CN104798818B - A kind of method for improving sod production drought resistance using garbage compost microbial bacterial agent is strengthened - Google Patents
A kind of method for improving sod production drought resistance using garbage compost microbial bacterial agent is strengthened Download PDFInfo
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Abstract
The invention discloses a kind of method for improving sod production drought resistance using garbage compost microbial bacterial agent is strengthened.It is by ratio of weight and number be 1:1:1 reinforcing bacillus cereus, strengthen lysine bacillus and strengthen rhodotorula glutinis and press 1:1:1 composition；Described reinforcing bacillus cereus, strengthen lysine bacillus and strengthen rhodotorula glutinis referring to carrying out using the method for being stepped up PEG6000 concentration, in strengthening process, depending on OD600Increase and step up PEG6000 concentration, for PEG6000 concentration with 50 g/L gradient increase, final goal is 250 g/L；PEG6000 concentration is respectively（w/w）5%th, 10%, 15%, 20% and 25%, often increase PEG6000 concentration, after thalline increment is stable, continues to increase PEG6000 concentration, strengthen drought-enduring mixed microorganism flora step by step.The present invention also discloses that the application for strengthening drought-enduring microbial bacterial agent in terms of the biomass of Festuca Arundinacea under improving drought stress simultaneously.
Description
Technical field
The invention belongs to environmental protection technical field, is related to a kind of using reinforcing garbage compost microbial bacterial agent raising turf
The method for producing drought resistance.
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
The influence of poor environment, such as arid, high temperature, low temperature, salt marsh adverse circumstance can all suppress the growth of turfgrass, make under turf quality
Drop.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.
When dry early stress refers to that the moisture that plant is consumed is higher than the moisture of plant absorption, cause plant internal water and lose
Lack, stress time increase causes the phenomenon that excessive water wanes.An important factor for drought stress is also limitation lawn plant growth.
Under drought stress conditions, the growth that the physiological indexes of plant are mainly reflected in plant is suppressed, including overground part
Increment, leaf area index, the height of plant etc.；The photosynthesis of plant is affected, i.e., photosynthetic rate reduces；Plant
Internal auxin GA content reduces, and ABA content increases；The substantial amounts of proline of plant interior accumulation；Plant
Internal superoxide dismutase, peroxidase and catalase activity reduces.Water partitioning occurs in plant
It is abnormal, suppress plant growth, more seriously cause plant mechanical injuries to cause plant dead.There are some researches show：Arid
The lower turfgrass leaf r elative water content of stress and chlorophyll content are remarkably decreased, MDA, soluble sugar and free proline
Content significantly rises.After coercing to a certain extent, water seasoning need to be carried out to it, prevent that its wilting is withered and yellow.For Arid Problem,
Research at present, which focuses primarily upon, chooses lawn plant, Water Saving Irrigation Mode and the Moisture conservation of strong drought resistance, using drought resistance agent
Seed soaking and water-loss reducer seed dressing etc., also have researcher using azophoska to improve turf grass drought resistance.With China city
The increase of green coverage, the contradiction between green area irrigation water and urban water supply.Lawn often occurs because of water shortage
Withered and yellow or even dead, this has become the bottleneck for restricting urban afforestation turf quality.Therefore how research preferably improves plant
Drought resistance be to alleviate urban water contradiction, improve the fundamental solution of urban afforestation quality.
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.
Effective strain is filtered out from producing fertilizer from refuse in daily life, then is configured to corresponding microorganism microbial inoculum, is had extensive
Application prospect.Some researchs show to find in composting municipal solid waste, during organic matter is decomposed, biota
Fall that also there occurs important change, mainly pathogenic bacteria largely to be reduced, and sporiferous bacillus then increasing number.House refuse exists
The acid change of compost obtained after hot fermentation is handled is big and salt content uprises, therefore micro- in hypertonic system environment
Biology has necessarily acidproof, salt tolerant and high-temperature stability.Whether in general microbial bacterial agent, or compost microbe microbial inoculum
The seed selection of middle strain, all it is directly from natural environment（Or compost）In filter out effective bacterial strain, then be configured to microbial bacterial agent
Applied to corresponding field.And by natural environment（Or compost）In microorganism pass through some specific directions reinforcing, and then
Research to some efficient enhancement microbiological microbial inoculums there is no report.
The content of the invention
Microbial bacterial agent has got the nod as a kind of bio-feritlizer efficiently, environmentally friendly, its disease-resistant growth-promoting functions, its drought resisting
Also there is certain research in terms of effect；But the research at present on enhancement microbiological microbial inoculum is also seldom, it is applied to carpet turf
Producing drought resisting application invention, there is not been reported.Gained strengthens drought-enduring microorganism, i.e. Bacillus cercus from consumer garbage compost,
Lysine bacillus and rhodotorula glutinis, and the complex micro organism fungicide for being configured to various concentrations is inoculated into carpet turf established by sod
In system, and then filter out the complex micro organism fungicide of optimal concentration, it is therefore an objective to screen excellent complex micro organism fungicide, improve
Carpet sod production drought resistance and water-saving result.
The invention discloses following technology contents to achieve the above object：
One kind strengthens drought-enduring microbial bacterial agent, it is characterised in that it by ratio of weight and number is 1 that it, which is,:1:The 1 waxy bud of reinforcing
Spore bacillus, strengthen lysine bacillus and strengthen rhodotorula glutinis and press 1:1:1 composition；Described reinforcing bacillus cereus, by force
Change lysine bacillus and strengthen rhodotorula glutinis and refer to carrying out using the method for being stepped up PEG6000 concentration, strengthening
During, depending on OD600Increase and step up PEG6000 concentration, PEG6000 concentration is with 50 g/L gradient increase, final mesh
Mark is 250 g/L；PEG6000 concentration is respectively（w/w）5%th, 10%, 15%, 20% and 25%, often increase PEG6000's
Concentration, after thalline increment is stable, continues to increase PEG6000 concentration, strengthen drought-enduring mixed microorganism flora step by step.
Of the present invention to strengthen drought-enduring microbial bacterial agent, it is will to screen obtained bacillus cereus and lysine gemma
Bacillus is at 30 DEG C, and 180r/min culture 32h, for rhodotorula glutinis at 28 DEG C, 220r/min cultivates 32h.From 600 nm（Fungi is used
560 nm）Wavelength carries out turbidimetric assay, and using the OD values of bacteria suspension as ordinate, incubation time is abscissa, draws microorganism
Growth curve, chooses the strain configuration complex micro organism fungicide of exponential phase, and complex microorganism bacterium solution is carried out by following processing
Prepare, bacillus cereus, lysine bacillus and rhodotorula glutinis in parts by weight 1:1:1 ratio is configured.Wherein 3
The clump count of strain exists（2.61±0.13）×109/mL-（3.15±0.03）×109Between/mL, the OD of Bacillus cercus600
Nm values are 0.673, the OD of lysine bacillus600 Nm values are 0.576, and the OD of rhodotorula glutinis560 Nm values are 0.912.
The present invention further discloses the biomass side for strengthening drought-enduring microbial bacterial agent Festuca Arundinacea in the case where improving drought stress
The application in face；Application particularly in terms of Testuca arundinacea chlorophyll content under improving drought stress.The reinforcing is drought-enduring
Bacteria agent refers to the reinforcing Salt-tolerant microbial agent of 100 times of dilution.
The present invention further discloses the preparation method for strengthening drought-enduring microbial bacterial agent, it is characterised in that by the steps
Carry out：
（1）The enrichment of strain：Compost sample is weighed into 10g to be placed in sterile conical flask, it is equal to add the vibration of 100mL sterilized waters
After even, 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）Strengthen drought-enduring Spawn incubation:Carried out using the method for being stepped up PEG6000 concentration, in strengthening process,
Depending on OD600Increase and step up PEG6000 concentration, for PEG6000 concentration with 50 g/L gradient increase, final goal is 250
g/L；PEG6000 concentration is respectively（w/w）5%th, 10%, 15%, 20% and 25%, often increase PEG6000 concentration, treat bacterium
After body increment is stable, continues to increase PEG6000 concentration, strengthen drought-enduring mixed microorganism flora step by step；
（4）Strengthen complex micro organism fungicide to prepare：Obtained bacillus cereus will be screened and lysine bacillus exists
30 DEG C, 180r/min culture 32h, rhodotorula glutinis is at 28 DEG C, and 220r/min cultivates 32h, from 600 nm（560 nm of fungi）
Wavelength carries out turbidimetric assay, and using the OD values of bacteria suspension as ordinate, incubation time is abscissa, and the growth for drawing microorganism is bent
Line, the strain configuration complex micro organism fungicide of exponential phase is chosen, complex microorganism bacterium solution is prepared by following processing, wax
Sample bacillus, lysine bacillus and rhodotorula glutinis in parts by weight 1:1:1 ratio is configured.
It is of the invention further to disclose using the side for strengthening garbage compost microbial bacterial agent raising sod production drought resistance
Method, it is characterised in that carried out by the steps：
（1）Experiment material：The Festuca Arundinacea of full seed, uniformity is chosen from turfgrass（Festuca arundinacea L .）Seed is test material；
Campus soil：PH is 7.44, salt content 0.29%, the % of the content of organic matter 4.68, the % of total nitrogen content 0.21, available phosphorus content
22.03mg·kg-1, the full gkg of potassium 45.61-1, the gL of unit weight 0.46-1, the gmL of saturation moisture content 0.58-1；
（2）Strengthen complex micro organism fungicide to prepare：Obtained bacillus cereus will be screened and lysine bacillus exists
30 DEG C, 180r/min culture 32h, rhodotorula glutinis is at 28 DEG C, and 220r/min cultivates 32h, from 600 nm（560 nm of fungi）
Wavelength carries out turbidimetric assay, and using the OD values of bacteria suspension as ordinate, incubation time is abscissa, and the growth for drawing microorganism is bent
Line, the strain configuration complex micro organism fungicide of exponential phase is chosen, complex microorganism bacterium solution is prepared by following processing, wax
Sample bacillus, lysine bacillus and rhodotorula glutinis in parts by weight 1:1:1 ratio is configured；
（3）Strengthen the application of complex micro organism fungicide：This experiment compels processing level using two arids：Medium drought
H1：60% ~ 40% FC, Severe drought stress H2：40%~20% FC；The complex micro organism fungicide application of various concentrations, corresponding
Processing mode is set under drought stress；
（4）Carpet turf drought stress planting
What is selected is the relatively common perennial Festuca Arundinacea of northern China, and matrix used is the campus soil prepared, 121
Sterilize 30min at DEG C, standby, and using sterile soil as matrix during experiment, planting carpet compounded turf matrix thickness is 15-18mm, is broadcast
Kind amount is 160-162 gm2, unify quantitative water supply daily, have a preferable water regime to keep culture matrix, and should be through
Each culture vessel position is often exchanged, to ensure that illumination is consistent, when plant strain growth highly reaches 6 ~ 7cm, adds various concentrations
Complex micro organism fungicide, drought stress processing is carried out after 3d, the measure of each index of plant is carried out after drought stress processing 30d, it is real
Test on hothouse plants culture platform and carry out, temperature is 22-30 DEG C, and relative humidity 35%-60%, illumination is natural light.
Needing special instruction is：
Reinforcing bacillus cereus used in the present invention, strengthen lysine bacillus and strengthen rhodotorula glutinis in the market
There is sale, method of the invention can both use commercially available reinforcing bacillus cereus, strengthen lysine gemma bar
Bacterium and reinforcing rhodotorula glutinis carry out domestication and reinforcing composite bacteria agent are made, and can also use method disclosed by the invention from house refuse
Isolated reinforcing bacillus cereus in compost, strengthen lysine bacillus and strengthen rhodotorula glutinis and then pass through domestication again
Composite bacteria agent is made.The biochemical characteristic of its 3 obtained bacterial strain is identical with commercially available therefore not in preservation.
More detailed description 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 drought-enduring 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
Drought-enduring strengthen of mixed microorganism flora is carried out using the method for being stepped up PEG6000 concentration, is being strengthened
Cheng Zhong, depending on OD600Increase and step up PEG6000 concentration, PEG6000 concentration is with 50 g/L gradient increase, final goal
It is 250 g/L；PEG6000 concentration is respectively（w/w）5%th, 10%, 15%, 20% and 25%, often PEG6000's of increase is dense
Degree, to continue to increase PEG6000 concentration, strengthen drought-enduring mixed microorganism flora step by step after thalline increment is stable.
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) mixed microorganism dilution is prepared：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 of μ g/ml.
3) it is coated with：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) cultivate：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 drought-enduring microorganism of reinforcing that screening obtains is expanded into culture in corresponding fluid nutrient medium.It is waxy
Bacillus and lysine bacillus are at 30 DEG C, and 180r/min culture 32h, at 28 DEG C, 220r/min is cultivated rhodotorula glutinis
32h.From 600 nm（560 nm of fungi）Wavelength carries out turbidimetric assay, using the OD values of bacteria suspension as ordinate, incubation time
For abscissa, the growth curve of microorganism is drawn.Choose the strain configuration complex micro organism fungicide of exponential phase, composite microbial
Thing bacterium solution is prepared by following processing, bacillus cereus, and lysine bacillus and rhodotorula glutinis press 1:1:1 ratio is entered
Row configuration.
1.4 complex micro organism fungicide applications
This experiment compels processing level using two arids：Medium drought H1：60%-40% FC（Field
Capacity, field capacity）, Severe drought stress H2：40%~20% FC；The complex micro organism fungicide application of various concentrations,
Processing mode is set under corresponding drought 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 carpet turf drought stress plantings
What experiment material was selected is the relatively common perennial Festuca Arundinacea of northern China.Matrix used is the campus prepared
Soil.Sterilize 30min at 121 DEG C, standby.It is about using sterile soil as matrix, planting carpet compounded turf matrix thickness during experiment
15mm, application rate are 160 gm2.Unified quantitative water supply daily, to keep culture matrix to have preferable water regime, and should
Each culture vessel position is often exchanged, to ensure that illumination is consistent, when plant strain growth highly reaches 6-7cm, adds various concentrations
Complex micro organism fungicide, respectively handle 4 repetitions, drought stress processing carried out after 3d, plant is carried out after drought stress processing 30d
The measure of each index.Experiment is carried out on hothouse plants culture platform, and temperature is 22-30 DEG C, relative humidity 35%-60%, illumination
For natural light.
1.6 testing index
1.6.1 biomass
The measure of biomass：Plant sample is put into electric drying oven with forced convection, after 105 DEG C of 15 min of fixing, 80 DEG C are dried to
Constant weight.
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：
Ca=12.7 OD663-2.69 OD645；
Cb=22.9 OD645-4.68 OD663；
CT= Ca + Cb =8.02 OD663 + 20.21 OD645；
1.7 data analysis
Handled using Microsoft Excel 2003 and SPSS11.7 softwares
2 development results are analyzed
2.1 bacterial screening
3 kinds of selected bacterium of this experiment are respectively Bacillus cercus, lysine bacillus and rhodotorula glutinis.Pass through survey
The growth curve of fixed withered Bacillus cercus, lysine bacillus and rhodotorula glutinis（Fig. 1）, can according to microbial growth curve
To judge that it grows logarithmic phase, then the strain of this phase is extracted, be made into different disposal, the inoculation for lawn plant.
By the Bacillus cercus in microbial bacterial agent, lysine bacillus and rhodotorula glutinis the method for plate culture count
Determine strain quantity.Measurement result such as table 1, the clump count of the strain of gained 3 exist（2.61±0.13）×109/mL~（3.15±
0.03）×109Between/mL, the OD of Bacillus cercus600Nm values are 0.673, the OD of lysine bacillus600 Nm values are
0.576, and the OD of rhodotorula glutinis560 Nm values are 0.912.
Clump count contained by the microbial bacterial agent of table 1
The influence of 2.2 complex micro organism fungicide alignment degrees and the lower turf biomass of Severe drought stress
Inoculating complex microorganism microbial inoculum significantly improves the biomass of Festuca Arundinacea under drought stress（Table 2）.Mild drought is coerced
Festuca Arundinacea ground fresh weight, ground dry weight and the underground dry weight for the treatment group for being vaccinated with 100 times of dilutions under compeling reach highest, respectively
It is 4 times, 2.37 times and 4.26 times of control.
Influence (g/m of the complex micro organism fungicide of the various concentrations of table 2 to Festuca Arundinacea biomass above and below the ground2)
Note：The different letters of data of going together represent significant differenceP<0.05 similarly hereinafter
Under Severe drought stress, also there is similar rule, be vaccinated with the treatment group most pronounced effects of 100 times of dilutions, point
It is not 4.63 times, 5.34 times and 2.42 times of control group.The treatment group effect for being vaccinated with 200 times of dilutions is also significantly better than control group
（P<0.05）.
The influence of 2.3 complex micro organism fungicide alignment degrees and the lower turf chlorophyll content of plant of Severe drought stress
Influence of the various concentrations complex micro organism fungicide for Testuca arundinacea chlorophyll content under drought stress is inoculated with to see
Table 3.The chlorophyll content for being vaccinated with the blade of the treatment group of complex micro organism fungicide is above nonvaccinated control.Mild drought
Under stress, leaf chlorophyll a, chlorophyll b and Chlorophyll content are all to reach peak value in 100 times of extension rates, are compared respectively
Control improves 36.42%, 85.71% and 41.82%.
Influence (the mgg of the complex micro organism fungicide alignment degree of table 3 and the lower Tall Fescue Leaves chlorophyll contents of Severe drought stress-1
FW)
Under Severe drought stress, the chlorophyll a, chlorophyll b and Chlorophyll that are vaccinated with complex micro organism fungicide blade contain
Amount is all remarkably higher than control group（P<0.05）.Effect is best when being especially vaccinated with 100 times of extension rates, is improved respectively than control group
63.89%, 181.18% and 41.82%.
The influence of 2.4 complex micro organism fungicide alignment degrees and the lower turf plant water use efficiency of Severe drought stress
During arid, total confluent and average confluent otherness between treatment group and control group be not notable.Only exist
Under Severe drought stress, the total water consumption of the treatment group of 100 times of dilutions is vaccinated with（Average consumption）Considerably less than other two
Group（P<0.05）.
Significant difference is also not present in whole conceptual phase, total confluent of each group.Total moisture utilization ratio exists notable
Sex differernce（P<0.05）.Under medium drought, the total moisture utilization ratio for being inoculated with the treatment group of 100 times of dilutions is up to
0.33, reduce 74.02% than control group.Under Severe drought stress, the water utilization for the treatment of group of 100 times of dilutions is inoculated with most
Height, 65.26% is reduced than control.This also just illustrates, can be with using water wisely using complex micro organism fungicide.
3 develop conclusion
Under drought stress, carpet turf established by sod is vaccinated with complex intensifying microbial bacterial agent, can be relieved drought stress
The injury brought to carpet turf established by sod, and significantly enhance under drought condition, the moisture profit in carpet sod production
Use efficiency.In complex intensifying microbial bacterial agent sod production using on dosage, typically it is better than with the significant effect for diluting 100 times
200 times of dilutions, and can be with more efficient raising water utilization, water saving efficiency, which improves, exceedes more than half.This is also just alleviation
Carpet sod production provides a new method under drought condition, while also provides a new think of for effective utilize of compost
Road.
Brief description of the drawings：
Fig. 1 is the growth curve of Bacillus cercus, lysine bacillus and rhodotorula glutinis.
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.Bacillus cercus, lysine bacillus and rhodotorula glutinis are by city
Sell, also refer to method disclosed by the invention and prepare.
Embodiment 1
Strengthen the preparation method of Salt-tolerant microbial agent：
（1）The enrichment of strain：Compost sample is weighed into 10g to be placed in sterile conical flask, it is equal to add the vibration of 100mL sterilized waters
After even, 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）Strengthen drought-enduring Spawn incubation:Carried out using the method for being stepped up PEG6000 concentration, in strengthening process,
Depending on OD600Increase and step up PEG6000 concentration, for PEG6000 concentration with 50 g/L gradient increase, final goal is 250
g/L；PEG6000 concentration is respectively（w/w）5%th, 10%, 15%, 20% and 25%, often increase PEG6000 concentration, treat bacterium
After body increment is stable, continues to increase PEG6000 concentration, strengthen drought-enduring mixed microorganism flora step by step；
（4）Strengthen complex micro organism fungicide to prepare：Obtained bacillus cereus will be screened and lysine bacillus exists
30 DEG C, 180r/min culture 32h, rhodotorula glutinis is at 28 DEG C, and 220r/min cultivates 32h, from 600 nm（560 nm of fungi）
Wavelength carries out turbidimetric assay, and using the OD values of bacteria suspension as ordinate, incubation time is abscissa, and the growth for drawing microorganism is bent
Line, the strain configuration complex micro organism fungicide of exponential phase is chosen, complex microorganism bacterium solution is prepared by following processing, wax
Sample bacillus, lysine bacillus and rhodotorula glutinis in parts by weight 1:1:1 ratio is configured.
Embodiment 2
The method for improving sod production drought resistance using garbage compost microbial bacterial agent is strengthened：
（1）Experiment material：The Festuca Arundinacea of full seed, uniformity is chosen from turfgrass（Festuca arundinacea L .）Seed is test material；
Campus soil：PH is 7.44, salt content 0.29%, the % of the content of organic matter 4.68, the % of total nitrogen content 0.21, available phosphorus content
22.03mg·kg-1, the full gkg of potassium 45.61-1, the gL of unit weight 0.46-1, the gmL of saturation moisture content 0.58-1；
（2）Strengthen complex micro organism fungicide to prepare：Obtained bacillus cereus will be screened and lysine bacillus exists
30 DEG C, 180r/min culture 32h, rhodotorula glutinis is at 28 DEG C, and 220r/min cultivates 32h, from 600 nm（560 nm of fungi）
Wavelength carries out turbidimetric assay, and using the OD values of bacteria suspension as ordinate, incubation time is abscissa, and the growth for drawing microorganism is bent
Line, the strain configuration complex micro organism fungicide of exponential phase is chosen, complex microorganism bacterium solution is prepared by following processing, wax
Sample bacillus, lysine bacillus and rhodotorula glutinis in parts by weight 1:1:1 ratio is configured；
（3）Strengthen the application of complex micro organism fungicide：This experiment compels processing level using two arids：Medium drought
H1：60% ~ 40% FC, Severe drought stress H2：40%~20% FC；The complex micro organism fungicide application of various concentrations, corresponding
Processing mode is set under drought stress；
（4）Carpet turf drought stress planting
What is selected is the relatively common perennial Festuca Arundinacea of northern China, and matrix used is the campus soil prepared, 121
Sterilize 30min at DEG C, standby, and using sterile soil as matrix during experiment, planting carpet compounded turf matrix thickness is 15-18mm, is broadcast
Kind amount is 160-162 gm2, unify quantitative water supply daily, have a preferable water regime to keep culture matrix, and should be through
Each culture vessel position is often exchanged, to ensure that illumination is consistent, when plant strain growth highly reaches 6 ~ 7cm, adds various concentrations
Complex micro organism fungicide, drought stress processing is carried out after 3d, the measure of each index of plant is carried out after drought stress processing 30d, it is real
Test on hothouse plants culture platform and carry out, temperature is 22 ~ 30 DEG C, and relative humidity is 35% ~ 60%, and illumination is natural light.
Claims (4)
1. one kind strengthens drought-enduring microbial bacterial agent, it is characterised in that it by ratio of weight and number is 1 that it, which is,:1:The 1 waxy gemma of reinforcing
Bacillus, strengthen lysine bacillus and strengthen rhodotorula glutinis and press 1:1:1 composition；Described reinforcing bacillus cereus, strengthen
Lysine bacillus and reinforcing rhodotorula glutinis refer to carrying out using the method for being stepped up PEG6000 concentration, are strengthening
Cheng Zhong, depending on OD600Increase and step up PEG6000 concentration, PEG6000 concentration is with 50 g/L gradient increase, final goal
It is 250 g/L；PEG6000 concentration is respectively（w/w）5%th, 10%, 15%, 20% and 25%, often PEG6000's of increase is dense
Degree, after thalline increment is stable, continues to increase PEG6000 concentration, strengthen drought-enduring mixed microorganism flora step by step；Mainly
It is that will screen obtained bacillus cereus and lysine bacillus at 30 DEG C, 180r/min culture 32h, rhodotorula glutinis is 28
DEG C, 220r/min cultures 32h；
From 600 nm（560 nm of fungi）Wavelength carries out turbidimetric assay, using the OD values of bacteria suspension as ordinate, incubation time
For abscissa, the growth curve of microorganism is drawn, chooses the strain configuration complex micro organism fungicide of exponential phase, composite microbial
Thing bacterium solution is prepared by following processing, bacillus cereus, lysine bacillus and rhodotorula glutinis in parts by weight 1:1:1
Ratio configured；The clump count of wherein 3 strains exists（2.61±0.13）×109/mL-（3.15±0.03）×109/ mL it
Between, the OD of Bacillus cercus600Nm values are 0.673, the OD of lysine bacillus600 Nm values are 0.576, and rhodotorula glutinis
OD560 Nm values are 0.912.
2. the biomass of Festuca Arundinacea, the raising in the case where improving drought stress of the drought-enduring microbial bacterial agent of reinforcing described in claim 1 are dry
Application in terms of the lower Testuca arundinacea chlorophyll content of drought stress, the drought-enduring bacteria agent of reinforcing refer to：100 times of dilution
Strengthen drought-enduring microbial bacterial agent.
3. strengthen the preparation method of drought-enduring 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）Strengthen drought-enduring Spawn incubation:Carried out using the method for being stepped up PEG6000 concentration, in strengthening process, depending on
OD600Increase and step up PEG6000 concentration, for PEG6000 concentration with 50 g/L gradient increase, final goal is 250
g/L；PEG6000 concentration is respectively（w/w）5%th, 10%, 15%, 20% and 25%, often increase PEG6000 concentration, treat bacterium
After body increment is stable, continues to increase PEG6000 concentration, strengthen drought-enduring mixed microorganism flora step by step；
（3）Strengthen complex micro organism fungicide to prepare：Obtained bacillus cereus and lysine bacillus will be screened at 30 DEG C,
180r/min cultivates 32h, and rhodotorula glutinis is at 28 DEG C, and 220r/min cultivates 32h, from 600 nm（560 nm of fungi）Wavelength enters
Row turbidimetric assay, using the OD values of bacteria suspension as ordinate, incubation time is abscissa, draws the growth curve of microorganism, is chosen
The strain configuration complex micro organism fungicide of exponential phase, complex microorganism bacterium solution are prepared by following processing, waxy gemma
Bacillus, lysine bacillus and rhodotorula glutinis in parts by weight 1:1:1 ratio is configured.
A kind of 4. method for improving sod production drought resistance using garbage compost microbial bacterial agent is strengthened, it is characterised in that by as follows
The step of carry out：
（1）Experiment material：The Festuca Arundinacea of full seed, uniformity is chosen from turfgrass（Festuca arundinacea L
.）Seed is test material；
Campus soil：PH is 7.44, salt content 0.29%, the % of the content of organic matter 4.68, the % of total nitrogen content 0.21, available phosphorus content
22.03mg·kg-1, the full gkg of potassium 45.61-1, the gL of unit weight 0.46-1, the gmL of saturation moisture content 0.58-1；
（2）Strengthen complex micro organism fungicide to prepare：Obtained bacillus cereus and lysine bacillus will be screened at 30 DEG C,
180r/min cultivates 32h, and rhodotorula glutinis is at 28 DEG C, and 220r/min cultivates 32h, from 600 nm（560 nm of fungi）Wavelength enters
Row turbidimetric assay, using the OD values of bacteria suspension as ordinate, incubation time is abscissa, draws the growth curve of microorganism, is chosen
The strain configuration complex micro organism fungicide of exponential phase, complex microorganism bacterium solution are prepared by following processing, waxy gemma
Bacillus, lysine bacillus and rhodotorula glutinis in parts by weight 1:1:1 ratio is configured；
（3）Strengthen the application of complex micro organism fungicide：This experiment compels processing level using two arids：Medium drought H1：
60%-40% FC, Severe drought stress H2：40%-20% FC；The complex micro organism fungicide application of various concentrations, does accordingly
The lower setting processing mode of drought stress；
（4）Carpet turf drought stress planting
What is selected is the relatively common perennial Festuca Arundinacea of northern China, and matrix used is the campus soil prepared, at 121 DEG C
Sterilize 30min, standby, and using sterile soil as matrix during experiment, planting carpet compounded turf matrix thickness is 15-18mm, application rate
For 160-162 gm2, unify quantitative water supply daily, to keep culture matrix to have preferable water regime, and should often adjust
Each culture vessel position is changed, to ensure that illumination is consistent, when plant strain growth highly reaches 6-7cm, adds the compound of various concentrations
Microbial bacterial agent, drought stress processing is carried out after 3d, the measure of each index of plant is carried out after drought stress processing 30d, is tested
Carried out on hothouse plants culture platform, temperature is 22-30 DEG C, and relative humidity 35%-60%, illumination is natural light.
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