CN104774769B - A method of low temperature sod production performance is improved using reinforcing compost microbial bacterial agent - Google Patents
A method of low temperature sod production performance is improved using reinforcing compost microbial bacterial agent Download PDFInfo
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Abstract
The invention discloses a kind of methods for improving low temperature sod production performance using reinforcing compost microbial bacterial agent, the low temperature resistant microbial bacterial agent of the reinforcing, it is reinforcing Pseudomonas stutzeri, the reinforcing bacillus subtilis, reinforcing by weight fraction ratio for 1:1:1:1pleosporaceaeIt is formed with cryptococcus is strengthened.The present invention from consumer garbage compost it is obtained strengthen low temperature resistant microorganism Pseudomonas stutzeri, bacillus subtilis,pleosporaceaeAnd cryptococcus, and the complex micro organism fungicide that they are configured to various concentration is inoculated into turf establishment system, purpose is the optimum concentration to filter out complex micro organism fungicide, to improve the comprehensive qualities such as turf low temperature resistivity and sod production performance, for turf winter production and store and overwintering provide technical support.
Description
Technical field
The invention belongs to environmental protection technical field, it is related to improving low temperature sod production using reinforcing compost microbial bacterial agent
Performance.
Background technique
Microbial bacterial agent is especially to grow up to have in recent years by the complex micro organism fungicide that multiple-microorganism forms
The biotechnology of wide application prospect.Microbial bacterial agent not only can be remedying oil-polluted soil and degradation water body in it is organic
Pollutant can also prevent and treat pest and disease damage, promote crop production and increasing both production and income, and the anti-of plant can be improved in some microbial bacterial agents
Since the 1970s, American-European Japan etc. has all succeeded in developing some composite bacteria agents in countries to inverse property in succession, very much
It has begun and is produced on a large scale, and form the product of seriation.Wherein the 1980s is by Japanese university of Ryukyu
Teach develop EM(effective microbiota) obtain great success, be widely used in a 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.Based on microbial bacterial agent in some prosperities
National research and extension obtains more early, and the relevant technologies are more mature, using also more commonly.In recent years, 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 object microbial inoculum, from the Application of composite for using multiple strains of single culture, also obtains in succession from theory into action
Some achievements.Agricultural University Of Nanjing's biological control product mainly has the waxy Bacillus of prevention and treatment Cucumber root-knot nematode disease evil
AT31 microbial inoculum (mission/line goes out, LS20120060), the probiotics (vegetable obtains health) for preventing and treating soil-borne diseases of vegetable, peaceful shield series
Product has obtained bio-feritlizer formal registration.Shandong biology institute trichoderma (prevention and treatment gray mold), Hunan Institute of Plant Protection photosynthetic bacteria
(promoting photosynthesis), the short kiss bacillus in Zhenjiang Runzhou (prevention and treatment lepidoptera pest) and Ningxia Nuo get Man biotech company root nodule
Bacterium (promoting leguminous plant growth) etc..There are much reports about composite bacteria agent development in recent years, the country, mainly utilize micro- life
Object environment purification, degradation organic contamination improve grain yield and raising plant resistance to environment stress etc..Some researches show that utilize from aquatic products
Isolated dominant bacteria develops water quality cleansing agent in the fine water of cultivation, is used in freshwater fish culturing pond, right
Reduce the COD, NH in water body3- N, nitrite etc. play ideal effect.There are also studies have shown that 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
Horizontal also in experimental study and Preliminary Applications, there are no be widely used in reality.Biocontrol microorganisms itself have fixed nitrogen, phosphorus decomposing, solution
The effect of potassium, stimulation plant generate appropriate auxin；Degradation larger molecular organics, improve organic fertilizer utilization rate；Microbial bacterial agent
It is like the appetizer of plant；It protects the integrality of cell membrane, maintain the root activity and chlorophyll content of higher level, this side
Face is just not only the effect of a growth-promoting, also plays the role of a kind of degeneration-resistant.Microbial bacterial agent induces a variety of degeneration-resistant bases in plant
The expression of cause significantly improves the activities of antioxidant enzymes of plant.Disease is generated after microbial bacterial agent can prevent crop from harvesting simultaneously
Evil, induction adversity gene expression, the generation of a variety of related substances such as induction SOD.So microbial bacterial agent exists for improving plant
Planting under the conditions of adverse circumstance, that is, salt stress, drought stress and low temperature stress has active influence.
Turfgrass beautifies the environment in addition to having, purify air, prevents erosion, keeps ecological balance, gives people's offer
Outside the functions such as rest and sports center, still play the role of adjusting miniclimate.However 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 inhibit the growth of turfgrass, make under turf quality
Drop.Especially saline and alkaline, low temperature (damaging to plants caused by sudden drop in temperature) and arid are 3 big abiotic factors of strong limitation lawn growth.
Chilling injury is one of main meteorological disaster in China, and the most frequent and severe with northern area.Chilling injury
Be during crop growth, although 0 DEG C of daily minimal tcmperature or more, 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 lower than making
The lower limit of object growth and development preference temperature, so that the growth and development for influencing crops or agricultural that is solid and causing the underproduction are certainly
Right disaster.Chilling injury also influences the growth and normal physiological biochemical process of this lawn plant to some extent, especially limits
The main reason for sod production period.Low temperature stress will lead to SOD(superoxide dismutase), CAT(catalase) etc. protect
The reduction of enzymatic activity is protected, chlorophyll decomposes, and proline and mda content increase i.e. relative permeability of membrane and increase, and then plant is thin
Born of the same parents are damaged.When influencing some researches show that low temperature stress on growth of Festuca elata, the reduction with temperature and stress time are obtained
Extend, the CAT activity reduction in Festuca Arundinacea blade is also faster, and chlorophyll content also decreases, and proline content increases 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
After~50d, when the coverage rate of the turfgrass on turf is up to 95% or more, that is, removable level ground.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 improves 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 lawn plant is improved 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 disease-resistant growth-promoting functions are
It gets the nod, will also have certain effect in terms of improving lawn plant winter resistance and shortening sod production.
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 treatment process, a large amount of heat can be generated, the organic matter in rubbish is decomposed completion, while also killing the disease in rubbish
Bacterium etc., and sporiferous bacillus can largely exist.Various microorganisms are phases not to the utmost to organic matter capacity of decomposition and decomposition rate
With, with temperature, the variation in season, the microbiologic population and quantity in composting process be not also identical；And existing research shows
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 provides foundation for the application of microbial bacterial agent and the improvement of composting process.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 experiments have shown 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 obviously 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 conducive to cow dung compost
It is decomposed.And be directed to and extract microorganism fungus kind from solid waste matrix, and be used in experiment and production, somebody's difference
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, this both provides foundation to the configuration of compost microbe microbial inoculum early period.Recent correlation
Document and production show: microbial bacterial agent be used as become a kind of novel biotechnology have been applied to our social life with
In production.Especially research of microbial bacterial agent in terms of promoting plant growth, improving is too numerous to enumerate.But
Source and preparation about microbial bacterial agent, most researchers are also only only limited to extract and screen from soil or sludge.
In relation to symbiosis and the relationship that influences 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 concentration
Change complex micro organism fungicide inoculation application, will have great importance.
Effective strain is filtered out from consumer garbage compost (hereinafter referred to as compost), then is configured to corresponding microorganism microbial inoculum,
It is with a wide range of applications.It is some research shows that being found in composting municipal solid waste, in the mistake that organic matter is decomposed
Important variation also has occurred in Cheng Zhong, biocoene, and mainly pathogenic bacteria largely reduce, and then quantity is acute for sporiferous bacillus
Increase.House refuse becomes larger in the compost acidity obtained after hot fermentation is handled and salt content is got higher, therefore in hypertonic
Microorganism in system environment has centainly acidproof, salt tolerant and high-temperature stability.Whether general microbial bacterial agent, still
The breeding of strain in compost microbe microbial inoculum is all directly to filter out effective bacterial strain from natural environment (or compost), then match
Microbial bacterial agent is made applied to corresponding field.And the microorganism in natural environment (or compost) is passed through into certain specific directions
Reinforcing, and then the research for obtaining some efficient enhancement microbiological microbial inoculums there is no report.
Main Factors one of of the temperature as plant growth, can influence transpiration, the flow of water, absorption and the metabolism of turfgrass
And almost all of enzymatic reaction, suspend mode and growth and development.Research in terms of stress resistance of plant at present mainly measures
Its physical signs, such as biomass, protective enzyme, malonaldehyde, soluble sugar index.But related green returning of lawn situation and after turning green
The research report of lawn appearance Comprehensive Assessment is considerably less.
Microbial bacterial agent is used frequently as alleviating adverse environmental factor to the physiological ecological harmful effect of lawn plant.But in low temperature
Under stress, strengthening low temperature resistant microbial bacterial agent has what to influence 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,.
Summary of the invention
From consumer garbage compost it is obtained strengthen low temperature resistant microorganism Pseudomonas stutzeri, bacillus subtilis,pleosporaceaeAnd cryptococcus, and the complex micro organism fungicide that they are configured to various concentration is inoculated into turf establishment body
In system, it is therefore an objective to be the optimum concentration for filtering out complex micro organism fungicide, to improve turf low temperature resistivity and sod production performance
Equal comprehensive qualities, for turf winter production and store and overwintering provide technical support.
To achieve the above object, the invention discloses following technology contents:
It is a kind of to strengthen low temperature resistant microbial bacterial agent, which is characterized in that it is applied by the reinforcing that weight fraction ratio is 1:1:1:1
Family name pseudomonad strengthens bacillus subtilis, strengthenspleosporaceaeIt is formed with cryptococcus is strengthened；The reinforcing Amur
Pseudomonad strengthens bacillus subtilis, strengthenspleosporaceaeIt is referred to cryptococcus is strengthened: by various concentration gradient
Complex microbial community be coated on beef-protein medium, on Gao Shi No. I culture medium and martin substratum, be placed on
It is cultivated 2 weeks in artificial climate incubator, temperature is 10 DEG C；The complex microbial community of the various concentration gradient refers to
10-1、10-2、10-3、10-4、10-5With 10-6µg/ml。
Pseudomonas stutzeri, bacillus subtilis and cryptococcus of the present invention are with flat-plate bacterial colony number in (1.51 ± 0.11)
×109/ mL-(2.85 ± 0.03) × 109Between/mL, the OD of Pseudomonas stutzeri600Nm value is 0.447, bacillus subtilis
OD600 Nm value is 0.356, cryptococcal OD560Nm value is 0.64.
The present invention further discloses the low temperature resistant microbial bacterial agents of reinforcing in the winter resistance for improving lawn, improves regrowth rate side
The application in face；Especially improve it is overwintering after Festuca Arundinacea regrowth rate, improve Festuca Arundinacea it is overwintering after quality, in terms of density and color
The application of application aspect；Wherein the reinforcing complex microbial community refer to Pseudomonas stutzeri, bacillus subtilis,
Pleosporaceae and the cryptococcus ratio of 1:1:1:1 by ratio of weight and the number of copies are 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 carries out:
(1) enrichment of strain: compost sample is weighed into 10g and is set in sterile conical flasks, it is equal that the oscillation of 100mL sterile water is added
After even, take 10mL suspension in the conical flask for filling 100mL enriched medium, at 28 DEG C, shaken cultivation 3d under 220r/min
, as mixed microorganism flora；
(2) complex microbial community of various concentration gradient the reinforcing of mixed microorganism flora: is coated on beef extract egg
It on white peptone culture medium, 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 complex microbial community to be strengthened；The complex microbial community of the various concentration 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 is cultivated,pleosporaceae
With cryptococcus at 28 DEG C, 220r/min is cultivated.Selecting 600 nm wavelength, (fungi carries out turbidimetric assay with 560 nm), with bacteria suspension
OD value be ordinate, incubation time is abscissa, draws the growth curve of microorganism, choose logarithmic growth phase strain configuration
Complex micro organism fungicide, complex microorganism bacterium solution by Pseudomonas stutzeri, bacillus subtilis,pleosporaceaeWith hidden ball
Bacterium is configured in the ratio of 1:1:1:1.
The present invention further discloses the method for improving low temperature sod production performance using reinforcing compost microbial bacterial agent,
It is characterized in that being carried out by following step:
(1) what experimental material was selected is the relatively common perennial Festuca Arundinacea of northern China, and matrix used is prepared
Campus soil, sterilize 30 min at 121 DEG C, spare；Sterile soil is placed in every culture vessel as matrix, planting carpet turf
Stromal thickness is 15-16mm, and application rate is 160 gm2, unify quantitative water supply, daily to keep culture substrate to have preferable water
Divide situation, and should often exchange each culture vessel position, to guarantee that illumination is consistent；
(2) after plant strain growth to 15-18d, 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 indoor natural light, after persistently handling 20d
Each index is measured, the plant height, biomass of individual tree and chlorophyll content of Festuca Arundinacea are measured.
(3) complex micro organism fungicide 15ml is added again after having measured These parameters, plant is then subjected to low temperature mistake
Degree processing, the temperature of excessive phase are 5-12oC excessively continues one week, then removes Festuca Arundinacea according to common lawn winterization
In hay, it is rotten grass, withered grass, then Festuca Arundinacea is cut to 4-6cm, is watered with overwintering water, finally by the related culture vessel of Festuca Arundinacea
It is put into and is stayed in foraminate sealing carton together, be put into the back without passing the winter in the room of heating, temperature is -6- during passing the winter
10oPlant is moved on to hot-house culture after continuing 40d, carries out processing of turning green by C, is measured it and is turned green and situation and evaluates its appearance and integrate
Quality.
The more detailed preparation method of the present invention
1 develops materials and methods
1.1 experimental material
Turfgrass choose full seed, uniform Festuca Arundinacea (Festuca arundinacea L) seed is
Test material.It is Tianjin Normal University's garden mould in the school, physicochemical property: pH 7.44, salt content 0.29%, organic matter for examination soil
4.68 % of content, total nitrogen content 0.21 %, available phosphorus content 22.03mgkg-1, complete 45.61 gkg of potassium-1, 0.46 g of bulk density
L-1, 0.58 gmL of saturation moisture content-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 at
Solid medium；
Beef-protein medium: beef extract 5g, peptone 10g, NaCl 5g, water 1000ml, pH 7.0-7.2 add
15g agar is at 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 add agar 20g at solid medium (when configuration, first with a small amount of cold water, by starch tune
It at paste, imports in the water boiled, is heated in fire, other compositions are added 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 at solid medium (0.03% streptomysin dilution is added before use
100ml makes every ml culture medium 30 μ g containing streptomysin).
The enrichment of strain
Compost sample is weighed 10g to set in sterile conical flasks, after 100mL sterile water shaken well is added, takes 10mL outstanding
Supernatant liquid is in the conical flask for filling 100mL enriched medium, and at 28 DEG C, shaken cultivation 3d under 220r/min as mixes micro- life
Object flora.
The reinforcing of microorganism:
The complex microbial community of various concentration gradient is coated on No. I beef-protein medium, Gao Shi culture medium
It in martin substratum, is placed in artificial climate incubator and cultivates 2 weeks, temperature is 10 DEG C；The various concentration 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) inverted plate: beef extract-peptone agar medium, No. I agar medium of Gao Shi, Martin (PDA) agar are trained
Base high-temperature sterilization is supported, when being cooled to 55-60 DEG C, in martin agar culture medium Streptomycin Solution being added, (whole mass concentration is
30 μ g/ml), it is mixed even rear inverted plate respectively.
2) it prepares mixed microorganism dilution: drawing the mixed microorganism bacteria suspension after 1ml strengthens with liquid-transfering gun and Sheng is added
It is mixed well in the Boiling tube for having 9ml sterile water, this is 10-1Dilution, and so on be made 10-2、10-3、10-4、10-5With 10-6The dilution of several concentration of μ g/ml.
3) be coated with: the dilution bacteria suspension for drawing 0.2ml various concentration respectively with liquid-transfering gun is accurately put into corresponding culture medium and puts down
Plate center, each various concentration gradient processing are repeated 3 times.It is lightly coated with uniformly with sterile glass rod in media surface.
4) cultivate: beef extract-peptone plate is inverted in 37 DEG C of incubators and cultivates, and will contain No. I culture medium of Gao Shi and Martin
The plate of family name's culture medium (PDA) is inverted in 28 DEG C of incubators and cultivates.
Plate streaking partition method
Choose bacterium colony: it is enterprising in new above-mentioned 3 kinds of culture mediums that the single bacterium colony grown after culture is picked them separately a little lawn
Row scribing 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 kit.The PCR system of predominant bacteria: 10 ×
Buffer(with MgCl2) 2 μ L, dNTP(10mmol/L) 0.4 μ L, 341f(10 μm of ol/L) 1 μ L, 534r(10 μm of ol/L) 1 μ
L, Taq enzyme (5u/ μ L) 0.4 μ L, 1 μ L of template DNA add ultrapure water to be settled to 20 μ L of final volume.PCR reaction condition: 94 DEG C of 5min
Initial denaturation, 94 DEG C of denaturation 1min, 55 DEG C of renaturation 45s, 72 DEG C of extension 45s, 30 recycle, 72 DEG C of extension 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 system 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(10 μm of ol/L)
0.4 μ L, GeoA2(10 μm of ol/L) 0.4 μ L, Taq enzyme (5u/ μ L) 0.2 μ L, 1 μ L of template DNA add ultrapure water to be settled to final volume
20µL.PCR reaction condition: 94 DEG C of 4min initial denaturations, 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 product is sent to Beijing Hua Da gene sequencing portion, according to sequencing
As a result, finding out corresponding strain in BLAST system.
The preparation of 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.It applies
Family name pseudomonad and bacillus subtilis are at 30 DEG C, 180r/min culture, and pleosporaceae and cryptococcus are at 28 DEG C, 220r/
Min culture.Selecting 600 nm wavelength, (fungi carries out turbidimetric assay with 560 nm), using the OD value of bacteria suspension as ordinate, cultivates
Time is abscissa, draws the growth curve of microorganism.The strain for choosing logarithmic growth phase configures complex micro organism fungicide, compound
Microbial inoculum is prepared by following processing.Pseudomonas stutzeri, bacillus subtilis, pleosporaceae and cryptococcus
It is configured in the ratio of 1:1:1:1.
1.4 the application of complex micro organism fungicide
The low temperature stress that this experiment uses was handled as entire winter, and temperature is about -6-10oC.Various concentration it is compound micro-
Bacteria agent application sets processing mode under corresponding low temperature stress: processing 1 is control, is inoculated with corresponding blank cultures
(CK)；Microbial inoculum (the CM of 100 times of dilutions is added in processing 21)；Microbial inoculum (the CM of 200 times of dilutions is added in processing 32).
1.5 turf establishment
What experimental material was selected is the relatively common perennial Festuca Arundinacea of northern China.Matrix used is prepared campus
Soil.Sterilize 30 min at 121 DEG C, spare.Sterile soil is placed when experiment in every culture vessel as matrix, planting carpetweed
Skin stromal thickness is about 15mm, and application rate is 160 gm2.Unified quantitative water supply daily, to keep culture substrate to have preferably
Water regime, and each culture vessel position should be often exchanged, to guarantee that illumination is consistent, respectively handle 4 repetitions.To plant strain growth
To after 15d, it is separately added into each 15ml of corresponding complex micro organism fungicide, same amount of aseptic culture medium is added in control group (CK).
During lawn plant is cultivated, temperature is 12-20 DEG C, relative humidity 25-45%, and illumination is to penetrate indoor natural light, lasting to locate
Each index is measured after reason 20d.Measure plant height, biomass of individual tree and the chlorophyll content of Festuca Arundinacea.
Complex micro organism fungicide 15ml is added again after having measured These parameters, then excessively locates plant progress low temperature
Reason, the temperature of excessive phase are 5-12oC.Excessively continue one week.Then remove in Festuca Arundinacea according to common lawn winterization
Hay, rotten grass, withered grass, then cut Festuca Arundinacea to 4cm or so, it is watered with overwintering water, finally by the related culture vessel one of Festuca Arundinacea
It rises and is put into and stays in foraminate sealing carton, be put into the back without passing the winter in the room of heating.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 measured and turns green and situation and evaluate its appearance comprehensive quality.
1.6 testing index
1.6.1 the measurement of regrowth rate:
Plant counts the total plant of Festuca Arundinacea in each container before passing the winter, record plant number of turning green daily after processing of turning green, will
Greenery, sprouting or being considered as young shoot protrusion is grown to turn green.Festuca Arundinacea regrowth rate is calculated as follows:
Festuca Arundinacea regrowth rate (%)=number/sum × 100% of turning green.
1.6.2 the measurement of the color of lawn quality, density and the calculating of homogeneity after turning green
Quality: measurement method measures the millimeter of blade the widest part after turning green.Density: each culture dish is small as one
Sample prescription calculates sample area and records the tiller number in subquadrat delineation area.Color: it is estimated after turning green.See Table 1 for details:
The observation of 1 Phenotypic traits of table and methods of marking
Homogeneity: the homogeneity on lawn refers to the uniformity coefficient of lawn appearance, it is that turf color, density, quality etc. refer to
Target concentrated expression.This research is using uniformity method measurement lawn homogeneity (Liu and east etc., 1999).In the turfgrass measured
Quality (T), density (D) and the color (C) of branches and leaves and on the basis of, the standard of three indexs is calculated with following statistical formula
Difference:
The analysis of 1.7 data
It is handled using Microsoft Excel 2003 and SPSS11.7 software
The analysis of 2 development results
2.1 bacterial screening
Selected 4 kinds of this experiment strengthen low temperature resistant microorganisms be respectively Pseudomonas stutzeri, bacillus subtilis,pleosporaceaeAnd cryptococcus.Pass through measurement Pseudomonas stutzeri, bacillus subtilis and cryptococcal growth curve (figure
1) it, then according to microbial growth curve may determine that it grows logarithmic phase, then the strain of this phase extracted, be made into and do not exist together
Reason, the inoculation for lawn plant.
Pseudomonas stutzeri, bacillus subtilis and cryptococcus in microbial bacterial agent is measured with the method for plate culture count
Strain quantity,pleosporaceaeThen measured with paper disk method.Measurement result such as table 2, the clump count of 4 strain of gained (1.51 ±
0.11) × 109/ mL-(2.85 ± 0.03) × 109Between/mL, the OD of Pseudomonas stutzeri600Nm value is 0.447, bacterium withered grass
The OD of bacillus600 Nm value is 0.356, cryptococcal OD560Nm value is 0.64.
Clump count contained by 2 microbial bacterial agent of table
Influence of 2.2 complex micro organism fungicides to Festuca Arundinacea regrowth rate after low temperature stress
Influence of the complex micro organism fungicide of various concentration to overwintering rear Festuca Arundinacea regrowth rate is shown in Table 3.After overwintering, control group
Festuca Arundinacea started to turn green at the about the 9th day, the Festuca Arundinacea of the processing group of 100 times of dilutions occurred as soon as sign of turning green at the 5th day, connect
The processing group of kind of 200 times of dilutions started to turn green at the about the 6th day, and there are significant difference (P < 0.05) between them.It is vaccinated with
After 30 days, regrowth rate has reached the standard turned green up to 81.57% to 100 times of dilution microbial bacterial agents, and comparison impinges upon in advance
5 days or so.And last regrowth rate be also significantly greater than group according to group and 200 times of dilutions processing group (P< 0.05).
Influence of the complex micro organism fungicide of 3 various concentration of table to Festuca Arundinacea regrowth rate after low temperature stress
The influence of quality, density and color of the 2.3 compound temperature microbial bacterial agents to Festuca Arundinacea after overwintering.
As shown in Table 4, quality, density and the color of processing group and control group Festuca Arundinacea there are significant difference (P<
0.05).And it is inoculated with the Festuca Arundinacea density maximum in the processing group of 100 times of dilutions, quality, color are best.It follows that inoculation
100 times of dilutions are affected to Festuca Arundinacea, are more advantageous to the raising of the overwintering rear quality of Festuca Arundinacea.
The complex micro organism fungicide of 4 various concentration of table is to the quality, density of Festuca Arundinacea after low temperature stress and the influence of color
The evaluation of 2.4 lawn appearance comprehensive qualities
Turfgrass comprehensive quality evaluation result is shown in Table 5.Evaluation result shows the height for being inoculated with the processing group of 100 times of dilutions
The grade of the quality of fescue grass, density and color is optimal；The homogeneity of control group is 0.76, is inoculated with the processing of 100 times of dilutions
The homogeneity of group is 0.90, and the homogeneity for being inoculated with the processing group of 200 times of dilutions is 0.87.I.e. at inoculating complex microorganism microbial inoculum
The comprehensive quality of the Festuca Arundinacea of reason group is better than not being inoculated with control group.And to be inoculated with the processing group Festuca Arundinacea of 100 times of dilutions
Comprehensive quality is optimal.
5 lawn comprehensive quality evaluation result of table
3 develop conclusion
The turf of enhancement microbiological microbial inoculum turning green earlier than nonvaccinated control group after overwintering is vaccinated in this technology, and
Regrowth rate is also significantly greater than nonvaccinated control group.And it is vaccinated with 100 times of last regrowth rates of diluted processing group and is up to
To 93.18%, this can also illustrate that inoculation strengthens low temperature resistant microorganism and the winter resistance of turf can be improved, and then improve it
Regrowth rate.The significant effect of 100 times of dilution complex micro organism fungicides is better than 200 times of dilutions；And help to improve Festuca Arundinacea
Lower temperature resistance, and then improve Festuca Arundinacea it is overwintering after regrowth rate and appearance comprehensive quality.In short, screening and culture are corresponding
Complex microorganism bacterial classification carries out artificial infection, and plant cold resistance can be improved, and then improves its regrowth rate and the comprehensive matter of appearance
Amount；This development and application for reinforcing compost high-effect bacterial, improves low temperature sod production performance；Apply composite microbial bacteria
Agent can effectively improve the appearance comprehensive quality of turf.
Detailed description of the invention:
Fig. 1 is Pseudomonas stutzeri, bacillus subtilis and cryptococcal growth curve.
Specific embodiment
In order to more fully explain implementation of the invention, following preparation method embodiments are provided.These embodiments are only
It is only to explain, be not intended to limit the scope of the invention.Needing to illustrate is: the complex microbial community that the present invention screens
Pseudomonas stutzeri, bacillus subtilis, pleosporaceae and cryptococcus have sale in the market, can also use this hair
Bright method isolated complex microbial community from consumer garbage compost, the biochemical characteristic of 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) enrichment of strain: compost sample is weighed into 10g and is set in sterile conical flasks, it is equal that the oscillation of 100mL sterile water is added
After even, take 10mL suspension in the conical flask for filling 100mL enriched medium, at 28 DEG C, shaken cultivation 3d under 220r/min
, as mixed microorganism flora；
(2) complex microbial community of various concentration gradient the reinforcing of mixed microorganism flora: is coated on beef extract egg
It on white peptone culture medium, 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 complex microbial community to be strengthened；The complex microbial community of the various concentration gradient refers to (μ g/ml)
10-1、10-2、10-3、10-4、10-5With 10-6。
(3) Pseudomonas stutzeri and bacillus subtilis will be strengthened at 30 DEG C, 180r/min is cultivated,pleosporaceae
With cryptococcus at 28 DEG C, 220r/min is cultivated.Selecting 600 nm wavelength, (fungi carries out turbidimetric assay with 560 nm), with bacteria suspension
OD value be ordinate, incubation time is abscissa, draws the growth curve of microorganism, choose logarithmic growth phase strain configuration
Complex micro organism fungicide, complex microorganism bacterium solution by Pseudomonas stutzeri, bacillus subtilis,pleosporaceaeWith hidden ball
Bacterium is configured in the ratio of 1:1:1:1.
Embodiment 2
Improve the method for low temperature sod production performance using reinforcing compost microbial bacterial agent:
(1) what experimental material was selected is the relatively common perennial Festuca Arundinacea of northern China, and matrix used is prepared
Campus soil, sterilize 30 min at 121 DEG C, spare；Sterile soil is placed in every culture vessel as matrix, planting carpet turf
Stromal thickness is 15-16mm, and application rate is 160 gm2, unify quantitative water supply, daily to keep culture substrate to have preferable water
Divide situation, and should often exchange each culture vessel position, to guarantee that illumination is consistent；
(2) after plant strain growth to 15d, each 15ml of corresponding complex micro organism fungicide, lawn plant culture are separately added into
Period, temperature are 12 DEG C, and relative humidity 35%, illumination is to penetrate indoor natural light, measure each finger after persistently handling 20d
Mark.Measure plant height, biomass of individual tree and the chlorophyll content of Festuca Arundinacea.
(3) complex micro organism fungicide 15ml is added again after having measured These parameters, plant is then subjected to low temperature mistake
Degree processing, the temperature of excessive phase are 5 ~ 12oC excessively continues one week, then removes Festuca Arundinacea according to common lawn winterization
In hay, it is rotten grass, withered grass, then Festuca Arundinacea is cut to 6cm, is watered with overwintering water, finally by the related culture vessel one of Festuca Arundinacea
It rises to be put into and stay in foraminate sealing carton, be put into the back without passing the winter in the room of heating, 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 measured and turns green and situation and evaluate its appearance comprehensive quality.
Claims (3)
1. application of the microbial bacterial agent in terms of improving the winter resistance on lawn, improving regrowth rate；The wherein microbial bacterial agent,
It is reinforcing Pseudomonas stutzeri, the reinforcing bacillus subtilis, reinforcing by weight fraction ratio for 1:1:1:1pleosporaceaeIt is formed with cryptococcus is strengthened；The reinforcing Pseudomonas stutzeri strengthens bacillus subtilis, strengthenspleosporaceaeCryptococcal preparation is as follows with strengthening:
(1) reinforcing of microorganism:
The complex microbial community by compost sample preparation of various concentration gradient is coated on beef-protein medium, height
It on family name No. I culture medium and martin substratum, is placed in artificial climate incubator and cultivates 2 weeks, temperature is 10 DEG C；It is described not
10 are referred to concentration gradient complex microbial community-1、10-2、10-3、10-4、10-5With 10-6µg/ml；
(2) separation and purifying of enhancement microbiological；
Dilution spread flat band method:
1) inverted plate: by beef extract-peptone agar medium, No. I agar medium of Gao Shi, Martin (PDA) agar medium
When being cooled to 55-60 DEG C, Streptomycin Solution is added in high-temperature sterilization in martin agar culture medium, and whole mass concentration is 30 μ g/
Ml is mixed even rear inverted plate respectively；
2) it prepares mixed microorganism dilution: drawing the complex microorganism bacteria suspension addition after 1ml strengthens with liquid-transfering gun and fill 9ml
It is mixed well in the Boiling tube of sterile water, this is 10-1Dilution, and so on be made 10-2、10-3、10-4、10-5With 10-6µg/
The dilution of several concentration of ml；
3) be coated with: the dilution bacteria suspension for drawing 0.2ml various concentration respectively with liquid-transfering gun is accurately put into corresponding culture medium flat plate
Centre, each various concentration gradient processing are repeated 3 times, are lightly coated with uniformly with sterile glass rod in media surface；
4) cultivate: beef extract-peptone plate is inverted in 37 DEG C of incubators and cultivates, and will contain No. I culture medium of Gao Shi and Martin training
The plate for supporting base (PDA), which is inverted in 28 DEG C of incubators, to be cultivated；
Plate streaking partition method:
It chooses bacterium colony: the single bacterium colony grown after culture being picked them separately into a little lawn on new above-mentioned 3 kinds of culture mediums and is drawn
Line purifying；Until on culture medium grow be pure culture, such as it is impure, still need to repeat the step；And gained microorganism is carried out
Strain idenfication；
(3) prepared by complex micro organism fungicide:
Spawn incubation: the low temperature resistant microorganism of reinforcing that step (2) screening obtains is expanded into training in corresponding fluid nutrient medium
It supports；Pseudomonas stutzeri and bacillus subtilis are at 30 DEG C, 180r/min culture, pleosporaceae and cryptococcus at 28 DEG C,
220r/min culture, selects 600 nm wavelength, and fungi carries out turbidimetric assay with 560 nm, using the OD value of bacteria suspension as ordinate,
Incubation time is abscissa, draws the growth curve of microorganism；The strain for choosing logarithmic growth phase configures complex micro organism fungicide,
Complex microorganism bacterium solution is prepared by following processing: being strengthened Pseudomonas stutzeri, is strengthened bacillus subtilis, strengthenspleosporaceaeIt is configured with cryptococcus is strengthened in the ratio of 1:1:1:1.
2. application of the microbial bacterial agent described in claim 1 in terms of improving overwintering rear Festuca Arundinacea regrowth rate.
3. a kind of method that microbial bacterial agent using described in claim 1 improves low temperature sod production performance, feature exist
It is carried out in by following step:
(1) what experimental material was selected is the perennial Festuca Arundinacea of northern China, and matrix used is prepared campus soil, 121
Sterilize 30 min at DEG C, spare；Sterile soil is placed in every culture vessel as matrix, planting carpet compounded turf matrix with a thickness of
15-16mm, application rate are 160 gm2, unify quantitative water supply daily, to keep culture substrate to have preferable water regime, and
And each culture vessel position is often exchanged, to guarantee that illumination is consistent；
(2) after plant strain growth to 15-18d, it is separately added into each 15ml of complex micro organism fungicide, during lawn plant is cultivated, temperature
Degree is 12 ~ 20 DEG C, and relative humidity is 25 ~ 45%, and illumination is to penetrate indoor natural light, measures each finger after persistently handling 20d
Mark；Measure plant height, biomass of individual tree and the chlorophyll content of Festuca Arundinacea；
(3) complex micro organism fungicide 15ml is added again after having measured These parameters, then excessively locates plant progress low temperature
Reason, the temperature of excessive phase are 5 ~ 12oC excessively continues one week, then removes in Festuca Arundinacea according to common lawn winterization
Hay, rotten grass, withered grass, then Festuca Arundinacea is cut to 4-6cm, is watered with overwintering water, together by the related culture vessel of Festuca Arundinacea finally
It is put into and stays in foraminate sealing carton, be put into the back without passing the winter in the room of heating, temperature is -6 ~ 10 during passing the winteroC is held
Plant is moved on into hot-house culture after continuous 40d, carries out processing of turning green, it is measured and turns green and situation and evaluate its appearance comprehensive quality.
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| CN102174403A (en) * | 2011-02-14 | 2011-09-07 | 天津师范大学 | Application of waste compost composite bacterial agent to improving drought resistance of lawn grass |
| 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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| CN102174403A (en) * | 2011-02-14 | 2011-09-07 | 天津师范大学 | Application of waste compost composite bacterial agent to improving drought resistance of lawn grass |
| 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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