CN104838842B - Strengthen the method for active nano garbage compost regulation salt stress turfgrass protective enzyme using salt tolerant - Google Patents
Strengthen the method for active nano garbage compost regulation salt stress turfgrass protective enzyme using salt tolerant Download PDFInfo
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Abstract
The invention discloses the method for strengthening active nano garbage compost regulation salt stress turfgrass protective enzyme using salt tolerant.It is that salt-durable microbe is extracted from compost, and screening is obtained into purebred salt-durable microbe, further strengthens to practice to be made using the concentration method for stepping up NaCl and strengthens salt tolerant microbial inoculum, is administered in combination with nanometer compost in saline-alkali soil lawn matrix.Experimental design is slight, the three kinds of salt stress processing of moderate and severe; realize the salt-resistance of enhancement microbiological joint nanometer compost enhancing turfgrass; purpose is development concentrated fertilizer; to reach the enhancing of defence enzyme activity under salt stress of regulation turfgrass, to reach the purpose for promoting saline-alkali soil turf establishment effect.
Description
Technical field
The invention belongs to environmental protection technical field, is related to a kind of using salt tolerant reinforcing active nano garbage compost regulation salt
The method for coercing turfgrass protective enzyme.
Background technology
Nanoscale science and technology（Nano-ST）It is the new science and technology to emerge phase late 1980s, it mainly studies knot
Structure yardstick is (10-7-10-9M) property of material and its application in the range of.Because nano material has interface surface interface effect
Should, the essential characteristic such as small-size effect, macro quanta tunnel effect and quantum size effect, so, there are many traditional materials
Expect the Strange properties not possessed.Nano material all shows to be different from tradition in terms of absorption, catalysis, magnetic effect and sensitivity characteristic
The characteristic of material, can with multiple fields height intersection, including physics, chemistry, electronics, material science and biology, in high-tech
Skill application above shows vast potentiality, so, applications to nanostructures is more and more paid close attention to by scientist.China's political affairs
The research of nanometer technology is attached great importance at mansion, and nanostructured and nano material are considered as into the key of nanometer technology and preferentially propped up
Hold.2012, Chinese industrial and information-based portion according to《National " 12 " emerging strategic industries development plan》Spirit,
Issue《New material industry " 12 " development plan》, nano material is classified as forward position field of new materials, and explicitly point out：China
Nanotechnology research will be increased, energetically promotes nano material to be led in new energy, environmental improvement, energy-saving and emission-reduction and bio-medical etc.
The research and utilization in domain.
The external research for nano material focuses primarily upon the research to safety of nano materials, and five mainly studied are big
Problem is：Absorption of the skin to nano material and its injury to skin；Drink the consequence of the water containing nano particle；Nano particle pair
The influence of animal lung；Influence of the nano particle of deposit in water to surrounding environment 1 to 4 May in 2005, Europe are become
Held " European nano biological effect meeting " in Bonn, Germany inside coenosarc.The session discussing formation of nano particle, release,
Motion and detection method in environment, its acute virus action, chronic toxic action and cellular level and molecular level
Influence, such as to haemocyte, lung cells, immunocyte, DNA damage, gene expression influence.The country there have been developed a nanometer material
Expect the influence to plant, nano fertilizer is proposed by soil and fertilizer research institute of the Chinese Academy of Agricultural Sciences Zhang Fudao researcher, and in country
Set up the project in " 863 " project.Nano fertilizer is a branch of nano biological science and technology, is to use nano material technology and medical microcapsules
Technique construction is modified and manufactured brand-new fertilizer, is divided into nano material coating, cementing sustained-release and controlled release fertilizer and nanostructured fertilizer.
It is well known that underground water pollution caused by abuse fertilizer is increasingly severe, utilization rate of fertilizer how is improved as solving the problems, such as
Emphasis, nano-material surface atom number accounts for more than the 80% of complete particle total atom number, because lacking phase around surface atom
Adjacent atom, containing many dangling bonds, so having unsaturation, it is easy to it is combined and settles out with other atoms, from
And show very high chemism.The utilization rate of fertilizer can be significantly improved with Fertilizer combination, is on the one hand due to a nanometer material
The magnetic effect of material, nutrient can be promoted to be absorbed by plants, stimulate growth and development of plants, and a variety of enzymes in plant can also be improved
Activity.On the other hand, it is because its skin effect, increases nano fertilizer surface energy and Surface binding energy, help it in soil
Absorbed in earth environment by root system of plant, the effect of Fertilizer application is improved.There are some researches show add nano-sized carbon fertilizer synergist
The decline of the available nutrient of soil can effectively be slowed down, improve rice yield, amount of increase in production is asked 1.9%-3.5%'s.Nano-sized carbon pair
The influence of rice yield and utilization rate of nitrogen fertilizer, also demonstrate and be remarkably improved rice yield using nano-carbon powder, meanwhile, suitable
Under the conditions of amount of nitrogen, being affixed by nano-carbon powder contributes to the raising of nitrogen fertilizer for paddy rice utilization rate.Nano material adhesive coating type slow/controlled release
The influence of Fertilizer on Crop Yields and quality, the results showed that, nano material adhesive coating type slowly/controlled releasing fertilizer each contributes to wheat
With the raising of maize kernel yield and protein output.The nano fertilizer studied at present is most for nano material synergy fertilizer or nanometer
Material coated fertilizer, and fertilizer is worked into the research that nanometer particle size is applied to lawn plant environment stress in itself, it there is no document report
Road.
Microbial manure is also known as bacterial manure, bio-feritlizer, and main function, which is shown, helps crop nutrient absorption, lifting chemical fertilizer
Utilization rate and quality of agricultural product, enhancing crop anti-adversity etc..Microbial manure generally can be divided into two classes, and one kind is microorganism
By the vital movement of itself, increase is available for the nutrient of plant absorption；It is another kind of, microbial life activity can be passed through
Secondary metabolism secreting hormone, come the resistance for promoting plant growth and improving plant.As nitrogen-fixing microorganism can be made by fixed nitrogen
With increase Soil Nitrogen Content, microorganism can improve the conversion and absorption of Nutrient Elements in Soil by vital movement, be big
The major function of most microbial manures.In addition, some beneficial microbes can produce a variety of sugar by the metabolic activity of itself
Class material, these glucides can not only organically combine with the gelatin substance in soil, while can also be with plant certainly
Mucus is combined together caused by body, plays a part of improved soil.Microbial bacterial agent also has improvement crop growth conditions, increases
The effects such as strong plant resistance to environment stress.Most of researchs are it has been shown that microorganism formulation can promote the suction of plant growth and nutrient at present
Receive.There is researcher to be applied to rice research, the results showed that, compared with conventional fertilizer application, applying bio-bacterial manure can effectively facilitate
The increase of rice yield.Also filter out seven kinds of plant growth-promoting fungies are applied to the culture studies hair of sunflower by someone, as a result
Prove, fungi can be obviously promoted its primary growth, and can effectively suppress the generation of downy mildew.In summary, it is relevant at present micro-
The research of biology lays particular emphasis on beneficial microorganism extraction, identification and application more, but is applied after beneficial microbe is further strengthened
In lawn plant adverse growth research there is no literature reported on.
Turf establishment system is the ecological work for integrating ecological regulation and control, beautifying the environment with the multiple functions such as entertainment
Journey.As the important component of urban landscaping, green grassland is one of the standard for weighing economic development level, Ye Shiheng
Measure one of standard of modern city, be one of modern city social development and economic strength it is strong embody, turf establishment body
The structure of system for improve City Investment Environment, invite outside investment, promotes travel etc. various aspects all play a part of can not ignore.
Lawn also plays an important role while landscape and landscaping effect is brought in terms of ecosystem balance is maintained.First, it
Air, sterilizing can be purified, lawn absorbs CO by photosynthesis2Release O2Keep CO in air2And O2Balance, grass
The Toxics such as toxic gas, carcinogen and some heavy metal gases that level ground can also dilute, decompose, absorb and fix in air
Matter, turned bane into boon by photosynthesis.Secondly, lawn can conserve water and soil, maintain the ecological balance.Lawn can fine and close covering earth's surface
Grass layer, and intensive root system can effectively set table soil, prevent the formation of rainwash, the possibility that reduction table soil is pulled away
Property.In addition, lawn can also slow down solar radiation, surface temperature, increase air humidity are effectively reduced.
Salt-soda soil（Soil）, it is that salinization is native, the general name of alkali soil and saline-alkali soil.In recent years, the salinization of soil constantly adds
Weight.At present, there is 20 × 106hm in China2Above salt-soda soil and 7 × 106hm2Above salinization soil, account for the 20% of cultivated area
.Whether in arid area, or water resource enrich coastal area, salt stress is all a kind of extremely serious abiotic side of body
Compel.And salt stress is one of critical environments factor for limiting Turfgrass Growth.Substantial amounts of salinization soil is fallen into disuse, greatly
Ground wastes soil resource, hinders agricultural production and urban afforestation process.Harm of the salinity to crop is mainly shown as, seed
Germination percentage, which reduces, suppresses decreased growth, root growth slows down, yellow leaf, injures plant tissue, causes plant cell membrane damage etc.
Phenomenon, growing for plant is caused to be severely limited.For a long time, the improvement of saline-alkali soil is mainly using the row of irrigation
Water, apply chemical improvement agent or mineral fertilizer and plant the plant of Salt And Alkali Tolerance.For problem above, research at present focuses primarily upon
Choose strong stress resistance lawn plant, using concentrated fertilizer, using measures such as water-loss reducer and modifying agents.Nano fertilizer is as a kind of new
The concentrated fertilizer of type, is received more and more attention in terms of Adversity-stressed Plant.
Nano fertilizer is a kind of special concentrated fertilizer, and the surface area of its particle, surface energy and Surface binding energy are all relative
It is larger, contribute to its root hair or tip of a root partial adsorbates in soil environment by plant, its characteristic property is possible to solve fertilizer
The problems such as utilization rate, crop yield and plant resistance to environment stress.At present on nano fertilizer research it is most for nano material synergy fertilizer or
Nano material coat fertilizer, and fertilizer is worked into the research that nanometer particle size is applied to lawn plant in itself, equally also have no related
Technology is reported.
Consumer garbage compost is a hypertonic system, and microorganism therein has certain adaptability to adverse circumstance, will wherein
It is significant that beneficial microbe microbial inoculum is applied to lawn adverse circumstance planting after further strengthening.Based on considerations above, in order to grind
The application effect of nanometer compost and its beneficial microbe in turf establishment is studied carefully, herein by garbage compost through nanofabrication technique
30nm or so is worked into, and separation and Extraction goes out suitable beneficial microbe strain from consumer garbage compost, by further resisting
Property domestication, obtain highly efficient enhancement microbiological microbial inoculum, after it is mixed by certain volume ratio, complex microorganism be made
Microbial inoculum, the turf establishment under the environment stresses such as stress is united and applied in nanometer compost, by exploring it to careless under environment stress
The influence of level ground plant physiological ecology characteristic, to combine application of the nanometer compost in adverse circumstance turf establishment for enhancement microbiological,
The planting of adverse circumstance environment lawn plant is promoted to provide theoretical foundation.
Microorganism can remarkably promote plant growth, promote the absorption of nutrient.In addition, fungi can effectively improve lawn plant
The resistances such as the winter resistance of thing, drought resistance, resistance biohazard.Some results of study show that nanometer compost has to plant growth
Obvious facilitation, but by nanometer compost be used for salt stress turf establishment aspect technology there is no literature reported on.
The content of the invention
The present invention extracts salt-durable microbe from compost, and screening is obtained into purebred salt-durable microbe, using stepping up
NaCl concentration method, which is further strengthened to practice to be made, strengthens salt tolerant microbial inoculum, is administered in combination with nanometer compost in saline-alkali soil lawn matrix
In.Experimental design is slight, the three kinds of salt stress processing of moderate and severe, realizes enhancement microbiological joint nanometer compost enhancing turfgrass
Salt-resistance, purpose for develop concentrated fertilizer, with reach regulation turfgrass the enhancing of defence enzyme activity under salt stress, with up to
To the purpose for promoting saline-alkali soil turf establishment effect.
The invention discloses following content to achieve the above object：
A kind of method for being strengthened active nano garbage compost regulation salt stress turfgrass protective enzyme using salt tolerant, its feature are existed
Carried out in by the steps：
（1）The preparation of nanometer compost：
By consumer garbage compost, 105 DEG C are dried to constant weight, and garbage compost processing grinding is made into 30-35nm nanometer compost,
It is standby；
（2）The preparation of Salt-tolerant microbial agent：
Compost sample is weighed into 10g to be placed in sterile conical flask, after adding 100mL sterilized water shaken wells, takes 10mL to hang
Supernatant liquid, at 28 DEG C, shaken cultivation 3d under 220r/min, as mixes micro- life in the conical flask for filling 100mL enriched mediums
Thing flora；Contain bacillus subtilis, lysine bacillus and penicillium chrysogenum in described mixed microorganism flora；
（3）Mixed microorganism flora salt tolerant is strengthened using the method for being stepped up salinity, highly concentrated to mitigate moment
Degree salt impacts and poisoned to caused by hybrid bacterial strain, and NaCl concentration increases to 3% with 0.5% gradient, often increases a NaCl
Concentration, could continue to increase NaCl concentration, tame out salt-durable microbe flora step by step after thalline increment is stable；
（4）The preparation of compound Salt-tolerant microbial agent：
Bacillus subtilis, lysine bacillus and penicillium chrysogenum by volume 1:1:1 ratio is configured to compound resistance to
Salt microbial bacterial agent, 100 times of dilution are standby；
（5）Turf established by sod：
Soil and saline-alkali soil sieving are cleaned, slight 0.3%, moderate 0.6% is made by a certain percentage and severe 0.9% is saline and alkaline
Soil, 30 min that the saline-alkali soil mixed is sterilized at 121 DEG C are standby；
Each stress sets 3 processing, and the set processing of three stress is identical, is respectively:
1）15 ml blank cultures as control matrix and are added using 100% soil
2）Soil and nanometer compost press 59：1 ratio is mixed and added into 15ml blank cultures
3）Soil and nanometer compost press 59：1 ratio is mixed and added into 15ml and strengthens Salt-tolerant microbial agent
（5）Lawn plant experiment in cultivation is carried out using the soil that mixes as matrix, and substrates quantity is 9400gm during experiment-2,
Compounded turf matrix thickness is 15 mm, and Festuca Arundinacea grass seeds application rate is 160gm-2, uniformly sowed in matrix table after seed is soaked into 24h
Layer, carries out dark processing first, and fully watering ensures to sprout, and is cultivated after sprouting, intensity of illumination is 450-700 μm of ol.m-2.s-1, a water is periodically during which poured sooner or later, often exchanges the position of culture turf to ensure that illumination is consistent, indoor is relatively wet
It is 20-25 DEG C to spend for 40-55%, temperature, and experiment carries out the measure of progress each index of plant after 50d.Wherein described is stepped up
The method of salinity refers to：NaCl concentration is respectively 0.5%, 1%, 1.5%, 2%, 2.5% and 3%, during domestication, depending on
OD600Increase and step up salinity, NaCl concentration increases to 3% with 0.5% gradient, material therefor：
（1）Festuca Arundinacea：Choose full seed, perennial Festuca Arundinacea of uniform size (Festuca arundinacea L.)
Seed is test material；
（2）Consumer garbage compost：From Tianjin little Dian composting plants, basic physical and chemical is：PH 7.62, it is saturated aqueous
Measure 0.76 ml/g, the g/ml of unit weight 0.85, full nitrogen 5.18%, the full g/kg of potassium 50.83, the mg/kg of available phosphorus 77.92, organic matter
12.12%, garbage compost is removed into the debris such as wood therein, plastics, metal, it is standby after air-drying；
（3）Soil：Impurities removing, 2mm sieves are crossed, be positioned over 2-3d at ventilating opening, air-dried under natural conditions, its physicochemical property
For：PH7.44, saturation moisture content 0.58mLg-1, organic matter 4.68%, full nitrogen 0.21%, salt content 0.29%；
（4）Saline-alkali soil:It is derived from Hebei province Huanghua City seashore pH 7.85, organic matter 2.21%, full nitrogen 0.258%, available phosphorus
15.26mgkg-1, full potassium 28.96gkg-1, salt content 1.58%.
The present invention further discloses strengthen active nano garbage compost regulation salt stress turfgrass protective enzyme using salt tolerant
Method improve Testuca arundinacea to the application in terms of salt stress adaptability.
More detailed description of the present invention：
1 develops materials and methods
1.1 experiment material
Choose full seed, perennial Festuca Arundinacea of uniform size (Festuca arundinaceaL.) seed is examination
Test material.
Consumer garbage compost comes from Tianjin little Dian composting plants, and basic physical and chemical is：PH 7.62, saturation moisture content
0.76 ml/g, the g/ml of unit weight 0.85, full nitrogen 5.18%, the full g/kg of potassium 50.83, the mg/kg of available phosphorus 77.92, organic matter
12.12%, garbage compost is removed into the debris such as wood therein, plastics, metal, it is standby after air-drying.
Soil matrix is derived from school institute of Tianjin Normal University, impurities removing, is crossed 2mm sieves, is positioned over 2-3d at ventilating opening, from
Air-dried under the conditions of so, its physicochemical property is：PH7.44, saturation moisture content 0.58mLg-1, organic matter 4.68%, full nitrogen 0.21%, contain
Salt amount 0.29%.
Saline-alkali soil is derived from Hebei province Huanghua City seashore pH 7.85, organic matter 2.21%, full nitrogen 0.258%, available phosphorus
15.26mgkg-1, full potassium 28.96gkg-1, salt content 1.58%.
The preparation of 1.2 nanometers of compost
By garbage compost, 105 DEG C are dried to constant weight, improved by Qinhuangdao Tai Ji ring nanometer products Co., Ltd, utilization
High-energy ball-milling process, moved by the quick multi-D swing of tank body, a nanometer compost is made in garbage compost processing grinding, it is standby
With.By granularmetric analysis under Electronic Speculum, nanometer compost particle size distribution is uniform, and average grain diameter is in 30nm or so（Fig. 1）.
The preparation of 1.3 Salt-tolerant microbial agents
1.3.1 the enrichment of strain
Compost sample is weighed into 10g to be placed in sterile conical flask, after adding 100mL sterilized water shaken wells, takes 10mL to hang
Supernatant liquid, at 28 DEG C, shaken cultivation 3d under 220r/min, as mixes micro- life in the conical flask for filling 100mL enriched mediums
Thing flora.
1.3.2 the reinforcing of salt-durable microbe
Mixed microorganism flora salt tolerant is strengthened using the method for being stepped up salinity, to mitigate moment high salt concentration
Impact and poison to caused by hybrid bacterial strain.Mixed bacterial without domestication is raw under conditions of being typically 0.5% in NaCl concentration
It is long preferable, and the target NaCl concentration this time studied is 3%, i.e., in our current research NaCl concentration be respectively 0.5%, 1%, 1.5%,
2%th, 2.5% and 3%.During domestication, depending on OD600Increase and step up salinity, NaCl concentration is increased with 0.5% gradient
3% is added to, often increases NaCl concentration, could be continued to increase NaCl concentration, step by step after thalline increment is stable
Tame out salt tolerant mixed microorganism flora.
1.3.3 the separation and purifying of salt-durable microbe are strengthened
1.3.3.1 dilution spread flat band method
1) it is down flat plate：By beef extract-peptone agar medium, Gao Shi No. I agar medium, Martin（PDA）Agar is trained
Base high-temperature sterilization is supported, when being cooled to 55-60 DEG C, Streptomycin Solution is added in martin agar culture medium（Final mass concentration
For 0.3%）, be mixed it is even after be down flat plate respectively.
2) mixed microorganism dilution is prepared：The mixed microorganism bacteria suspension after 1ml strengthens is drawn with liquid-transfering gun and adds Sheng
Have in the Boiling tube of 9ml sterilized waters and fully mix, this is 10-1Dilution, 10 are made by that analogy-2、10-3、10-4、10-5With 10-6The dilution of several concentration.
3) it is coated with：The dilution bacteria suspension for drawing 0.2ml various concentrations respectively with liquid-transfering gun is accurately put into corresponding culture medium and put down
Plate center, each various concentrations gradient processing are repeated 3 times.Lightly it is coated with uniformly in media surface with sterile glass rod.
4) cultivate：Beef extract-peptone flat board is inverted in 37 DEG C of incubators and cultivated, and will contain No. I culture medium of Gao Shi and Martin
Family name's culture medium（PDA）Flat board be inverted in 28 DEG C of incubators and cultivate 3-5 d.
1.3.3.2 plate streaking partition method
Choose bacterium colony：The single bacterium colony grown after culture the difference a little lawn of picking is enterprising in new above-mentioned 3 kinds of culture mediums
Row line purifying.Until on culture medium grow be it is purebred, such as it is impure, still need to repeat the step, finally obtain 3 strains.
1.3.4 the identification of enhancement microbiological
The DNA of dominant bacteria is extracted according to the operation manual of kit.The PCR system of predominant bacteria：10×Buffer
（with MgCl2）2 μ L, dNTP（10mmol/L）0.4 μ L, 341f（10µmol/L）1 μ L, 534r（10µmol/L）1 μ L, Taq
Enzyme（5u/µL）0.4 μ L, the μ L of template DNA 1, ultra-pure water is added to be settled to the μ L of final volume 20.PCR reaction conditions：94 DEG C of 5min become in advance
Property, 1min, 55 DEG C of renaturation 45s, 72 DEG C of extension 45s of 94 DEG C of denaturation, 30 circulations, 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 systems of dominant fungi：10×Buffer
（without MgCl2）2 μ L, MgCl2（25mmol/L）1.6 μ L, dNTP（10mmol/L）0.4 μ L, Geo11（10µmol/L）
0.4 μ L, GeoA2（10µmol/L）0.4 μ L, Taq enzyme（5u/µL）0.2 μ L, the μ L of template DNA 1, add ultra-pure water to be settled to final volume
20µL.PCR reaction conditions：94 DEG C of 4min pre-degenerations, 94 DEG C of denaturation 1min, 54 DEG C of renaturation 1min, 72 DEG C of extension 2min, 30 are followed
Ring, 72 DEG C of extension 7min.Primer is GeoA2（5′-CCA GTA GTC ATA TGC TTG TCT C-3′）And Geo11（5′-
ACC TTG TTA CTT TTA CTT CC-3′）.Obtained PCR primer is sent to Beijing Hua Da gene sequencing portion, according to sequencing
Result, corresponding strain is found out in BLAST systems.
Above-mentioned 3 strain is passed through to its colony morphological observation, binding molecule qualification result（PCR sequence similarities）Compare,
Qualification result is respectively：Bacillus subtilis, lysine bacillus and penicillium chrysogenum.
Table 1 strengthens salt-durable microbe qualification result
1.3.5 prepared by complex micro organism fungicide
The reinforcing salt-durable microbe for screening to obtain in previous experiments is expanded into culture on corresponding fluid nutrient medium.Withered grass
Bacillus and lysine bacillus are at 30 DEG C, and 180r/min culture 1d, at 28 DEG C, 220r/min is cultivated penicillium chrysogenum.Training
Bacillus subtilis after supporting, lysine bacillus and penicillium chrysogenum press 1:1:1 ratio is configured to complex micro organism fungicide, dilute
Release 100 times it is standby.
1.4 experimental designs and turf established by sod
Campus soil and saline-alkali soil sieving are cleaned, are made by a certain percentage slight（0.3%）, moderate（0.6%）And severe
（0.9%）Saline-alkali soil, 30 min that the saline-alkali soil mixed is sterilized at 121 DEG C are standby.Each stress sets 3 processing, three sides of body
It is identical to compel set processing, is respectively:
（1）15 ml blank cultures as control matrix and are added using 100% soil（CK）
（2）Soil and nanometer compost press 59：1 ratio is mixed and added into 15ml blank cultures（M1
（3）Soil and nanometer compost press 59：1 ratio is mixed and added into 15ml and strengthens Salt-tolerant microbial agent（M2）
Lawn plant experiment in cultivation is carried out using the soil that mixes as matrix, and substrates quantity is 9400gm during experiment-2, turf
Stromal thickness is 15 mm, and Festuca Arundinacea grass seeds application rate is 160gm-2.Uniformly sowed in matrix top layer after seed is soaked into 24h, it is first
Dark processing is first carried out, fully watering ensures to sprout, and is cultivated after sprouting, intensity of illumination is 450-700 μm of ol.m-2.s-1,
Period periodically pours a water sooner or later, often exchanges the position of culture turf to ensure that illumination is consistent.Indoor relative humidity is 40-
55%, temperature is 20-25 DEG C, and experiment carries out the measure of progress each index of plant after 50d.
The measure of 1.5 indexs
1.5.1 proline, the measure of MDA (MDA)
Using acid ninhydrine colorimetric method：Weigh 0.5 g blades to shred, be respectively placed in Boiling tube, be then respectively adding
10 mL 3% sulfosalicylic acid, extracts 10min in boiling water bath（Extraction process will often shake）, in 3000 r after cooling
min-110min is centrifuged, 2mL extract solutions is drawn in another clean test tube with ground stopper, adds 2 mL glacial acetic acid and 2 mL acidity indenes
Triketone, 30 min, cooling 4 mL toluene extraction, 3000 rmin are heated in boiling water-15 min are centrifuged, take upper liquid in 520
Light absorption value is surveyed under nm.As a result calculate：Standard curve finds proline content in 2 mL measure liquid（ug/mL）, then calculate sample
Middle proline content percentage.Formula is as follows：
Using thiobarbituricacidα- method；0.5 g blades are weighed, are extracted with 10%TCA, constant volume 10 mL, 4000 rmin-1
Centrifuge 10 min, take the mL of upper liquid 2 to add 2 mL 0.6%TBA, 15min in boiling water, centrifuge 10 min, take supernatant in
Light absorption value is surveyed under 532 nm and 450 nm.
1.5.2 the measure of protective enzyme
Crude enzyme liquid extracts：0.5 g sample leaves accurately are weighed, use phosphate buffer（PBS, pH7.8）Ice bath milling and extracting, constant volume
25 mL, 10 mL are taken in centrifuge tube, 10000 rmin-1The min of EPPENDOFF centrifuges 20, supernatant are that thick enzyme carries
Take liquid.
POD determinations of activity use guaiacol method, 3mL reaction mixture（PH6.0 phosphate buffer 50mL, add
The μ L of guaiacol 28, heating stirring, until guaiacol dissolves, after solution cooling, add the μ L of 30% hydrogen peroxide 19, mixing
Uniformly, it is stored in refrigerator）, the μ L of enzyme liquid 100 are added, manual time-keeping is opened immediately, absorbance is measured under 470 nm wavelength,
Every 0.5 min readings once.The size of enzymatic activity is represented with OD value changes per minute, i.e., with △ OD470/ min is represented.And with
△ A per minute470Change 0.01 is a peroxidase activity unit.
SOD determinations of activity use NBT methods, are filling 3mL reaction mixtures（54mL 14.5mmol/L dl- methionines
In be separately added into 3 μm of ol/L EDTA, 2.25mmol/L NBT, 60 μm of each 2mL of ol/L riboflavin solutions）Test tube in, add 50
μ L crude enzyme liquid, it is placed on after mixing on transparent rack for test tube, accurate irradiation is anti-under 4000 Lx fluorescent lamps in illumination box
20 min are answered, dark place is placed in as light control, while by control tube using the irradiation pipe for being not added with enzyme liquid（Enzyme liquid is replaced with buffer solution）,
It is used as zeroing.SOD active units are to suppress the 50% of NBT photochemical reductions as a unit of enzyme activity, be surveyed under 560nm wavelength
Determine absorbance, calculate and react repressed percentage.
CAT determinations of activity use ultraviolet spectrophotometry, and reaction mixture is added in test tube（PH7.8 phosphoric acid buffer
Liquid 1.5mL, enzyme liquid 0.2mL, distilled water 1.0mL）, control tube replaces enzyme liquid with buffer solution, then adds 0.3mL in pipe is determined
Concentration is 0.1mol/L hydrogen peroxide（30% hydrogen peroxide 1.13mL distilled water constant volume is to 100mL）, while timing immediately, it is fast
Speed measures absorbance under 240nm wavelength, every 0.5 min readings once.With △ A240 per minute changes 0.01 for one
Catalase activity unit.
1.6 data processing
Handled using the software of Microsoft Excel and SPSS 17.0.
2 development results are analyzed
2.1 enhancement microbiologicals combine influence of the nanometer compost to Festuca Arundinacea proline, mda content under salt stress
Osmotic adjustment is a kind of important Physiological protection mechanism that plant adapts to stress, and proline is considered as preferably to permeate
Auto-regulator.The Festuca Arundinacea proline content of processing, compared with the control, three are administered in combination with nanometer compost for enhancement microbiological microbial inoculum
39.0%, 40.4% and 38.9% is reduced respectively under kind concentration.Singly apply nanometer compost treatment also has conspicuousness poor compared with the control
It is different.
MDA is one of primary product of lipid peroxidation, and its accumulation is the performance of toxicity of biological active oxygen effect, and its content is normal
By as a leading indicator for judging peroxidation of membrane lipids.As can be seen from Table 2, under different degrees of salt stress, strengthen micro-
Bacteria agent combines nanometer compost treatment group, and lawn plant leaf malondialdehyde content is substantially less than adjoining tree, in 0.6% salt
Under stress, 56.7% is reduced compared with the control, illustrates that enhancement microbiological microbial inoculum joint nanometer compost inhibits the conjunction of MDA
Into, alleviate stress caused by level of lipid peroxidation.
The enhancement microbiological microbial inoculum of table 2 combines influence of the nanometer compost to Festuca Arundinacea MDA and proline content under salt stress
2.2 enhancement microbiologicals combine influence of the nanometer compost to lawn plant defence enzyme activity under salt stress
With the increasing of salt stress, the activity of protective enzyme is substantially suppressed（Table 3）, singly apply nanometer compost and nanometer compost
Handled with enhancement microbiological combined administration, under same salt stress degree, Festuca Arundinacea SOD, POD and CAT activity is all significantly high
In control, it is the most obvious to add the treatment effect of enhancement microbiological, in severe salt stress, adds the micro- fertile place of reinforced nano
Reason, SOD, POD and CAT content are 7.6 times, 2.1 times and 3.2 times of control respectively, illustrate that enhancement microbiological can significantly improve height
Adaptation reaction of the fescue grass to salt stress, enzymatic activity enhancing is shown as, the injury of cell membrane lipid peroxidating mitigates, plant salt tolerant
Property strengthen.There is different degrees of decline with the reinforcement defence enzyme activity of salt stress.
The enhancement microbiological of table 3 combines influence of the nanometer compost to Festuca Arundinacea defence enzyme activity under salt stress
3 develop conclusion
Test result indicates that enhancement microbiological joint nanometer compost can significantly improve lawn plant salt tolerance, ensure lawn
Plant still upgrowth situation when salt content is 0.9% is good.Show as enhancement microbiological joint nanometer compost and effectively facilitate height
The raising of fescue grass defence enzyme activity, the injury of cell membrane lipid peroxidating is reduced, so as to improve Testuca arundinacea to salt stress
Adaptability.
Brief description of the drawings：
Granularmetric analysis under Fig. 1 nanometer compost Electronic Speculum.
Embodiment
In order to more fully explain the implementation of the present invention, there is provided following preparation method embodiments.These embodiments are only
Only it is to explain rather than limit the scope of the present invention.Needing special instruction is：The complex micro organism fungicide that present invention screening obtains
Middle bacillus subtilis, lysine bacillus and penicillium chrysogenum in the market have sale, the method that can also use the present invention
The isolated complex micro organism fungicide from consumer garbage compost, the biochemical characteristic of its obtained flora it is identical with commercially available thus not
In preservation.
Embodiment 1
Material therefor：
（1）Festuca Arundinacea：Choose full seed, perennial Festuca Arundinacea of uniform size (Festuca arundinacea L.)
Seed is test material；
（2）Consumer garbage compost：From Tianjin little Dian composting plants, basic physical and chemical is：PH 7.62, it is saturated aqueous
Measure 0.76 ml/g, the g/ml of unit weight 0.85, full nitrogen 5.18%, the full g/kg of potassium 50.83, the mg/kg of available phosphorus 77.92, organic matter
12.12%, garbage compost is removed into the debris such as wood therein, plastics, metal, it is standby after air-drying；
（3）Soil：Impurities removing, 2mm sieves are crossed, be positioned over 2-3d at ventilating opening, air-dried under natural conditions, its physicochemical property
For：PH7.44, saturation moisture content 0.58mLg-1, organic matter 4.68%, full nitrogen 0.21%, salt content 0.29%；
（4）Saline-alkali soil:It is derived from Hebei province Huanghua City seashore pH 7.85, organic matter 2.21%, full nitrogen 0.258%, available phosphorus
15.26mgkg-1, full potassium 28.96gkg-1, salt content 1.58%.
（1）The preparation of nanometer compost：
By consumer garbage compost, 105 DEG C are dried to constant weight, and garbage compost processing grinding is made into 30-35nm nanometer compost,
It is standby；
（2）The preparation of Salt-tolerant microbial agent：
Compost sample is weighed into 10g to be placed in sterile conical flask, after adding 100mL sterilized water shaken wells, takes 10mL to hang
Supernatant liquid, at 28 DEG C, shaken cultivation 3d under 220r/min, as mixes micro- life in the conical flask for filling 100mL enriched mediums
Thing flora；Contain bacillus subtilis, lysine bacillus and penicillium chrysogenum in described mixed microorganism flora；
（3）Mixed microorganism flora salt tolerant is strengthened using the method for being stepped up salinity, highly concentrated to mitigate moment
Degree salt impacts and poisoned to caused by hybrid bacterial strain, and NaCl concentration increases to 3% with 0.5% gradient, often increases a NaCl
Concentration, could continue to increase NaCl concentration, tame out salt-durable microbe flora step by step after thalline increment is stable；
（4）The preparation of compound Salt-tolerant microbial agent：
Bacillus subtilis, lysine bacillus and penicillium chrysogenum by volume 1:1:1 ratio is configured to compound resistance to
Salt microbial bacterial agent, 100 times of dilution are standby；
（5）Turf established by sod：
Soil and saline-alkali soil sieving are cleaned, slight 0.3%, moderate 0.6% is made by a certain percentage and severe 0.9% is saline and alkaline
Soil, 30 min that the saline-alkali soil mixed is sterilized at 121 DEG C are standby；
Each stress sets 3 processing, and the set processing of three stress is identical, is respectively:
1）15 ml blank cultures as control matrix and are added using 100% soil
2）Soil and nanometer compost press 59：1 ratio is mixed and added into 15ml blank cultures
3）Soil and nanometer compost press 59：1 ratio is mixed and added into 15ml and strengthens Salt-tolerant microbial agent
（5）Lawn plant experiment in cultivation is carried out using the soil that mixes as matrix, and substrates quantity is 9400gm during experiment-2,
Compounded turf matrix thickness is 15 mm, and Festuca Arundinacea grass seeds application rate is 160gm-2, uniformly sowed in matrix table after seed is soaked into 24h
Layer, carries out dark processing first, and fully watering ensures to sprout, and is cultivated after sprouting, intensity of illumination is 450-700 μm of ol.m-2.s-1, a water is periodically during which poured sooner or later, often exchanges the position of culture turf to ensure that illumination is consistent, indoor is relatively wet
It is 20-25 DEG C to spend for 40-55%, temperature, and experiment carries out the measure of progress each index of plant after 50d.Wherein described is stepped up
The method of salinity refers to：NaCl concentration is respectively 0.5%, 1%, 1.5%, 2%, 2.5% and 3%, during domestication, depending on
OD600Increase and step up salinity, NaCl concentration increases to 3% with 0.5% gradient.
Claims (2)
- A kind of 1. method of reinforced nano garbage tankage regulation salt stress turfgrass protective enzyme, it is characterised in that enter by the steps OK：（1）Soil and saline-alkali soil sieving are cleaned, slight 0.3%, moderate 0.6% is made by a certain percentage and severe 0.9% is saline and alkaline Soil, 30 min that the saline-alkali soil mixed is sterilized at 121 DEG C are standby；（2）By step（1）The saline-alkali soil and 30-35nm nanometer compost mixed is by 59：1 ratio is mixed and added into 15ml reinforcings Salt-tolerant microbial agent；Described reinforcing Salt-tolerant microbial agent NaCl concentration increases to 3% with 0.5% gradient；Salt tolerant is micro- Biology refers to：Bacillus subtilis, lysine bacillus and penicillium chrysogenum by volume 1:1:1 ratio is configured to；（3）The soil matrix of the step of to mix (2) carries out lawn plant experiment in cultivation, and substrates quantity is during experiment 9400g·m-2, compounded turf matrix thickness is 15 mm, and Festuca Arundinacea grass seeds application rate is 160gm-2, will be uniform after seed immersion 24h Sow in matrix top layer, carry out dark processing first, fully watering ensures to sprout, and is cultivated after sprouting, intensity of illumination is 450-700μmol·m-2·s-1, a water is periodically during which poured sooner or later, often exchanges the position of culture turf to ensure illumination one Cause, indoor relative humidity is 40-55%, and temperature is 20-25 DEG C, and experiment carries out the measure of progress each index of plant after 50d.
- 2. the method for reinforced nano garbage tankage regulation salt stress turfgrass protective enzyme described in claim 1, wherein（1）The preparation of nanometer compost：By consumer garbage compost, 105 DEG C are dried to constant weight, and garbage compost processing grinding is made into 30-35nm nanometer compost, standby；（2）The preparation of Salt-tolerant microbial agent：Compost sample is weighed into 10g to be placed in sterile conical flask, after adding 100mL sterilized water shaken wells, takes 10mL suspension In the conical flask for filling 100mL enriched mediums, in 28 DEG C, shaken cultivation 3d under 220r/min, as mixed microorganism bacterium Group；Contain bacillus subtilis, lysine bacillus and penicillium chrysogenum in described mixed microorganism flora；（3）Mixed microorganism flora salt tolerant is strengthened using the method for being stepped up salinity, to mitigate moment high salt concentration Impact and poison to caused by hybrid bacterial strain, NaCl concentration increases to 3% with 0.5% gradient, and often NaCl's of increase is dense Degree, it could continue to increase NaCl concentration, tame out salt-durable microbe flora step by step after thalline increment is stable；（4）The preparation of compound Salt-tolerant microbial agent：Bacillus subtilis, lysine bacillus and penicillium chrysogenum by volume 1:1:It is micro- that 1 ratio is configured to compound salt tolerant Bacteria agent, 100 times of dilution are standby；（5）Turf established by sod：Soil and saline-alkali soil sieving are cleaned, slight 0.3%, moderate 0.6% and the saline-alkali soil of severe 0.9% are made by a certain percentage, will The saline-alkali soil mixed 30 min that sterilized at 121 DEG C are standby；Each stress sets 3 processing, and the set processing of three stress is identical, is respectively:1）15 ml blank cultures as control matrix and are added using 100% soil；2）The saline-alkali soil and nanometer compost mixed is by 59：1 ratio is mixed and added into 15ml blank cultures；3）The saline-alkali soil and nanometer compost mixed is by 59：1 ratio is mixed and added into 15ml and strengthens Salt-tolerant microbial agent；（6）Lawn plant experiment in cultivation is carried out using the saline-alkali soil that mixes as matrix, and substrates quantity is 9400gm during experiment-2, Compounded turf matrix thickness is 15 mm, and Festuca Arundinacea grass seeds application rate is 160gm-2, uniformly sowed in matrix after seed is soaked into 24h Top layer, dark processing is carried out first, fully watering ensures to sprout, and is cultivated after sprouting, intensity of illumination is 450-700 μ mol·m-2·s-1, a water is periodically during which poured sooner or later, often exchanges the position of culture turf to ensure that illumination is consistent, it is indoor Relative humidity is 40-55%, and temperature is 20-25 DEG C, and experiment carries out the measure of progress each index of plant after 50d.
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| CN110896842B (en) * | 2019-12-27 | 2021-07-30 | 华中农业大学 | Efficient cultivation method of citrus virus-free etiolation stock and application thereof |
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