,20,80,120,200 μ g/mL is respectively with HPLC level methanol configuration concentration.Each
Concentration sample introduction 3 times, results averaged are mapped to concentration with peak area, draw standard curve.
2nd, ZS3 strains for degrading DDT and HHO abilities are determined
Bacterial strain ZS3 is accessed into 40mL Nutrient medium amplification culture 2 days, 4000r/min is centrifuged 5 minutes, precipitation with 1mL without
Bacterium aqueous suspension, determines bacteria suspension concentration using colony counting method.Take l00uL bacterium solutions access 10mL and respectively contain DDT and HHO 200ug's
In inorganic salt culture fluid, shaking table culture 5 days is measured by sampling the content of two allelochemicals.
The extracting process of DDT and HHO:The dissolubility of DDT and HHO in water is very low, tests the concentration for adopting and is far longer than
Its dissolubility, therefore be distributed with uneven crystal state in the solution, the measure of its concentration must whole bottle sample whole
Extraction.Selection normal hexane is extractant, is comprised the following steps that:Culture fluid is transferred to into 50mL separatory funnels, with 1/2 culture liquid
Long-pending normal hexane washing triangular flask, washing liquid are also transferred in separatory funnel, horizontal oscillations 5min, stratification.Subnatant (is had
Machine phase) received by anhydrous sodium sulfate dehydration (in a little Cotton Gossypii of little funnel bottom plug, putting about 2g anhydrous sodium sulfates above) afterwards
Combine in Cor Gigeriae Galli bottle；Upper liquid (water phase) is extracted once according to front method again with the normal hexane of 1/2 nutrient solution volume again, equally by lower floor
Fluid dewatering, is collected in Cor Gigeriae Galli bottle.40 DEG C of liquid rotary evaporator in Cor Gigeriae Galli bottle is evaporated under reduced pressure, be concentrated into 2mL, and mistake-
0.45 μm of microporous filter membrane, takes 1mL to chromatography column feed materials bottle.Determined with Ultra Performance Liquid Chromatography instrument.The degradation rate of bacterial strain with added with
In the process of ZS3 bacterial strains allelochemical content relative to the reduction percentage ratio of allelochemical content in control treatment representing, i.e.,
(VControl-VZS3)/VControl×100。
As a result：When the initial concentration of allelochemical DDT and HHO is 100 μ g/mL, after 5 days, arthrobacterium ZS3 is to DDT and HHO
Degradation rate be respectively 49.8% and 56.6%.
The effect of 1 arthrobacterium ZS3 of table degraded allelochemical DDT and HHO
| Allelochemical | Experiment process | Initial content (μ g/mL) | 5 days residual quantities (μ g/mL) | Degradation rate % |
| DDT | ZS3 | 100 | 48.4±84.3 | 49.8±8.6 |
| Blank | 100 | 96.5±2.7 | ||
| HHO | ZS3 | 100 | 28.9±3.6 | 56.6±5.5 |
| Blank | 100 | 66.6±3.2 |
It is prepared by embodiment III, ZS3 biocontrol agents
By biocontrol bacterial strain ZS3, activated with activation medium；Will be the amplification culture base of sterilizing quantitatively standby；After activation
Strain be seeded to amplification culture in the amplification culture base that sterilization treatment is crossed, shaken cultivation 1～5 day respectively；By bacterium solution 4000
～6000r/min is centrifuged 10 minutes, and precipitation aquesterilisa is diluted to ZS3 viable bacterias total concentration for 1 × 109～1 × 1010CFU/ml's
Microbial inoculum.
Culture medium used in microbial inoculum preparation：
Activation medium：Ammonium nitrate 3g；Potassium dihydrogen phosphate 0.5g；Dipotassium hydrogen phosphate 0.5g；Trace element storing solution 2ml；
Yeast extract 1g；Water 1L；DTD 1g；HHO 1g；PH 7.0 or so.Trace element storing solution is included：Magnesium sulfate 4g, copper sulfate
1g, manganese sulfate 1g, iron sulfate 1g, calcium chloride 1g, water 1L.
Amplification culture base：Peptone 5g, yeast extract 1g, NaCl 2g；DTD 2g；HHO 2g；pH 7.0.
Embodiment IV, application experiment
With the ZS3 biocontrol agents for preparing, (concentration is 109～1010CFU/ml 50g is sprayed application to the amount of 4mL/100g soil)
In the raw-soil, then on soil sow recipient plant Chinese cabbage seed.Meanwhile, setting is not added with the control treatment of biocontrol agent.Each
Process 8 repetitions, 30 Chinese cabbage seeds of each repeat sowing.10% Eupatorium cannabinum L. (Eupatorium coelestinum L.) blade leaching liquor 20mL is watered after 24h, with
And water same volume water be blank.After 5 days, the sprouting number of Chinese cabbage is recorded.Allelopathy with Eupatorium cannabinum L. (Eupatorium coelestinum L.) leaching liquor at
The percentage ratio that Chinese cabbage seed germination rate is reduced relative to the control that adds water after reason representing, i.e. E=(VWater-VLeaching liquor)/VWater×100。
As a result show, after spraying ZS3 microbial inoculums, the allelopathy of Eupatorium cannabinum L. (Eupatorium coelestinum L.) reduces 46.8%.ZS3 bacterial strains are imitated to allelopathic
The impact answered represented by being inoculated with biological and ecological methods to prevent plant disease, pests, and erosion soil bacteria relative to the reduction percentage ratio of soil is not inoculated with, i.e. (EControl-EZS3)/EControl
×100。
Table 2 adds and is not added with arthrobacterium ZS3 and processes lower allelopathy effect of the Eupatorium cannabinum L. (Eupatorium coelestinum L.) to Chinese cabbage
| Process | Allelopathy effect (germination percentage reduces percentage ratio %) |
| ZS3 | 34.7±8.2 |
| Blank | 65.3±8.2 |
| Allelopathy effect reduces percentage ratio (%) | 46.8 |
Claims (3)
1. it is a kind of reduce Eupatorium cannabinum L. (Eupatorium coelestinum L.) allelopathy biocontrol bacterial strain, it is characterised in that：The bacterial strain is arthrobacterium
(Arthrobacter sp.) ZS3, preserving number are CGMCC No.9283.
2. the using method of the biocontrol bacterial strain described in claim 1, it is characterised in that：Arthrobacterium ZS3 is expanded after fermentation culture
Concentration is made for 109～1010The microbial inoculum of CFU/ml, is then sprayed application in soil with the amount of 4mL/100g soil.
3. the using method of biocontrol bacterial strain according to claim 2, it is characterised in that described expansion fermentation culture is concrete
For：Arthrobacterium ZS3 is activated with activation medium；Will be the amplification culture base of sterilizing quantitatively standby；Strain after activation point
Amplification culture in the amplification culture base that sterilization treatment is crossed, shaken cultivation 1～5 day are not seeded to；By bacterium solution in 4000～6000r/
Min is centrifuged 10 minutes, and it is 1 × 10 that precipitation aquesterilisa is diluted to ZS3 viable bacteria concentrations sum9～1 × 1010The microbial inoculum of CFU/ml.
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Non-Patent Citations (2)
| Title |
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| Trichoderma harzianum SQR-T037 rapidly degrades allelochemicals in rhizospheres of continuously cropped cucumbers;Lihua Chen et al.;《Applied Microbiology and Biotechnology》;20101024;第89卷(第5期);1653-1663 * |
| 胜红蓟的主要化感物质早熟素Ⅱ降解菌的筛选、鉴定及其降解特性研究;张桂花;《中国优秀硕士论文全文数据库 2011 Agriculture Science and Technology》;20110115(第01期);D046-213 * |
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