CN104762233A - Preparation method and application of lawn drought-resisting enhanced complex microbial community in garbage compost - Google Patents
Preparation method and application of lawn drought-resisting enhanced complex microbial community in garbage compost Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a lawn drought-resisting enhanced complex microbial community in garbage compost, wherein a drought-resisting microbial strain is obtained by reinforcing microbes in compost by simulating drought stress with PEG 6000. The enhanced complex microbial community comprises bacillus cereus, lysine bacillus and rhodotorula glutinis which are allocated according to a ratio in parts by weight of 1:1:1. The invention aims at obtaining enhanced compost microbes and providing a technical support for lawn drought-resisting application of an enhanced urban garbage compost microbial agent.
Description
Technical field
The invention belongs to environmental protection technical field, relate to preparation method and the application of drought-enduring strengthening complex microbial community in a kind of garbage compost.
Background technology
The complex micro organism fungicide that microbiobacterial agent is especially made up of multiple-microorganism is the biotechnology that development in recent years gets up to have wide application prospect.The soil that microbiobacterial agent not only can be remedying oil-polluted and the organic pollutant of degrading in water body, all right prevention and elimination of disease and pests, promote crop production and increasing both production and income, some microbiobacterial agent can improve the resistance of plant since 20 century 70s, the countries such as American-European Japan have succeeded in developing some composite fungus agents all in succession, much start to produce on a large scale, and defined the product of seriation.The EM(effective microorganism wherein developed by Japanese Ryukyu college professor the eighties in 20th century) obtain great success, be widely used in more than 90 country to relate to and plant industry aquaculture and environmental purification aspect, and achieve significant economic benefit and social benefit.Must comparatively early in some developed country's research and extensions based on microbiobacterial agent, correlation technique is comparatively ripe, applies also more general.In recent years, there is not the microbiobacterial agent that all can cause very large effect as EM in the whole world abroad more yet.
Relative to the research of developed countries related microorganisms microbial inoculum, China be from the eighties in 20th century just the correlative study of microbiobacterial agent, from theory into action, from the Application of composite using multiple bacterial classification of single culture, also in succession achieve some achievements.Agricultural University Of Nanjing's biological control product mainly contains the harmful waxy Bacillus AT31 microbial inoculum of control Cucumber root-knot nematode disease, and (mission/line goes out, LS20120060), prevent and treat the probiotics (vegetables obtain health) of soil-borne diseases of vegetable, peaceful shield series product, obtained bio-feritlizer and formally registered.Biological institute's trichoderma (preventing and treating gray mold) in Shandong, Hunan Institute of Plant Protection photosynthetic bacterium (promotion photosynthesis), Runzhou, Zhenjiang short kiss bacillus (control lepidoptera pest) and Ningxia Nuo get Man biotech company root nodule bacterium (promoting leguminous plants growth) etc.Recent years, domestic have much about the report of composite fungus agent development, mainly utilizes microbially decontaminate environment, the aspects such as degraded organic contamination, raising grain yield and raising plant resistance to environment stress.There are some researches show that utilization is separated the dominant bacteria obtained and develops water quality cleansing agent from the fine water of aquaculture, use it in freshwater fish culturing pond, to the COD reduced in water body, NH
3-N, nitrite etc. serve desirable effect.Also there are some researches show, microbial-bacterial fertilizer all can the growth of promotion romaine lettuce in various degree under suitable concentration, improves lettuce quality, reduces nitrate content.But these are mostly also in experimental study and Preliminary Applications level, are not also widely used in reality.Biocontrol microorganisms itself has the effect of fixed nitrogen, phosphorus decomposing, potassium decomposing, and stimulating plant produces appropriate growth hormone; Degraded larger molecular organics, improves fertilizer utilization ratio; Microbiobacterial agent similarly is the appetizer of plant; The integrity of Cell protection film, the improving activity of root system maintaining higher level and chlorophyll content, the effect of a this respect just not only growth-promoting, also has a kind of degeneration-resistant effect.In microbiobacterial agent inducing plant body, the expression of multiple adversity gene, significantly improves the activities of antioxidant enzymes of plant materials.Produce disease after microbiobacterial agent can prevent crop harvesting simultaneously, induction adversity gene is expressed, the generation of the multiple related substanceses such as induction SOD.So microbiobacterial agent has active influence for improving the planting of plant under adverse circumstance and salt stress, drought stress and low temperature stress condition.
Turfgrass is beautified the environment except having, purify air, prevent erosion, keep ecological balance, provide the functions such as rest and sports center to people except, still have the effect regulating miniclimate.But turfgrass is the same with other plant, often suffer the impact of poor environment, as the adverse circumstances such as arid, high temperature, low temperature, salt marsh all can suppress the growth of turfgrass, turf quality is declined.Especially saline and alkaline, low temperature (damaging to plants caused by sudden drop in temperature) and arid are the 3 large abiotic factors strongly limiting lawn growth.
Coerce dry morning when referring to moisture higher than plant absorption of moisture that plant consumes, cause water deficit in plant materials, the phenomenon that stress time increase causes excessive water to wane.Drought stress is also the important factor of restriction lawn plant growth.Under drought stress conditions, the growth that the physiological indexes of plant materials is mainly reflected in plant is suppressed, and comprises the height etc. of the increment of overground part, leaf area index, plant; The photosynthesis of plant is affected, and namely photosynthetic rate reduces; Growth hormone GA content in plant materials reduces, and ABA content increases; The a large amount of proline(Pro) of plant interior accumulation; Superoxide-dismutase in plant materials, peroxidase and catalase activity all reduce.In plant materials, water partitioning occurs abnormal, suppresses plant-growth, more seriously causes plant mechanical injuries to cause plant dead.There are some researches show: under drought stress, turfgrass leaf r elative water content and chlorophyll content significantly decline, mda, soluble sugar and free proline content significantly rise.Coerce to a certain extent, water seasoning need be carried out to it, prevent its wilting withered and yellow.For Arid Problem, current research mainly concentrates on the aspect such as lawn plant, Water Saving Irrigation Mode and Moisture conservation, the seed soaking of application drought resistance agent and water-holding agent seed dressing choosing strong drought resistance, also has investigator to utilize azophoska to improve turf grass drought resistance.Along with the increase of China's city's green areas, the contradiction between green area irrigation water and Urban water supply.Lawn often occurs withered and yellow because of lack of water, even dead, and this has become the bottleneck of restriction urban afforestation turf quality.Therefore studying the drought resistance improving plant how is better the fundamental solution alleviated town water contradiction, improve urban afforestation quality.
Producing fertilizer from refuse in daily life is the microorganism of occurring in nature, such as is bacterium and fungi etc., by their physiological metabolism, can accelerate organic decomposition rate, and then be become the process of humic acids.In producing fertilizer from refuse in daily life aerobic treating processes, can produce a large amount of heats, the organism in rubbish has been decomposed, and also kill the germ etc. in rubbish, and sporiferous bacillus can exist in a large number simultaneously.Various microorganism is not quite similar to organic substance decomposing ability Sum decomposition speed, and along with the change in temperature, season, the microflora in composting process and quantity are also not identical; And there are some researches show, the microflora in compost is quite complicated.The many factors such as the interaction in compost between microbe population and Species structure and compost organic material component and content, microorganism are closely related.Someone studies the impact of inoculation external source microbial inoculum on microbe population in compost and Enzyme activities, for the application of microbiobacterial agent and the improvement of composting process provide foundation.Pig manure straw compost complex micro organism fungicide can more effectively promote and optimize composting process and improves rapidly heap temperature raising top temperature prolongation time pliotherm period and more effectively can improve heap body nutrient levels.Cow dung compost test shows, adds origin microbial inoculum and can improve compost temperature fast, promotes heap body fermentation maturity, shortens the composting time; From Mierocrystalline cellulose and hemicellulose degradation rate, obviously strong than the control treatment of not adding microbial inoculum after adding microbial inoculum process, as can be seen here, interpolation microbiobacterial agent is conducive to cow dung compost and becomes thoroughly decomposed.And for extracting microbial strains from solid waste matrix, and use it in experiment and production, somebody filters out efficient degrading bacteria respectively and is mixed with corresponding complex micro organism fungicide from chicken manure fermenting and cow dung, certain basis has been established in application for complex micro organism fungicide, and this both provides foundation to the configuration of compost microbe microbial inoculum in early stage.Recent pertinent literature and production show: microbiobacterial agent as become a kind of novel biotechnology be applied to we social life and produce in the middle of.Especially the research of microbiobacterial agent in Promoting plant growth, raising plant resistance to environment stress etc. is too numerous to enumerate.But about source and the preparation of microbiobacterial agent, most researchers is also only only limited to be extracted and screening from soil or mud.About in compost between microbe colony structure, compost bacterial classification symbiosis and the relation of influencing each other there is ambiguity and complicacy.Microbial strains in garbage compost is prepared in strengthening, using reinforcing compost microbial strains as Inoculant, is mixed with the strengthening complex micro organism fungicide inoculation application of different concns, will has great importance.
From consumer garbage compost (hereinafter referred to as compost), filter out effective strain, then be mixed with corresponding microorganism microbial inoculum, be with a wide range of applications.Some researchs show to find in composting municipal solid waste, and in the process that organism is decomposed, coenosis also there occurs important change, and mainly pathogenic bacterium reduce in a large number, sporiferous bacillus then increasing number.Domestic refuse becomes large and saltiness uprises in the compost acidity obtained after thermophilic fermentation process, is therefore in the height microorganism of oozing in system environment and has certain acidproof, salt tolerant and high-temperature stability.No matter be general microbiobacterial agent, or the seed selection of bacterial classification in compost microbe microbial inoculum, be all directly from physical environment (or compost), filter out effective bacterial strain, then be mixed with microbiobacterial agent to be applied to corresponding field.And by the strengthening of the microorganism in physical environment (or compost) through some specific direction, and then the research obtaining some efficient enhancement microbiological microbial inoculums there is no report.
Summary of the invention:
What the acquisition of drought-enduring microorganism strains adopted is the microorganism that PEG6000 Drought stress simulation comes in reinforcing compost.The present invention is intended to obtain reinforcing compost microorganism, and provides technical support for the drought-enduring application in lawn of strengthening producing fertilizer from refuse in daily life microbiobacterial agent.
summary of the invention
For achieving the above object, the invention discloses following technology contents:
A preparation method for drought-enduring strengthening complex microbial community in garbage compost, is characterized in that being undertaken by following step:
(1) material: consumer garbage compost takes from Tianjin little Dian garbage compost factory,
rubbishthe pH of compost is 7.62, organic content 12.12 %, total nitrogen content 5.18 %, available phosphorus content 77.92 mgkg
-1, full potassium 50.83 gkg
-1, unit weight 0.85 gL
-1, saturation moisture content 66.58 gmL
-1;
(2) substratum:
1) enrichment medium: extractum carnis 5g, peptone 10g, NaCl 5g, water 1000ml, pH 7.4-7.6, add 15g agar and become solid medium;
2) beef-protein medium: extractum carnis 5g, peptone 10g, NaCl 5g, water 1000ml, pH 7.0-7.2, add 15g agar and become solid medium;
3) Gao Shi No. I substratum: Zulkovsky starch 20g, KNO
320g, K
2hPO
30.5g, MgSO
40.5g, FeSO
40.01g, water 1000ml, pH 7.2-7.4, add agar 20g and become solid medium, during configuration, first use a small amount of cold water, and by starch furnishing pasty state, import in the water boiled, heat in fire, add other compositions while stirring, after dissolving, moisturizing is to 1000ml;
4) Ma Dingshi substratum: glucose 10g, peptone 5g, KHPO
31g, MgSO (7H
2o) 0.5g, the 1% rose-bengal aqueous solution, 3.3ml, water 1000ml, pH nature, adds agar 15g and becomes solid medium, add 0.03% Streptomycin sulphate diluent 100ml before use, makes every ml substratum containing Streptomycin sulphate 30 μ g;
(3) enrichment of bacterial classification:
Garbage compost sample is taken 10g and is placed in aseptic Erlenmeyer flask, after adding 100mL sterilized water shaken well, get 10mL suspension in the Erlenmeyer flask filling 100mL enrichment medium, at 28 DEG C, shaking culture 3d under 220r/min, is complex microbial community;
(4)
drought-enduringthe strengthening of complex microbial community:
What the drought-enduring strengthening of mixing microorganisms flora adopted is that the method progressively increasing PEG6000 concentration is carried out, in strengthening process, depending on OD
600increase and progressively improve PEG6000 concentration, the concentration of PEG6000 increases with the gradient of 50 g/L, and ultimate aim is 250 g/L; The concentration of PEG6000 is respectively (w/w) 5%, 10%, 15%, 20% and 25%, often increases the concentration of a PEG6000, after thalline increment is stable, will continues the concentration increasing PEG6000, strengthen step by step and drought-enduring mixing microorganisms flora;
(5) separation and the purifying of complex microorganism is strengthened:
1) plate is down flat: by beef extract-peptone nutrient agar, Gao Shi No. I nutrient agar, Ma Dingshi (PDA) nutrient agar high-temperature sterilization, when being cooled to 55 ~ 60 DEG C, Streptomycin Solution is added in martin agar substratum, whole mass concentration is 30 μ g/ml, is mixed evenly to be down flat plate respectively afterwards;
2) mixing microorganisms diluent is prepared: add in the Boiling tube filling 9ml sterilized water with the mixing microorganisms bacteria suspension after liquid-transfering gun absorption 1ml strengthening and fully mix, this is 10
-1diluent, makes (μ g/ml) 10 by that analogy
-2, 10
-3, 10
-4, 10
-5with 10
-6the diluent of several concentration of μ g/ml;
3) be coated with: the dilution bacteria suspension drawing 0.2ml different concns with liquid-transfering gun respectively accurately puts into corresponding culture medium flat plate central authorities, and the process of each different concns gradient repeats 3 times, is coated with evenly with sterile glass rod lightly in media surface;
4) cultivate: beef extract-peptone flat-plate inverted is placed in 37 DEG C of incubators and cultivates, and the flat-plate inverted containing Gao Shi No. I substratum and Ma Dingshi substratum (PDA) is placed in 28 DEG C of incubators and cultivates;
(6) plate streaking partition method
Choose bacterium colony: by the single bacterium colony that grows after cultivating respectively a little lawn of picking on new above-mentioned 3 kinds of substratum, carry out line purifying, until longer on substratum is pure culture, as impure, still need to repeat this step, finally the complex microorganism of strengthening is identified; To be strengthened complex microbial community through purifying of repeatedly ruling.The complex microbial community that wherein strengthened refers to: bacillus cereus, Methionin genus bacillus and rhodotorula glutinis.
The strengthening complex microbial community that the present invention openly adopts this method to prepare further coerces the application in lower turfgrass drought-resistant ability in raising moderate and Severe drought; Particularly coerce lower Festuca Arundinacea defence enzyme activity in moderate and Severe drought, coerce the application in lower Festuca Arundinacea blade mda and proline content in reduction moderate and Severe drought; Wherein said strengthening complex microbial community refers to bacillus cereus, and Methionin genus bacillus and rhodotorula glutinis are by ratio of weight and the number of copies for the ratio of 1:1:1 is configured.
The more detailed preparation method of the present invention is as follows:
1 development materials and methods
1.1 materials:
Consumer garbage compost takes from Tianjin little Dian garbage compost factory.The pH of compost is 7.62, organic content 12.12 %, total nitrogen content 5.18 %, available phosphorus content 77.92 mgkg
-1, full potassium 50.83 gkg
-1, unit weight 0.85 gL
-1, saturation moisture content 66.58 gmL
-1.
1.2 substratum
Enrichment medium: extractum carnis 5g, peptone 10g, NaCl 5g, water 1000ml, pH 7.4-7.6, add 15g agar and become solid medium;
Beef-protein medium: extractum carnis 5g, peptone 10g, NaCl 5g, water 1000ml, pH 7.0-7.2, add 15g agar and become solid medium;
Gao Shi No. I substratum: Zulkovsky starch 20g, KNO
320g, K
2hPO
30.5g, MgSO
40.5g, FeSO
40.01g, water 1000ml, pH 7.2-7.4, add agar 20g and become solid medium (during configuration, first use a small amount of cold water, by starch furnishing pasty state, import in the water boiled, heat in fire, add other compositions while stirring, after dissolving, moisturizing is to 1000ml);
Ma Dingshi substratum: glucose 10g, peptone 5g, KHPO
31g, MgSO (7H
2o) 0.5g, the 1% rose-bengal aqueous solution, 3.3ml, water 1000ml, pH nature, adds agar 15g and becomes solid medium (add 0.03% Streptomycin sulphate diluent 100ml before use, make every ml substratum containing Streptomycin sulphate 30 μ g).
The enrichment of 1.3 bacterial classifications
Compost sample is taken 10g and be placed in aseptic Erlenmeyer flask, after adding 100mL sterilized water shaken well, get 10mL suspension in the Erlenmeyer flask filling 100mL enrichment medium, at 28 DEG C, shaking culture 3d under 220r/min, is mixing microorganisms flora.
The strengthening of 1.4 microorganisms
What the drought-enduring strengthening of mixing microorganisms flora adopted is the method progressively increasing PEG6000 concentration.Mixed bacterial without strengthening generally grows better, in strengthening process, depending on OD under without the condition of PEG6000
600increase and progressively improve PEG6000 concentration, the concentration of PEG6000 increases with the gradient of 50 g/L, and ultimate aim is 250 g/L; Namely the concentration of PEG6000 is respectively (w/w) 5%, 10%, 15%, 20% and 25% in this experiment.Often increase the concentration of a PEG6000, after thalline increment is stable, will could continues the concentration increasing PEG6000, strengthen step by step and drought-enduring mixing microorganisms flora.
1
.the separation of 5 enhancement microbiologicals and purifying
1.5.1 dilution spread flat band method
1) plate is down flat: by beef extract-peptone nutrient agar, Gao Shi No. I nutrient agar, Ma Dingshi (PDA) nutrient agar high-temperature sterilization, when being cooled to 55 ~ 60 DEG C, in martin agar substratum, add Streptomycin Solution (whole mass concentration is 30 μ g/ml), being mixed evenly is down flat plate respectively afterwards.
2) mixing microorganisms diluent is prepared: add in the Boiling tube filling 9ml sterilized water with the mixing microorganisms bacteria suspension after liquid-transfering gun absorption 1ml strengthening and fully mix, this is 10
-1diluent, makes 10 by that analogy
-2, 10
-3, 10
-4, 10
-5with 10
-6the diluent of several concentration.
3) be coated with: the dilution bacteria suspension drawing 0.2ml different concns with liquid-transfering gun respectively accurately puts into corresponding culture medium flat plate central authorities, and the process of each different concns gradient repeats 3 times.Be coated with lightly evenly in media surface with sterile glass rod.
4) cultivate: beef extract-peptone flat-plate inverted is placed in 37 DEG C of incubators and cultivates, and the flat-plate inverted containing Gao Shi No. I substratum and Ma Dingshi substratum (PDA) is placed in 28 DEG C of incubators and cultivates.
1.5.2 plate streaking partition method
Choose bacterium colony: by the single bacterium colony that grows after cultivating respectively a little lawn of picking on new above-mentioned 3 kinds of substratum, carry out line purifying.Until longer on substratum is pure culture, as impure, still need to repeat this step.
The qualification of 1.6 enhancement microbiologicals
The DNA of dominant bacteria is extracted according to the operational manual of Ezup pillar test kit.The PCR system of predominant bacteria: 10 × Buffer(with MgCl
2) 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, template DNA 1 μ L, add ultrapure water and be settled to final volume 20 μ L.PCR reaction conditions: 94 DEG C of 5min denaturations, 94 DEG C of sex change 1min, 55 DEG C of renaturation 45s, 72 DEG C extend 45s, 30 circulations, and 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 system of dominant fungi: 10 × Buffer (without MgCl
2) 2 μ L, MgCl
2(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, template DNA 1 μ L, add ultrapure water and be settled to final volume 20 μ L.PCR reaction conditions: 94 DEG C of 4min denaturations, 94 DEG C of sex change 1min, 54 DEG C of renaturation 1min, 72 DEG C extend 2min, 30 circulations, and 72 DEG C extend 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 ').The PCR primer obtained is delivered to Hua Da gene sequencing portion, Beijing, according to the result of order-checking, in BLAST system, find out corresponding bacterial classification.
2 development results analyses
The mensuration of the drought-enduring microorganism curve of 2.1 strengthening
To the flora cultivated in the substratum of each PEG6000 concentration, survey its OD value when just switching respectively, be the OD initial value of thalline.At regular intervals later (8 hours, the bacterial classification interval of PEG6000 concentration below 15%, 20% and 25%PEG6000 concentration under bacterial classification interval 24 hours) survey the OD value of a thalline, drafting OD-time plot.
(1) microorganism species growth curve under PEG6000 concentration is directly raised
As seen from Figure 1, without under the concentration of PEG6000, the adaptive phase of flora is very short, reaches logarithmic phase soon, and maximum growth value is also the highest.Along with the concentration of PEG6000 raises, the adaptive phase of flora increases gradually, and reach the time also corresponding growth needed for maximum growth value, maximum growth value also decreases.Under high PEG6000 concentration, flora needs an adaptive phase relatively grown just can reach logarithmic phase, and the time reaching maximum growth value is also the longest, and maximum growth value is also minimum.
Result illustrates, the mixed bacterial in compost is deteriorated (adaptive phase growth) gradually along with the rising upgrowth situation of PEG6000 concentration, and reach the time also corresponding prolongation of maximum growth value, maximum growth value also decreases.This is because the PEG6000 concentration of initial inoculation flora is very low, along with the rising of PEG6000 concentration, thalline will bear the pressure that PEG6000 concentration is brought, and needs an adaptive process.Growth due to thalline is a process slowly, and the rising of PEG6000 concentration is the process of a drastic change, and the change of flora to PEG6000 concentration adapts to gradually.Along with the rising of PEG6000 concentration, know from experience dead for a large amount of immalleable, now there will be an adaptive phase, the flora that can adapt to high PEG6000 concentration will survive, and continues amount reproduction, so there will be logarithmic phase after the adaptive phase.
(2) microorganism species growth curve under PEG6000 concentration is progressively improved
As can be seen from Figure 2, after flora through strengthening under above-mentioned low PEG6000 concentration is transferred, the adaptive phase can shorten (with without compared with strengthening), enters into logarithmic phase soon, the time reached required for maximum growth value also shortens, and maximum growth value also increases.
Through progressively improving the mixed bacterial of the method strengthening of PEG6000 concentration, adapt to the environment of previous relatively high PEG6000 concentration.And due to switching be to carry out at the logarithmic phase of flora, and the rear new substratum of switching is nutritious, the growth of flora is stable, rapid, compared with directly raising the method for PEG6000 concentration, adaptive phase is corresponding shortening also, enter logarithmic phase soon, the time reaching maximum growth value also substantially reduces.In addition, the growth maximum value of mixed bacterial under high PEG6000 concentration through progressively strengthening also increases.
Drought-enduring microorganism is separated and purification result in 2.2 strengthening
This experiment carries out drought-enduring strengthening to the microorganism in compost, and eventually pass through 3 kinds of purebred microorganisms that purifying of repeatedly ruling obtains, be 2 kinds of bacteriums and a kind of fungi, result as shown in Figure 2.
Carry out preliminary evaluation according to cultural characteristic and thalli morphology, 2 kinds of bacteriums that can will obtain, be classified as genus bacillus, and fungi are yeast. detailed microbe colony form is in table 1:
The drought-enduring microbial molecules qualification result of 2.3 strengthening
Pass through its colony morphological observation above-mentioned 3 bacterial classifications, binding molecule qualification result (PCR sequence similarity) compares, and identify 2 kinds of genus bacillus and be respectively: bacillus cereus and Methionin genus bacillus, 1 Yeasts is rhodotorula glutinis:
3 development conclusions
This research finally becomes work hardening, separation, Purification and Characterization to obtain 3 kinds of drought-enduring compost microbes, i.e. bacillus cereus, Methionin genus bacillus and rhodotorula glutinis.Be made up of wherein a kind or several enhancement microbiological and strengthen complex micro organism fungicide accordingly; And the enhancement microbiological microbial inoculum be prepared into is applied in the middle of drought environment turfgrass planting system, the upgrowth situation of lawn plant can be improved.
Accompanying drawing illustrates:
Fig. 1 is the growth curve of mixed bacterial under different PEG6000 concentration;
Fig. 2 is the drought-enduring predominant bacteria of strengthening and fungi.
Embodiment
In order to explain enforcement of the present invention more fully, provide following preparation method's embodiment.These embodiments are only explain instead of limit the scope of the invention.Special instruction is needed to be: the present invention screens the complex microbial community bacillus cereus and Methionin genus bacillus that obtain, all there is sale in market to rhodotorula glutinis, also method of the present invention can be adopted to be separated from consumer garbage compost and obtain complex microbial community, the biochemical characteristic of its flora obtained is identical with commercially available therefore not in preservation.
Embodiment 1
The preparation method of drought-enduring strengthening complex microbial community in garbage compost:
(1) material: consumer garbage compost takes from Tianjin little Dian garbage compost factory,
rubbishthe pH of compost is 7.62, organic content 12.12 %, total nitrogen content 5.18 %, available phosphorus content 77.92 mgkg
-1, full potassium 50.83 gkg
-1, unit weight 0.85 gL
-1, saturation moisture content 66.58 gmL
-1;
(2) substratum:
1) enrichment medium: extractum carnis 5g, peptone 10g, NaCl 5g, water 1000ml, pH 7.4-7.6, add 15g agar and become solid medium;
2) beef-protein medium: extractum carnis 5g, peptone 10g, NaCl 5g, water 1000ml, pH 7.0-7.2, add 15g agar and become solid medium;
3) Gao Shi No. I substratum: Zulkovsky starch 20g, KNO
320g, K
2hPO
30.5g, MgSO
40.5g, FeSO
40.01g, water 1000ml, pH 7.2-7.4, add agar 20g and become solid medium, during configuration, first use a small amount of cold water, and by starch furnishing pasty state, import in the water boiled, heat in fire, add other compositions while stirring, after dissolving, moisturizing is to 1000ml;
4) Ma Dingshi substratum: glucose 10g, peptone 5g, KHPO
31g, MgSO (7H
2o) 0.5g, the 1% rose-bengal aqueous solution, 3.3ml, water 1000ml, pH nature, adds agar 15g and becomes solid medium, add 0.03% Streptomycin sulphate diluent 100ml before use, makes every ml substratum containing Streptomycin sulphate 30 μ g;
(3) enrichment of bacterial classification:
Garbage compost sample is taken 10g and is placed in aseptic Erlenmeyer flask, after adding 100mL sterilized water shaken well, get 10mL suspension in the Erlenmeyer flask filling 100mL enrichment medium, at 28 DEG C, shaking culture 3d under 220r/min, is complex microbial community;
(4)
drought-enduringthe strengthening of complex microbial community:
What the drought-enduring strengthening of mixing microorganisms flora adopted is that the method progressively increasing PEG6000 concentration is carried out, in strengthening process, depending on OD
600increase and progressively improve PEG6000 concentration, the concentration of PEG6000 increases with the gradient of 50 g/L, and ultimate aim is 250 g/L; The concentration of PEG6000 is respectively (w/w) 5%, 10%, 15%, 20% and 25%, often increases the concentration of a PEG6000, after thalline increment is stable, will continues the concentration increasing PEG6000, strengthen step by step and drought-enduring mixing microorganisms flora;
(6) separation and the purifying of complex microorganism is strengthened:
1) plate is down flat: by beef extract-peptone nutrient agar, Gao Shi No. I nutrient agar, Ma Dingshi (PDA) nutrient agar high-temperature sterilization, when being cooled to 55 DEG C, in martin agar substratum, add Streptomycin Solution, whole mass concentration is 30 μ g/ml, is mixed evenly to be down flat plate respectively afterwards;
2) mixing microorganisms diluent is prepared: add in the Boiling tube filling 9ml sterilized water with the mixing microorganisms bacteria suspension after liquid-transfering gun absorption 1ml strengthening and fully mix, this is 10
-1diluent, makes 10 by that analogy
-2, 10
-3, 10
-4, 10
-5with 10
-6the diluent of several concentration of μ g/ml;
3) be coated with: the dilution bacteria suspension drawing 0.2ml different concns with liquid-transfering gun respectively accurately puts into corresponding culture medium flat plate central authorities, and the process of each different concns gradient repeats 3 times, is coated with evenly with sterile glass rod lightly in media surface;
4) cultivate: beef extract-peptone flat-plate inverted is placed in 37 DEG C of incubators and cultivates, and the flat-plate inverted containing Gao Shi No. I substratum and Ma Dingshi substratum (PDA) is placed in 28 DEG C of incubators and cultivates;
(6) plate streaking partition method
Choose bacterium colony: by the single bacterium colony that grows after cultivating respectively a little lawn of picking on new above-mentioned 3 kinds of substratum, carry out line purifying, until longer on substratum is pure culture, as impure, still need to repeat this step, finally the complex microorganism of strengthening is identified; To be strengthened complex microbial community through purifying of repeatedly ruling.
Embodiment 2
Application example
Complex micro organism fungicide alignment degree and Severe drought coerce the impact of lower Festuca Arundinacea mda and proline content
Described strengthening complex microbial community refers to bacillus cereus, and Methionin genus bacillus and rhodotorula glutinis are by ratio of weight and the number of copies for the ratio of 1:1:1 is configured.
(1) moderate and Severe drought coerce lower complex micro organism fungicide process Festuca Arundinacea blade mda and proline content is all remarkable in nonvaccinated adjoining tree (table 1).Under medium drought condition, what apply that Festuca Arundinacea blade mda and proline content in the treatment group of the complex micro organism fungicide of dilution 100 times reduce is the most obvious, reduces 38.93% and 65.03% respectively than control group.
The complex micro organism fungicide of table 1 different concns is on the impact of Festuca Arundinacea mda and proline content
And under Severe drought stress conditions, after the complex micro organism fungicide process of dilution 100 times, Festuca Arundinacea blade mda and proline content with control group reduce all very remarkable, reduce 47.65% and 66.37% respectively.The treatment group of diluting 200 times also exist compared with control group significant difference (
p<0.05).
(2) complex micro organism fungicide alignment degree and Severe drought coerce the impact of lower Festuca Arundinacea defence enzyme activity
Inoculating complex microorganism microbial inoculum significantly improves moderate and Severe drought coerces lower Festuca Arundinacea foliar SOD, POD and CAT activity (table 2).When being applied to 100 times of diluents under medium drought, Festuca Arundinacea foliar SOD, POD and CAT activity reach the highest, are 6.74,1.88 and 2.41 times of contrast respectively.When to strengthen drought-enduring microbiobacterial agent diluent multiple be 100, Severe drought coerces lower Festuca Arundinacea foliar SOD, POD and CAT activity for the highest, is 2.12,2.17 and 5.43 times of control group respectively.Compared with control group, 200 times of dilution process groups exist significant difference (
p<0.05).
The complex micro organism fungicide of table 2 different concns is on the impact of Festuca Arundinacea defence enzyme activity
Development conclusion:
Under drought stress, Festuca Arundinacea is vaccinated with complex micro organism fungicide, can significantly improve defence enzyme activity, thus alleviates the injury that drought stress brings Festuca Arundinacea, contributes to the raising of the drought-resistant ability promoting turfgrass.Print from the consumption of complex intensifying microbiobacterial agent, the Be very effective of 100 times of diluents is better than 200 times of diluents.This technological method alleviates turfgrass injury effect under can be drought condition has important using value.
Claims (5)
1. the preparation method of the drought-enduring strengthening complex microbial community in lawn in garbage compost, is characterized in that being undertaken by following step:
(1) material: consumer garbage compost takes from Tianjin little Dian garbage compost factory,
rubbishthe pH of compost is 7.62, organic content 12.12 %, total nitrogen content 5.18 %, available phosphorus content 77.92 mgkg
-1, full potassium 50.83 gkg
-1, unit weight 0.85 gL
-1, saturation moisture content 66.58 gmL
-1;
(2) substratum:
1) enrichment medium: extractum carnis 5g, peptone 10g, NaCl 5g, water 1000ml, pH 7.4-7.6, add 15g agar and become solid medium;
2) beef-protein medium: extractum carnis 5g, peptone 10g, NaCl 5g, water 1000ml, pH 7.0-7.2, add 15g agar and become solid medium;
3) Gao Shi No. I substratum: Zulkovsky starch 20g, KNO
320g, K
2hPO
30.5g, MgSO
40.5g, FeSO
40.01g, water 1000ml, pH 7.2-7.4, add agar 20g and become solid medium, during configuration, first use a small amount of cold water, and by starch furnishing pasty state, import in the water boiled, heat in fire, add other compositions while stirring, after dissolving, moisturizing is to 1000ml;
4) Ma Dingshi substratum: glucose 10g, peptone 5g, KHPO
31g, MgSO (7H
2o) 0.5g, the 1% rose-bengal aqueous solution, 3.3ml, water 1000ml, pH nature, adds agar 15g and becomes solid medium, add 0.03% Streptomycin sulphate diluent 100ml before use, makes every ml substratum containing Streptomycin sulphate 30 μ g;
(3) enrichment of bacterial classification:
Garbage compost sample is taken 10g and is placed in aseptic Erlenmeyer flask, after adding 100mL sterilized water shaken well, get 10mL suspension in the Erlenmeyer flask filling 100mL enrichment medium, at 28 DEG C, shaking culture 3d under 220r/min, is complex microbial community;
(4)
drought-enduringthe strengthening of complex microbial community:
What the drought-enduring strengthening of mixing microorganisms flora adopted is that the method progressively increasing PEG6000 concentration is carried out, in strengthening process, depending on OD
600increase and progressively improve PEG6000 concentration, the concentration of PEG6000 increases with the gradient of 50 g/L, and ultimate aim is 250 g/L; The concentration of PEG6000 is respectively (w/w) 5%, 10%, 15%, 20% and 25%, often increases the concentration of a PEG6000, after thalline increment is stable, will continues the concentration increasing PEG6000, strengthen step by step and drought-enduring mixing microorganisms flora;
(5) separation and the purifying of complex microorganism is strengthened:
1) plate is down flat: by beef extract-peptone nutrient agar, Gao Shi No. I nutrient agar, Ma Dingshi (PDA) nutrient agar high-temperature sterilization, when being cooled to 55-60 DEG C, Streptomycin Solution is added in martin agar substratum, whole mass concentration is 30 μ g/ml, is mixed evenly to be down flat plate respectively afterwards;
2) mixing microorganisms diluent is prepared: add in the Boiling tube filling 9ml sterilized water with the mixing microorganisms bacteria suspension after liquid-transfering gun absorption 1ml strengthening and fully mix, this is 10
-1diluent, makes 10 by that analogy
-2, 10
-3, 10
-4, 10
-5with 10
-6the diluent of several concentration of μ g/ml;
3) be coated with: the dilution bacteria suspension drawing 0.2ml different concns with liquid-transfering gun respectively accurately puts into corresponding culture medium flat plate central authorities, and the process of each different concns gradient repeats 3 times, is coated with evenly with sterile glass rod lightly in media surface;
4) cultivate: beef extract-peptone flat-plate inverted is placed in 37 DEG C of incubators and cultivates, and the flat-plate inverted containing Gao Shi No. I substratum and Ma Dingshi substratum (PDA) is placed in 28 DEG C of incubators and cultivates;
(6) plate streaking partition method
Choose bacterium colony: by the single bacterium colony that grows after cultivating respectively a little lawn of picking on new above-mentioned 3 kinds of substratum, carry out line purifying, until longer on substratum is pure culture, as impure, still need to repeat this step, finally the complex microorganism of strengthening is identified; To be strengthened complex microbial community through purifying of repeatedly ruling.
2. the preparation method of drought-enduring strengthening complex microbial community in garbage compost described in claim 1, the complex microbial community that wherein strengthened refers to: bacillus cereus, Methionin genus bacillus and rhodotorula glutinis.
3. the strengthening complex microbial community that described in employing claim 1 prepared by method coerces the application in lower turfgrass drought-resistant ability in raising moderate and Severe drought; Wherein said strengthening complex microbial community refers to bacillus cereus, and Methionin genus bacillus and rhodotorula glutinis are by ratio of weight and the number of copies for the ratio of 1:1:1 is configured.
4. the strengthening complex microbial community that described in employing claim 1 prepared by method coerces the application in lower Festuca Arundinacea defence enzyme activity in raising moderate and Severe drought; Wherein said strengthening complex microbial community refers to bacillus cereus, and Methionin genus bacillus and rhodotorula glutinis are by ratio of weight and the number of copies for the ratio of 1:1:1 is configured.
5. the strengthening complex microbial community that described in employing claim 1 prepared by method coerces the application in lower Festuca Arundinacea blade mda and proline content in reduction moderate and Severe drought; Wherein said strengthening complex microbial community refers to bacillus cereus, and Methionin genus bacillus and rhodotorula glutinis are by ratio of weight and the number of copies for the ratio of 1:1:1 is configured.
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Citations (2)
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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 |
| CN103947683A (en) * | 2014-05-08 | 2014-07-30 | 天津师范大学 | Method for improving festuca arundinacea drought resistance by adopting dried sludge fungicide |
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2015
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103947683A (en) * | 2014-05-08 | 2014-07-30 | 天津师范大学 | Method for improving festuca arundinacea drought resistance by adopting dried sludge fungicide |
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| LEI BING ET AL: "Overexpression of the AtSTK Gene Increases Salt, PEG and ABA Torerance in Arabidopsis", 《J. PLANT BIOL》 * |
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