CN104798819B - A kind of method for improving turfgrass anti-seismic design using low temperature resistant microbial bacterial agent is strengthened - Google Patents
A kind of method for improving turfgrass anti-seismic design using low temperature resistant microbial bacterial agent is strengthened Download PDFInfo
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Abstract
The invention discloses a kind of method for improving turfgrass anti-seismic design using low temperature resistant microbial bacterial agent is strengthened.The low temperature resistant microbial bacterial agent of described reinforcing, it be by ratio of weight and number be 1:1:1:1 reinforcing Pseudomonas stutzeri, strengthen bacillus subtilis, strengthen pleosporaceae and strengthen cryptococcus composition；Described reinforcing Pseudomonas stutzeri, reinforcing bacillus subtilis, reinforcing pleosporaceae and reinforcing cryptococcus refers to：The complex microbial community of various concentrations gradient is coated on beef-protein medium, on Gao Shi No. I culture medium and martin substratum, is placed in artificial climate incubator and cultivates 2 weeks, temperature is 10 DEG C.The acquisition that the present invention strengthens low temperature resistant microbial strains is to cultivate producing fertilizer from refuse in daily life microorganism under the conditions of 10 DEG C.It is contemplated that obtaining reinforcing compost microorganism, and technical support is provided to strengthen the application of the turfgrass low-temperature resistance of producing fertilizer from refuse in daily life microbial bacterial agent.
Description
Technical field
The invention belongs to environmental protection technical field, be related to using strengthen low temperature resistant microbial bacterial agent improve turfgrass resist it is low
The method of warm ability.
Background technology
The complex micro organism fungicide that microbial bacterial agent is especially made up of multiple-microorganism is to grow up to have in recent years
The biotechnology of wide application prospect.Microbial bacterial agent not only can be with organic in remedying oil-polluted soil and degraded water body
Pollutant, pest and disease damage can also be prevented and treated, promote crop production and increasing both production and income, some microbial bacterial agents can improve the anti-of plant
Inverse property is since the 1970s, and American-European Japan etc. has all succeeded in developing some composite bacteria agents in country in succession, a lot
Have begun to be produced on a large scale, and form the product of seriation.Wherein the 1980s is by Japanese university of Ryukyu
Teach the EM developed（CEM）Obtain great success, be widely used in individual country more than 90 be related to plant industry support
In terms of growing industry and the depollution of environment, and achieve significant economic benefit and social benefit.It is flourishing at some based on microbial bacterial agent
National research and extension obtains more early, and correlation technique is more ripe, using also more commonly.In recent years, it is external also not occur picture again
EM can cause the microbial bacterial agent of very big effect in the whole world like that.
Relative to the research of developed countries related microorganisms microbial inoculum, China is micro- life since the 1980s is just
The correlative study of thing microbial inoculum, from theory into action, from the Application of composite for using multiple strains of single culture, also obtain in succession
Some achievements.Agricultural University Of Nanjing's biological control product mainly has the waxy Bacillus of preventing and treating Cucumber root-knot nematode disease evil
AT31 microbial inoculums（Mission/line goes out, LS20120060）, preventing and treating soil-borne diseases of vegetable probiotics（Vegetable obtains health）, peaceful shield series
Product, bio-feritlizer formal registration is obtained.Shandong biology institute trichoderma（Prevent and treat gray mold）, Hunan Institute of Plant Protection photosynthetic bacteria
（Promote photosynthesis）, the short kiss bacillus in Zhenjiang Runzhou（Prevent and treat lepidoptera pest）And Ningxia Nuo get Man biotech companies root nodule
Bacterium（Promote legume growth）Deng.There are much reports on composite bacteria agent development recent years, the country, mainly utilize micro- life
Thing environment purification, degraded organic contamination, improve grain yield and improve plant resistance to environment stress etc..There are some researches show utilize from aquatic products
Isolated dominant bacteria develops water quality cleansing agent in the fine water of cultivation, uses it in freshwater fish culturing pond, right
Reduce the COD, NH in water body3- N, nitrite etc. serve preferable effect.Also there are some researches show the equal energy of microbial-bacterial fertilizer
The growth of different degrees of promotion romaine lettuce, improves lettuce quality, reduces nitrate content under suitable concentration.But these are mostly
It is horizontal also in experimental study and Preliminary Applications, it is not widely used in reality also.Biocontrol microorganisms have fixed nitrogen, phosphorus decomposing, solution in itself
The effect of potassium, plant is stimulated to produce appropriate auxin；Degraded larger molecular organicses, improve organic fertilizer utilization rate；Microbial bacterial agent
It is like the appetizer of plant；Protect the integrality of cell membrane, maintain the root activity and chlorophyll content of higher level, this side
The face just not just effect of a growth-promoting, also plays the role of a kind of degeneration-resistant.A variety of degeneration-resistant bases in microbial bacterial agent induction plant
The expression of cause, significantly improve the activities of antioxidant enzymes of plant.Microbial bacterial agent produces disease after can preventing crop harvesting simultaneously
The generation of a variety of related substanceses such as evil, induction adversity gene expression, induction SOD.So microbial bacterial agent exists for improving plant
Adverse circumstance is that the planting under the conditions of salt stress, drought stress and low temperature stress has active influence.
Turfgrass is except with beautifying the environment, purify air, prevent erosion, keep ecological balance, provided to people
Outside the function such as rest and sports center, still play the role of to adjust miniclimate.But turfgrass is the same with other plants, often by not
The influence of good environment, such as arid, high temperature, low temperature, salt marsh adverse circumstance can all suppress the growth of turfgrass, decline turf quality.
Especially saline and alkaline, low temperature（Damage to plants caused by sudden drop in temperature）It is 3 big abiotic factors of strong limitation lawn growth with arid.
Chilling injury is one of main meteorological disaster in China, and the most frequent and severe with northern area.Chilling injury
It is during crop growth, although more than 0 DEG C of daily minimal tcmperature, weather is warmer, continuing for long period occurs
Property microthermal climate, or occur short-term strong microthermal climate process during being also crop reproductive growth, daily mean temperature is less than making
Thing grows the lower limit of preference temperature, so as to influence crops grow or agricultural that is solid and causing the underproduction from
Right disaster.Chilling injury also influences growth and the normal physiological biochemical process of this lawn plant to some extent, especially limits
The main reason for sod production cycle.Low temperature stress can cause SOD（Superoxide dismutase）、CAT（Catalase）Deng guarantor
The reduction of enzymatic activity is protected, chlorophyll decomposes, and proline and mda content increase are relative permeability of membrane increase, and then plant is thin
Born of the same parents sustain damage.When being influenceed there are some researches show low temperature stress on growth of Festuca elata, the reduction with temperature and stress time are drawn
Extend, the CAT activity reductions in Festuca Arundinacea blade are also faster, and chlorophyll content also decreases, and proline content increase is obvious.
The cool-season grasses optimum sowing time is It is the end of summer, just turning into autumn or early spring, and warm season turf is in the end of spring and the beginning of summer.After planting 40
It is removable level ground after~50d, when the coverage rate of the turfgrass on turf is up to more than 95%.It is so general in first northern roll of sod
It can just be listed in five, June.If the annual period of sod production can be shortened, ahead of time turf Time To Market, can greatly improve
The market competitiveness of roll of sod, improve the income of people.The method of the cold resistance of traditional raising lawn plant has, and sprays
Growth regulator, such as cryoprotective agent；Carry out Cold Acclimation.And improve lawn plant using complex micro organism fungicide is applied
Anti-cold research have not been reported.Microbial bacterial agent is as a kind of bio-feritlizer efficiently, environmentally friendly, and its disease-resistant growth-promoting functions is
Get the nod, it will also have certain effect in raising lawn plant winter resistance and shortening sod production week etc..
Producing fertilizer from refuse in daily life is the microorganism in nature, for example bacterium and fungi etc., passes through their life
Reason metabolism, can accelerate the decomposition rate to organic matter, and then become the process of humic acid.Producing fertilizer from refuse in daily life has
In oxygen processing procedure, substantial amounts of heat can be produced, the organic matter in rubbish is decomposed completion, while also kills the disease in rubbish
Bacterium etc., and sporiferous bacillus can largely be present.Various microorganisms are phase not to the utmost to organic matter capacity of decomposition and decomposition rate
With, with temperature, the change in season, microbiologic population and quantity in composting process also differ；And there are some researches show,
Microbiologic population in compost is considerably complicated.In compost micro organism quantity and Species structure and compost organic material component and
The many factors such as the interaction between content, microorganism are closely related.Someone studies inoculation external source microbial inoculum to microorganism in compost
The influence of quantity and Enzyme activities, the improvement of application and composting process for microbial bacterial agent provide foundation.Pig manure stalk
Compost complex micro organism fungicide more effectively can promote and optimize composting process and improve heap temperature raising maximum temperature rapidly
Extend time megathermal period and can more effectively improve heap body nutrient levels.Cow dung compost experiment shows that addition origin microbial inoculum can be fast
Speed improves compost temperature, promotes heap body fermentation maturity, shortens the composting time；In terms of cellulose and hemicellulose degradation rate, addition
It is obvious stronger than being not added with the control treatment of microbial inoculum after microbial inoculum processing, it can be seen that, addition microbial bacterial agent is advantageous to cow dung compost
It is decomposed.And for extracting microorganism fungus kind from solid waste matrix, and use it for testing and produce, somebody distinguishes
Efficient degrading bacteria is filtered out from chicken manure fermenting and cow dung and is configured to corresponding complex micro organism fungicide, is composite microbial bacteria
Certain basis has been established in the application of agent, and this configuration to compost microbe microbial inoculum early stage both provides foundation.Recent correlation
Document and production show：Microbial bacterial agent as a kind of new biotechnology have been applied to our social life and
Among production.Especially microbial bacterial agent is promoting the research of plant growth, raising plant resistance to environment stress etc. too numerous to enumerate.But
Source and preparation on microbial bacterial agent, most researchers are also only only limited to extract and screen from soil or sludge.
About symbiosis and the relation of influencing each other have ambiguity and complexity between microbe colony structure, compost strain in compost.By force
Change prepares microorganism fungus kind in garbage compost, using reinforcing compost microorganism fungus kind as Inoculant, is configured to the strong of various concentrations
Change complex micro organism fungicide inoculation application, will have great importance.
From consumer garbage compost（Hereinafter referred to as compost）In filter out effective strain, then be configured to corresponding microorganism microbial inoculum,
It is with a wide range of applications.Some researchs show to find in composting municipal solid waste, in the mistake that organic matter is decomposed
Cheng Zhong, also there occurs important change, mainly pathogenic bacteria largely to reduce for biocoene, and then quantity is acute for sporiferous bacillus
Increase.The acid change of compost that house refuse obtains after being handled by hot fermentation is big and salt content uprises, therefore in hypertonic
Microorganism in system environment has necessarily acidproof, salt tolerant and high-temperature stability.Whether in general microbial bacterial agent, still
The seed selection of strain in compost microbe microbial inoculum, all it is directly from natural environment（Or compost）In filter out effective bacterial strain, then match somebody with somebody
Microbial bacterial agent is made and is applied to corresponding field.And by natural environment（Or compost）In microorganism pass through some specific directions
Reinforcing, and then the research for obtaining some efficient enhancement microbiological microbial inoculums there is no report.
One of the Main Factors of temperature as plant growth, transpiration, the flow of water, absorption and the metabolism of turfgrass can be influenceed
And almost all of enzymatic reaction, dormancy and grow.Research in terms of stress resistance of plant at present mainly determines
Its physical signs, such as biomass, protective enzyme, MDA, soluble sugar index.But relevant green returning of lawn situation and after turning green
The research report of lawn outward appearance Comprehensive Assessment is considerably less.
Microbial bacterial agent is used frequently as alleviating physiological ecological injury effect of the adverse environmental factor to lawn plant.But in low temperature
Under stress, strengthen low temperature resistant microbial bacterial agent has what to influence on turfgrass, rare people's research, and which kind of concentration microbial inoculum can make
It is even more that research is very few that the turfgrass handled under low temperature stress, which reaches optimum growh situation,.
The content of the invention
From consumer garbage compost obtained by the low temperature resistant microorganism Pseudomonas stutzeri of reinforcing, bacillus subtilis,pleosporaceaeAnd cryptococcus, and the complex micro organism fungicide that they are configured to various concentrations is inoculated into turf establishment body
In system, study under low temperature stress, the change of Turfgrass Growth index, it is therefore an objective to filter out the most suitable of complex micro organism fungicide
Concentration, improve lawn plant low temperature resistivity.The acquisition for strengthening low temperature resistant microbial strains is by the micro- life of producing fertilizer from refuse in daily life
Thing is cultivated under the conditions of 10 DEG C.This technology is intended to obtain reinforcing compost microorganism, and to strengthen the micro- life of producing fertilizer from refuse in daily life
The turfgrass low-temperature resistance application of thing microbial inoculum provides technical support.]
To achieve the above object, the invention discloses following technical scheme：
Strengthen low temperature resistant microbial bacterial agent, it is characterised in that it be by ratio of weight and number be 1:1:1:1 reinforcing Amur is false
Monad, strengthen bacillus subtilis, strengthenpleosporaceaeFormed with cryptococcus is strengthened；Described reinforcing Amur is false single
Born of the same parents bacterium, strengthen bacillus subtilis, strengthenpleosporaceaeReferred to cryptococcus is strengthened：By answering for various concentrations gradient
Close microorganism species and be coated on beef-protein medium, on Gao Shi No. I culture medium and martin substratum, be placed on artificial
Cultivated 2 weeks in weather incubator, temperature is 10 DEG C；The complex microbial community of described various concentrations gradient refers to 10-1、
10-2、10-3、10-4、10-5With 10-6µg/ml。
At 30 DEG C, 180r/min is cultivated for the reinforcing Pseudomonas stutzeri and bacillus subtilis of the present invention,pleosporaceaeWith cryptococcus at 28 DEG C, 220r/min is cultivated.From 600 nm wavelength（560 nm of fungi）Carry out than turbid
Measure, using the OD values of bacteria suspension as ordinate, incubation time is abscissa, draws the growth curve of microorganism, chooses logarithm life
Long-term strain configuration complex micro organism fungicide, complex microorganism bacterium solution by Pseudomonas stutzeri, bacillus subtilis,pleosporaceae1 is pressed with cryptococcus:1:1:1 ratio is configured.
The present invention further discloses strengthen low temperature resistant microbial bacterial agent improving the winter resistance on lawn, improving regrowth rate side
The application in face；Application particularly in terms of Festuca Arundinacea regrowth rate, the quality of Festuca Arundinacea, density and color and luster after raising is survived the winter.Its
Described in reinforcing complex microbial community refer to Pseudomonas stutzeri, bacillus subtilis, pleosporaceae and hidden ball
Bacterium by ratio of weight and the number of copies 1:1:1:1 ratio is configured.
The present invention further discloses the preparation method for strengthening low temperature resistant microbial bacterial agent, it is characterised in that by following
Step is carried out：
（1）The enrichment of strain：
Compost sample is weighed into 10g to be placed in sterile conical flask, after adding 100mL sterilized water shaken wells, takes 10mL to hang
Supernatant liquid, at 28 DEG C, shaken cultivation 3d under 220r/min, as mixes micro- life in the conical flask for filling 100mL enriched mediums
Thing flora；
（2）The reinforcing of mixed microorganism flora：The complex microbial community of various concentrations gradient is coated on beef extract egg
On white peptone culture medium, Gao Shi No. I culture medium and martin substratum, it is placed in artificial climate incubator and cultivates 2 weeks, temperature is
10 DEG C, the complex microbial community to be strengthened；The complex microbial community of described various concentrations gradient refers to 10-1、10-2、10-3、10-4、10-5With 10-6µg/ml。
（3）Pseudomonas stutzeri and bacillus subtilis will be strengthened at 30 DEG C, 180r/min cultures,pleosporaceae
With cryptococcus at 28 DEG C, 220r/min is cultivated.From 600 nm wavelength（560 nm of fungi）Turbidimetric assay is carried out, with bacteria suspension
OD values be ordinate, incubation time is abscissa, draws the growth curve of microorganism, choose exponential phase strain configuration
Complex micro organism fungicide, complex microorganism bacterium solution by Pseudomonas stutzeri, bacillus subtilis,pleosporaceaeWith hidden ball
Bacterium presses 1:1:1:1 ratio is configured.
It is of the invention further to disclose using the side for strengthening low temperature resistant microbial bacterial agent raising turfgrass anti-seismic design
Method, it is characterised in that carried out by the steps：
（1）Material：What is selected is the relatively common perennial Festuca Arundinacea of northern China, and matrix used is the campus prepared
Soil, sterilize 30 min at 121 DEG C, standby；
（2）Per placing sterile soil in culture vessel as matrix, planting carpet compounded turf matrix thickness is 15mm, application rate
For 160 gm-2, unify quantitative water supply daily, to keep culture matrix to have preferable water regime, and should often exchange each
Culture vessel position, to ensure that illumination is consistent；
（3）After plant to be planted grows into 15-20d, each 15ml of corresponding complex micro organism fungicide, lawn plant training are separately added into
During supporting, temperature is 12-20 DEG C, relative humidity 25-45%, and illumination is to penetrate the natural light of interior, after persistently handling 20d
Each index is determined, determines the plant height, biomass of individual tree and chlorophyll content of Festuca Arundinacea.
The more detailed preparation method of the present invention is as follows：
1 develops materials and methods
1.1 experiment material
Turfgrass choose full seed, uniformity Festuca Arundinacea (Festuca arundinacea L) seed is
Test material.It is Tianjin Normal University's garden mould in the school for examination soil, physicochemical property：PH is 7.44, salt content 0.29%, organic matter
The % of content 4.68, total nitrogen content 0.21 %, available phosphorus content 22.03mgkg-1, the full gkg of potassium 45.61-1, the g of unit weight 0.46
L-1, the gmL of saturation moisture content 0.58-1。
Reinforcing, separation, purifying and the identification of low temperature resistant microorganism in 1.2 garbage composts.
Culture medium
Enriched medium：Beef extract 5g, peptone 10g, NaCl 5g, water 1000ml, pH 7.4-7.6, add 15g agar into
Solid medium；
Beef-protein medium：Beef extract 5g, peptone 10g, NaCl 5g, water 1000ml, pH 7.0-7.2, add
15g agar is into solid medium；
No. I culture medium of Gao Shi：Soluble starch 20g, KNO3 20g, K2HPO30.5g, MgSO40.5g, FeSO4
0.01g, water 1000ml, pH 7.2-7.4, adds agar 20g into solid medium（During configuration, first with a small amount of cold water, starch is adjusted
Into pasty state, import in the water boiled, heated in fire, add other compositions while stirring, after dissolving, moisturizing to 1000ml）；
Martin substratum：Glucose 10g, peptone 5g, KHPO3 1g, MgSO (7H2O) 0.5g, 1% rose-bengal water
Solution, 3.3ml, water 1000ml, pH naturally, plus agar 15g into solid medium（0.03% streptomysin dilution is added before use
100ml, make every ml culture mediums μ g containing streptomysin 30）.
The enrichment of strain
Compost sample is weighed into 10g to be placed in sterile conical flask, after adding 100mL sterilized water shaken wells, takes 10mL to hang
Supernatant liquid, at 28 DEG C, shaken cultivation 3d under 220r/min, as mixes micro- life in the conical flask for filling 100mL enriched mediums
Thing flora.
The reinforcing of microorganism
The complex microbial community of various concentrations gradient is coated on No. I beef-protein medium, Gao Shi culture medium
In martin substratum, it is placed in artificial climate incubator and cultivates 2 weeks, temperature is 10 DEG C；Described various concentrations gradient
Complex microbial community refer to 10-1、10-2、10-3、10-4、10-5With 10-6µg/ml。
The separation and purifying of enhancement microbiological
Dilution spread flat band method
1）It is down flat plate：By beef extract-peptone agar medium, Gao Shi No. I agar medium, Martin（PDA）Agar is trained
Base high-temperature sterilization is supported, when being cooled to 55 ~ 60 DEG C, Streptomycin Solution is added in martin agar culture medium（Whole mass concentration is
30µg/ml）, be mixed it is even after be down flat plate respectively.
2）Prepare mixed microorganism dilution：The mixed microorganism bacteria suspension after 1ml strengthens is drawn with liquid-transfering gun and adds Sheng
Have in the Boiling tube of 9ml sterilized waters and fully mix, this is 10-1Dilution, 10 are made by that analogy-2、10-3、10-4、10-5With 10-6The dilution of several concentration.
3）Coating：The dilution bacteria suspension for drawing 0.2ml various concentrations respectively with liquid-transfering gun is accurately put into corresponding culture medium and put down
Plate center, each various concentrations gradient processing are repeated 3 times.Lightly it is coated with uniformly in media surface with sterile glass rod.
4）Culture：Beef extract-peptone flat board is inverted in 37 DEG C of incubators and cultivated, and will contain No. I culture medium of Gao Shi and Martin
Family name's culture medium（PDA）Flat board be inverted in 28 DEG C of incubators and cultivate.
Plate streaking partition method
Choose bacterium colony：The single bacterium colony grown after culture the difference a little lawn of picking is enterprising in new above-mentioned 3 kinds of culture mediums
Row line purifying.Until on culture medium grow be pure culture, such as it is impure, still need to repeat the step.
The identification of enhancement microbiological
The DNA of dominant bacteria is extracted according to the operation manual of Ezup pillar kits.The PCR system of predominant bacteria：10×
Buffer（with MgCl2）2 μ L, dNTP（10mmol/L）0.4 μ L, 341f（10µmol/L）1 μ L, 534r（10µmol/L）1µ
L, Taq enzyme（5u/µL）0.4 μ L, the μ L of template DNA 1, ultra-pure water is added to be settled to the μ L of final volume 20.PCR reaction conditions：94℃5min
Pre-degeneration, 94 DEG C of denaturation 1min, 55 DEG C of renaturation 45s, 72 DEG C of extension 45s, 30 circulations, 72 DEG C extend 10min.Primer is 341f
（5′-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG
CAG-3′）And 534r（5′-ATT ACC GCG GCT GCT GG-3′）.The PCR reaction systems of dominant fungi：10×Buffer
（without MgCl2）2 μ L, MgCl2（25mmol/L）1.6 μ L, dNTP（10mmol/L）0.4 μ L, Geo11（10µmol/L）
0.4 μ L, GeoA2（10µmol/L）0.4 μ L, Taq enzyme（5u/µL）0.2 μ L, the μ L of template DNA 1, add ultra-pure water to be settled to final volume
20µL.PCR reaction conditions：94 DEG C of 4min pre-degenerations, 94 DEG C of denaturation 1min, 54 DEG C of renaturation 1min, 72 DEG C of extension 2min, 30 are followed
Ring, 72 DEG C of extension 7min.Primer is GeoA2（5′-CCA GTA GTC ATA TGC TTG TCT C-3′）And Geo11（5′-
ACC TTG TTA CTT TTA CTT CC-3′）.Obtained PCR primer is sent to Beijing Hua Da gene sequencing portion, according to sequencing
Result, corresponding strain is found out in BLAST systems.
It is prepared by 1.3 complex micro organism fungicides
Spawn incubation:The low temperature resistant microorganism of reinforcing that screening obtains is expanded into culture in corresponding fluid nutrient medium.Apply
At 30 DEG C, 180r/min is cultivated for family name pseudomonad and bacillus subtilis,pleosporaceaeWith cryptococcus at 28 DEG C, 220r/
Min is cultivated.From 600 nm wavelength（560 nm of fungi）Turbidimetric assay is carried out, using the OD values of bacteria suspension as ordinate, culture
Time is abscissa, draws the growth curve of microorganism.The strain configuration complex micro organism fungicide of exponential phase is chosen, it is compound
Microbial inoculum is prepared by following processing.Pseudomonas stutzeri, bacillus subtilis,pleosporaceaeAnd cryptococcus
By 1:1:1:1 ratio is configured.
The application of 1.4 complex micro organism fungicides
The low temperature stress that this experiment uses was handled as whole winter, and temperature is about -6-10oC.Various concentrations it is compound micro-
Bacteria agent application, processing mode is set under corresponding low temperature stress：Processing 1 is control, is inoculated with corresponding blank cultures
（CK）；Processing 2 adds the microbial inoculum of 100 times of dilutions（CM1）；Processing 3 adds the microbial inoculum of 200 times of dilutions（CM2）.
1.5 turf establishment
What experiment material was selected is the relatively common perennial Festuca Arundinacea of northern China.Matrix used is the campus prepared
Soil.Sterilize 30 min at 121 DEG C, standby.Sterile soil is placed as matrix, planting carpetweed in every culture vessel during experiment
Skin stromal thickness is about 15mm, and application rate is 160 gm-2.Unified quantitative water supply daily, to keep culture matrix to have preferably
Water regime, and each culture vessel position should be often exchanged, to ensure that illumination is consistent, respectively handle 4 repetitions.
After plant to be planted grows into 15d, each 15ml of corresponding complex micro organism fungicide, control group are separately added into（CK）Add phase
The aseptic culture medium of same amount.During lawn plant is cultivated, temperature is 12-20 DEG C, relative humidity 25-45%, and illumination is to penetrate room
Interior natural light, each index is determined after persistently handling 20d.Plant height, biomass of individual tree and the chlorophyll of measure Festuca Arundinacea contain
Amount.
Add complex micro organism fungicide 15ml again after These parameters have been determined, then excessively locate plant progress low temperature
Reason, the temperature of excessive phase is 5-12oC.Excessively continue one week.Then remove according to common lawn winterization in Festuca Arundinacea
Hay, rotten grass, withered grass, then cut Festuca Arundinacea to 4cm or so, water of surviving the winter is poured, finally by the related culture vessel one of Festuca Arundinacea
Rise to be put into and stay in foraminate sealing carton, be put into room of the back without heating and pass the winter.Temperature is -6-10 during passing the winteroC。
Plant moved on into hot-house culture after continuing 40d, carries out processing of turning green, it is determined and turns green and situation and evaluate its outward appearance comprehensive quality.
1.6 testing index
1.6.1 Festuca Arundinacea plant height and biomass of individual tree
Plant height is determined for the first time after applying microbial inoculum 10d, it is rear to be determined once per 5d, totally 3 times.
After applying microbial inoculum 20d, 10 plants of Testuca arundinaceas are randomly selected from individual processing, are weighed, record fresh weight and dry weight.With
Its average biomass of individual tree as the processing.
1.6.2 the measure of chlorophyll content：
1.6.2 the measure of chlorophyll content
The measure of chlorophyll content uses AAS, that is, takes 0.2g lawn plant aerial part to be placed in mortar,
A small amount of quartz sand, calcium carbonate and 80% acetone are added, carries out being fully ground extraction, homogenate is transferred in 15mL centrifuge tubes, and with suitable
80% acetone washing bowl is measured, is transferred in the lump in centrifuge tube, precipitation is abandoned after centrifugation, take supernatant to be transferred in 25mL volumetric flasks, use
80% acetone is settled to graduation mark；Using 80% acetone as control, produce UV-1700 types ultraviolet specrophotometer with SHIMADZU and distinguish
Determine the absorbance at 663nm, 645nm wavelength.
Calculation formula is as follows：
1.7 data analysis
Handled using Microsoft Excel 2003 and SPSS11.7 softwares
2 development results are analyzed
2.1 bacterial screening
Selected 4 kinds of this experiment strengthen low temperature resistant microorganisms be respectively Pseudomonas stutzeri, bacillus subtilis,pleosporaceaeAnd cryptococcus.By determining Pseudomonas stutzeri, bacillus subtilis and cryptococcal growth curve（Figure
1）, then according to microbial growth curve may determine that its grows logarithmic phase, then the strain of this phase is extracted, be made into and do not exist together
Reason, the inoculation for lawn plant.
Pseudomonas stutzeri in microbial bacterial agent, bacillus subtilis and cryptococcus are determined with the method for plate culture count
Strain quantity,pleosporaceaeThen determined with paper disk method.Measurement result such as table 1, the clump count of the strain of gained 4 exist（1.51±
0.11）×109/mL- （2.85±0.03）×109Between/mL, the OD of Pseudomonas stutzeri600Nm values are 0.447, bacterium withered grass
The OD of bacillus600 Nm values are 0.356, cryptococcal OD560Nm values are 0.64.
Clump count contained by the microbial bacterial agent of table 1
Influence of 2.2 complex micro organism fungicides to Festuca Arundinacea plant height
Festuca Arundinacea dynamic plant height is shown in Table 2 after being vaccinated with the complex micro organism fungicide of various concentrations.As can be seen from the table, connect
, significant difference between treatment group and control group be present in kind microbial inoculum 10d, 15d and 20d.With 100 times of complex microorganism of dilution
Microbial inoculum application group treatment effect is best, compared with control group, has been respectively increased 24.30%, 9.51% and 9.85%.
Influence (cm) of the complex micro organism fungicide of the various concentrations of table 2 to Festuca Arundinacea plant height
Note：The different letters of data of going together represent significant differenceP<0.05Similarly hereinafter
Influence of 2.3 complex micro organism fungicides to Festuca Arundinacea ground biomass
As can be known from Table 3, complex micro organism fungicide significantly improves Festuca Arundinacea single-strain fresh weight, dry weight and fresh and dried ratio.It is different
Significant difference all be present between the complex micro organism fungicide of concentration and between treatment group and control group.Wherein, 100 times are inoculated with
Complex micro organism fungicide is diluted compared with control group, and 105%, 198.48% and has been respectively increased in fresh weight, dry weight and fresh and dried ratio
38.46%.Compared with control group, fresh weight, dry weight and fresh and dried ratio are respectively increased 200 times of dilution complex micro organism fungicides of inoculation
56.26%th, 100.38% and 23.08%.
Influence (mg/ strain) of the complex micro organism fungicide of the various concentrations of table 3 to Festuca Arundinacea ground biomass
Influence of 2.4 complex micro organism fungicides to Tall Fescue Leaves chlorophyll contents
As known from Table 4, complex micro organism fungicide processing can significantly improve Tall Fescue Leaves chlorophyll contents.Wherein composite microbial
100 times of dilutions processing of thing microbial inoculum is the most notable, and its chlorophyll a, chlorophyll b and Chlorophyll content are respectively what is compareed
1.22 times, 1.34 times and 1.27 times（P<0.05）.
Influence (mgg of the complex micro organism fungicide of the various concentrations of table 4 to Tall Fescue Leaves chlorophyll contents-1 FW)
3 develop conclusion
The use of complex micro organism fungicide significantly improves Tall Fescue Leaves chlorophyll contents in this research.Festuca Arundinacea inoculation is compound
After microbial bacterial agent, its individual plant is fresh and dried than increase, just illustrates the increase of Festuca Arundinacea dry-matter accumulation, is reduced with respect to water content, cold-resistant
Property relative also just raise.Compound micro- enhanced biological microbial inoculum is inoculated with this technology, the biomass of individual tree of Festuca Arundinacea can be significantly improved
And fresh and dried ratio, chlorophyll content, so as to effectively alleviate the injury that low temperature stress is brought to Festuca Arundinacea, it is favorably improved height
The lower temperature resistance of fescue grass.The significant effect of 100 times of dilution complex micro organism fungicides is better than 200 times of dilutions.So screening and
Cultivate corresponding complex microorganism bacterial classification and carry out artificial infection, plant cold resistance can be improved, so improve its regrowth rate and
Outward appearance comprehensive quality.This development and application and turfgrass low-temperature resistance for reinforcing compost high-effect bacterial is all most important.
Brief description of the drawings
Fig. 1 Pseudomonas stutzeris, bacillus subtilis and cryptococcal growth curve.
Embodiment
In order to more fully explain the implementation of the present invention, there is provided following preparation method embodiments.These embodiments are only
Only it is to explain rather than limit the scope of the present invention.Needing special instruction is：The complex microbial community that present invention screening obtains
Pseudomonas stutzeri, bacillus subtilis, pleosporaceae and cryptococcus in the market have sale, can also use this hair
Bright method isolated complex microbial community from consumer garbage compost, the biochemical characteristic of its obtained flora with it is commercially available
It is identical thus not in preservation.
Embodiment 1.
Strengthen the preparation method of low temperature resistant microbial bacterial agent：
（1）The enrichment of strain：
Compost sample is weighed into 10g to be placed in sterile conical flask, after adding 100mL sterilized water shaken wells, takes 10mL to hang
Supernatant liquid, at 28 DEG C, shaken cultivation 3d under 220r/min, as mixes micro- life in the conical flask for filling 100mL enriched mediums
Thing flora；
（2）The complex microbial community of various concentrations gradient is coated on No. I beef-protein medium, Gao Shi culture
On base and martin substratum, it is placed in artificial climate incubator and cultivates 2 weeks, temperature is 10 DEG C, and what is strengthened is compound micro-
Biological flora；The complex microbial community of described various concentrations gradient refers to 10-1、10-2、10-3、10-4、10-5With 10-6µ
g/ml。
（3）Pseudomonas stutzeri and bacillus subtilis will be strengthened at 30 DEG C, 180r/min cultures,pleosporaceae
With cryptococcus at 28 DEG C, 220r/min is cultivated.From 600 nm wavelength（560 nm of fungi）Turbidimetric assay is carried out, with bacteria suspension
OD values be ordinate, incubation time is abscissa, draws the growth curve of microorganism, choose exponential phase strain configuration
Complex micro organism fungicide, complex microorganism bacterium solution by Pseudomonas stutzeri, bacillus subtilis,pleosporaceaeWith hidden ball
Bacterium presses 1:1:1:1 ratio is configured.
Embodiment 2
The method for improving turfgrass anti-seismic design using low temperature resistant microbial bacterial agent is strengthened：
（1）Material：What is selected is the relatively common perennial Festuca Arundinacea of northern China, and matrix used is the campus prepared
Soil, sterilize 30 min at 121 DEG C, standby；
（2）Per placing sterile soil in culture vessel as matrix, planting carpet compounded turf matrix thickness is 15mm, application rate
For 160 gm-2, unify quantitative water supply daily, to keep culture matrix to have preferable water regime, and should often exchange each
Culture vessel position, to ensure that illumination is consistent；
（3）After plant to be planted grows into 15-20d, each 15ml of corresponding complex micro organism fungicide, lawn plant training are separately added into
During supporting, temperature is 18 DEG C, relative humidity 35%, and illumination is to penetrate the natural light of interior, is persistently determined after processing 20d each
Index, determine the plant height, biomass of individual tree and chlorophyll content of Festuca Arundinacea.
Claims (3)
1. one kind strengthens low temperature resistant microbial bacterial agent, it is characterised in that it by ratio of weight and number is 1 that it, which is,:1:1:1 reinforcing Amur
Pseudomonad, strengthen bacillus subtilis, strengthenpleosporaceaeFormed with cryptococcus is strengthened；Described reinforcing Amur is false
Monad, strengthen bacillus subtilis, strengthenpleosporaceaeReferred to cryptococcus is strengthened：By various concentrations gradient
Complex microbial community is coated on beef-protein medium, on Gao Shi No. I culture medium and martin substratum, is placed on people
Cultivated 2 weeks in work weather incubator, temperature is 10 DEG C；The complex microbial community of described various concentrations gradient refers to 10-1、
10-2、10-3、10-4、10-5With 10-6µg/ml；The reinforcing Pseudomonas stutzeri and bacillus subtilis are at 30 DEG C, 180r/min
Culture,pleosporaceaeWith cryptococcus at 28 DEG C, 220r/min is cultivated；
From 600 nm wavelength, fungi carries out turbidimetric assay with 560 nm, and using the OD values of bacteria suspension as ordinate, incubation time is
Abscissa, the growth curve of microorganism is drawn, choose the strain configuration complex micro organism fungicide of exponential phase, complex microorganism
Bacterium solution by Pseudomonas stutzeri, bacillus subtilis,pleosporaceae1 is pressed with cryptococcus:1:1:1 ratio is configured.
2. strengthen application of the low temperature resistant microbial bacterial agent in terms of the winter resistance on lawn is improved, improve regrowth rate；It is wherein described
Strengthen complex microbial community and refer to Pseudomonas stutzeri, bacillus subtilis, pleosporaceae and cryptococcus by weight
Portion rate 1:1:1:1 ratio is configured.
3. strengthen the preparation method of low temperature resistant microbial bacterial agent described in claim 1, it is characterised in that carry out by the steps：
（1）The enrichment of strain：Compost sample is weighed into 10g to be placed in sterile conical flask, adds 100mL sterilized water shaken wells
Afterwards, 10mL suspension is taken in the conical flask for filling 100mL enriched mediums, at 28 DEG C, shaken cultivation 3d under 220r/min,
As mixed microorganism flora；
（2）The reinforcing of mixed microorganism flora：The complex microbial community of various concentrations gradient is coated on beef extract-peptone
On culture medium, Gao Shi No. I culture medium and martin substratum, it is placed in artificial climate incubator and cultivates 2 weeks, temperature 10
DEG C, the complex microbial community to be strengthened；The complex microbial community of described various concentrations gradient refers to 10-1、10-2、
10-3、10-4、10-5With 10-6µg/ml；
（3）Pseudomonas stutzeri and bacillus subtilis will be strengthened at 30 DEG C, 180r/min cultures,pleosporaceaeWith it is hidden
At 28 DEG C, 220r/min is cultivated coccus；
From 600 nm wavelength, fungi carries out turbidimetric assay with 560 nm, and using the OD values of bacteria suspension as ordinate, incubation time is
Abscissa, the growth curve of microorganism is drawn, choose the strain configuration complex micro organism fungicide of exponential phase, complex microorganism
Bacterium solution by Pseudomonas stutzeri, bacillus subtilis,pleosporaceae1 is pressed with cryptococcus:1:1:1 ratio is configured.
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| CN102612944A (en) * | 2012-03-26 | 2012-08-01 | 天津师范大学 | Method adopting lanthanum for improving low-temperature resistance of ryegrass turf taking garbage compost as base material |
| CN103931664A (en) * | 2014-05-08 | 2014-07-23 | 天津师范大学 | Low-temperature resisting method for festuca arundinacea by using complex microbial inoculants |
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| CN102612944A (en) * | 2012-03-26 | 2012-08-01 | 天津师范大学 | Method adopting lanthanum for improving low-temperature resistance of ryegrass turf taking garbage compost as base material |
| CN103931664A (en) * | 2014-05-08 | 2014-07-23 | 天津师范大学 | Low-temperature resisting method for festuca arundinacea by using complex microbial inoculants |
Non-Patent Citations (3)
| Title |
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| Plant-derived compatible solutes proline betaine;Bashir Abdallah etal.;《Microbiology》;20141031;第160卷;第2283-2294页 * |
| Survey of Plant Drought-Resistance Promoting Bacteria;Shanshan Wang etal.;《Indian J Microbiol》;20140613;第54卷(第4期);第419-426页 * |
| 植物抗寒性研究进展;彭筱娜 等;《生物技术通报》;20070826(第4期);第15-17页 * |
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