CN102577806A - Method for using Bacillus thruingiensis fermentation broth to improve salt resistance of lawn plants - Google Patents
Method for using Bacillus thruingiensis fermentation broth to improve salt resistance of lawn plants Download PDFInfo
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Abstract
The invention discloses a method for using Bacillus thruingiensis fermentation broth to improve salt resistance of lawn plants, which includes: adding 200g of saline-alkali soil different in concentration into disposable plastic cups 7cm in diameter; sowing 0.5g of seeds of Perennial ryegrass, and 0.5g of seeds of Festuca arundinacea in each cup; repeating testing for three times; adding 1ml of various types of inoculants into the soil when the lawn plants grow about 5cm high; adding sterile liquid medium into a control group; moving the plastic cups to a test land and burying the cups deep in the soil to keep plant growth environment to be consistent to the test land soil environment; and quantitatively watering the plants every day to guarantee consistent treatments; testing indexes after the plants grow for 2 months, transplanting the plants to a field and burying deep in the soil when emergent seedlings are stable (10d) so as to keeping the plant growth environment being consistent to the test land soil environment. Test results show that adding the Bacillus thruingiensis inoculant relieves stressed degree of the lawn plants to a certain degree, and growth of the lawn plants is promoted.
Description
Technical field
The invention belongs to environmental protection technical field, relate to application with the probiotics that extracts in the producing fertilizer from refuse in daily life.A kind of employing bacillus thuringiensis of saying so is more specifically improved lawn plant salt-resistance method.
Background technology
Composted (Composting) is meant to be had under the condition of control, makes organic waste under microorganism (being mainly bacterium) effect, degrade, and the process that organic matter is transformed to stable humus direction.Product after the composted is referred to as compost (Compost).Composting process can reduce 40%-50% effectively with the volume of mixture, and can rely on the heat that metabolism in the hot stage produces and kill the cause of disease material in the rubbish.Compost is not a new technology, but handles urban solid garbage in this way, meets the interests of environment, because in composting process, remove or reduced the toxicity in the urban solid garbage and made that final product can be used to utilize again.The program of compost comprises preliminary treatment, fermenting raw materials and the post processing of raw material, and composting process receives the influence of factors such as moisture, oxygen content, C/N ratio, temperature, pH and ventilation situation.
The essence of composted process is microorganism metabolism under optimum conditions; In the composted process; Solubilized organic substance in the house refuse sees through cells of microorganisms wall and cell membrane and is absorbed by Institute of Micro-biology; And microorganism changes into inorganic matter to the organic matter that absorbs through the vital movement process of self.Microorganism is playing the part of important role in composting process, closely related with compost cycle and compost quality.Therefore, the research of microorganism is quickened composting process to exploitation compost microbe resource in the compost, shortens the compost cycle, improves compost quality and all has great importance.
Microbe species is various, and occurring in nature only has the microorganism of only a few to obtain identifying that the kind of can survive, cultivating is few especially, is at most the l% of microorganism total amount.At present, carried out the research of series of theories and practice both at home and abroad for the microorganism in the compost.The environmental problem that possibly occur for the application compost has also caused people's attention; But how to address these problems and also lack careful deep research; At present; How to improve the quality of producing fertilizer from refuse in daily life, issuable environmental problem is the focus that the related scientific research personnel study concern always in the solution compost application process.The research that changes for the microorganism in composition of the microorganism in the compost and the composting process has many reports, but for the rare report of research that microorganism in the compost is separated, is applied to other matrix.
Consumer garbage compost as lawn matrix, not only can be avoided food chain, solved the problem of outlet of rubbish simultaneously again.But contain materials such as heavy metal in the garbage compost and possibly cause adverse influence environment; This is the major issue during compost is used always, from garbage compost, separates and extracts useful microorganism fungus kind, is mixed with different microbial bacterial agents and is inoculated in the turf establishment system; Both can improve the utilization ratio of compost; Solve simultaneously the environmental threat that compost heavy metal etc. possibly constitute again, embodied the superiority of biologic product, met the requirement of sustainable development; Through studying of the influence of different compost microbe microbial inoculums to the lawn plant salt-resistance; Purpose is in order to screen suitable microbial bacterial agent, improves quality and the resistance of lawn plant, and the quality of improving lawn soil matrix provides theoretical foundation.
Bacillus thuringiensis is present maximum of output in the world and the microorganism insecticide that is successfully used to prevent and treat farming, woods, pest of stored grain and some sanitary insect pests; Bacillus thuringiensis is when forming gemma; Can form a kind of parasporal crystal of being made up of insecticidal crystal protein, its main active insecticidal components is a parasporal crystal protein.Discover that this albumen has the specific killing effect to multiple agriculture and forestry injurious insect.Behind the insect's food-taking gemma mixed crystal; Be degraded under the effect of midgut alkaline environment and the protease of crystal in insect bodies and form insecticidal activity albumen; Result of study shows that synergy has toxic action in various degree to insect between independent effect of these toxalbumin or different albumen; Further strengthen the research of bacillus thuringiensis aspect control and Pest Control harm, realize that for the protection environment for human survival sustainable development is significant.Bacillus thuringiensis,Bt (Bacillus thuringiensis is called for short Bt) is the biological insecticides that current production rate is maximum, use is the widest.About adopting method that the bacillus thuringiensis ferment filtrate is used for improving the lawn plant salt-resistance still for seeing bibliographical information.
The soil salt damage is the key factor of limiting plant growth and crop yield, and the agricultural land in the whole world about 20% receives the influence of high salt.The soil salt damage almost influences plant physiology and biochemical movable various aspects, and it mainly shows as: plant physiology property lack of water, nutrient absorption is unbalance and the toxic action etc. that receives ion.Discover that microorganism can be alleviated salt stress, promote plant growing; Microorganism can be improved the root structure of plant, reduces the cytoplasm membrane permeability of plant leaf blade, improves the activity of superoxide dismutase (SOD) in its root system vigor and the blade; Promote the growth of plant and the accumulation of dry matter; Thereby improve the salt resistance of plant, in addition, microbe inoculation can change carbon cycle and the photosynthesis of plant; Improve gas exchange capacity, the raising plant Net Photosynthetic Rate of plant leaf blade, reduce the extent of injury of salt stress plant.This research will join in the lawn soil matrix of moderate and severe salt stress by the microbial inoculum of isolated bacillus thuringiensis from compost; Discussion is under field condition; Different compost microbe microbial inoculums provide reference to improving the influence of lawn plant salt-resistance for further improving the lawn plant salt-resistance.
Summary of the invention
The bacillus thuringiensis that the present invention adopts (latin name:
Bacillus thuringiensis) culture presevation number: CFCC10206, depositary institution: China Forest microorganism fungus kind preservation administrative center externally can provide.
Physio-biochemical characteristics: cell is Gram-positive, and is shaft-like, and size is 1.0-1.2 * 3-5 μ m.Form half spore crystal, arabinose mannitol is not produced acid produce amylase, nitrate reduction is to nitrite.
Bacillus thuringiensis toxicity: to the people, animal low toxicity, the oral acute LD of rat
50852.7-856.7 milligram/kilogram is to low toxicities such as poultry, birds, fish, poultrys.
The bacillus thuringiensis ferment filtrate that the present invention will be mixed with variable concentrations is inoculated in the turf establishment system.Improve lawn plant salt-resistance method through studying a kind of employing bacillus thuringiensis, for the saline-alkali soil planting of lawn plant provides foundation.
For realizing above-mentioned purpose, the present invention provides following technical scheme:
A kind of
Adopt the bacillus thuringiensis zymotic fluid to improve the method for lawn plant salt-resistance, it is characterized in that being undertaken by following step:
(1)The separation of bacillus thuringiensis: bacillus thuringiensis itself has commercially available; The consumer garbage compost that also can separate Xiao Dian garbage compost treatment plant from Tianjin; The present invention is identical with commercially available bacillus thuringiensis from the resulting bacillus thuringiensis Physiology and biochemistry of consumer garbage compost character, and the method for separation is following:
1) fresh compost sample is taken by weighing 10g, put into the 90ml sterile water triangular flask that is added with 20 beades, vibration; Make in the sample microorganism fully and be uniformly distributed in the liquid; In triangular flask, get the lml mother liquor with liquid-transfering gun, add abundant mixing in the Boiling tube that fills the 9ml sterile water, this is l0
-1The dilute solution of concentration according to said method is diluted to l0 respectively
-2, l0
-3, l0
-4, l0
-5, l0
-6The compost dilution of several kinds of concentration; Use liquid-transfering gun to draw 0.1 ml concn respectively and be l0
-2, l0
-3, l0
-4, l0
-5, l0
-6Dilution is injected on the beef extract-peptone solid plate medium, and each dilution factor triplicate, sterile water are blank; With aseptic glass slicker bacteria suspension is evenly spread upon on the whole beef extract-peptone solid culture medium, microorganism is evenly distributed, the flat board that will contain the beef extract-peptone solid culture medium is inverted in 37 ℃ of constant incubators to be cultivated 1 day;
2) bacillus thuringiensis preparation of fermentation liquid:
A. isolated bacillus thuringiensis bacterial classification purifying was cultivated for 2 generations; Insert then 180 r/min37 ℃ cultivation is housed in the 250 mL triangular flasks of 50mL beef extract-peptone liquid nutrient medium; Selecting for use the 600nm wavelength to carry out turbidimetric assay, is ordinate with the OD value of bacteria suspension, and incubation time is an abscissa, draws the microbial growth curve; According to the growth curve acquisition time of growth of microorganism to stationary phase, the bacterial classification of getting stationary phase adopts the microscope direct counting method to calculate the viable count in every mL bacterium liquid;
B. get purifying bacillus thuringiensis bacterial classification; Access is equipped with in the 250 mL triangular flasks of 50mL Gause I liquid nutrient medium; Cultivate 8～12 h to stationary phase for 180 r/min37 ℃, the bacterial classification of getting stationary phase adopts the microscope direct counting method to calculate the viable count in every mL bacterium liquid, and the bacterium liquid of stationary phase is carried out suction filtration; Be the miillpore filter of 0.22um with bacterium liquid through the aperture behind the suction filtration, the filtered fluid of acquisition then is the filtered fluid behind the microbial fermentation; The clump count of bacillus thuringiensis (individual) wherein
Conclusion: the clump count situation through the bacillus thuringiensis that on beef-protein medium, grows can find out that the bacillus thuringiensis clump count in the compost is higher than the soil contrast, because l0
-4In the soil under times concentration and l0
-5, l0
-6Concentration under bacterium colony number average in compost and the soil be less than 30, therefore can't carry out statistical counting.
3) dilution bacillus thuringiensis zymotic fluid:
Get a part of filtered fluid and be diluted to 4 times of filtratings and 8 times of filtratings respectively, subsequent use;
4) a kind of employing bacillus thuringiensis is improved the lawn plant salt-resistance:
Method for potted is adopted in experiment, in diameter is the disposal plastic cup of 7cm, adds 200g, the saline-alkali soil of 0.4-0.8% concentration; In each cup, sow perennial ryegrass, Festuca Arundinacea grass seeds 0.5g respectively, experiment is 3 repetitions; When treating the about 5cm of lawn plant growth, in soil, add the 1ml zymotic fluid respectively, control group adding aseptic liquid nutrient medium; It is buried in soil that plastic cup is moved to sample plot; To keep plant growth environment with experimental field soil environment is consistent, every day, quantitative water supply guaranteed that each is consistent between handling; Treat to measure each item index after it was grown 2 months; Emerging, to be transplanted to the field buried in soil in stable back (10 d), to keep plant growth environment with experimental field soil environment is consistent;
Purifying cultivation 2 wherein is commissioned to train to support and is referred to: directly provoke bacterium colony to be purified with connecing collarium; Be inoculated in the preprepared Gause I flat-plate solid medium; Line gently from left to right on flat board; Culture dish is inverted in 37 ℃ of constant incubators cultivated 1 day, this process is a purifying generation, and to repeat this process again be two generations of purifying to picking colony from the bacterial classification of a purifying generation.
The prepared bacillus thuringiensis of the present invention with from consumer garbage compost prepared bacillus thuringiensis Physiology and biochemistry character and commercially available identical, so not preservation.
Qualification result about bacillus thuringiensis
The picking bacterium colony similar with bacillus thuringiensis on beef-protein medium finds that the bacterium colony that on medium, forms is circle, white; The edge is smooth, and is opaque, and Gram is positive; It is shaft-like that thalline is examined under a microscope ovalize, and gemma is oval, carries out parasporal crystal dyeing with bromophenol blue and sarranine dye liquor; Observed red thalline under the mirror, colourless gemma and blue parasporal crystal, it is bacillus thuringiensis (with commercially available identical) for a Preliminary Identification.
The Physiology and biochemistry experimental result of bacterium
With Preliminary Identification is that the bacterial strain of bacillus thuringiensis is decided to be bacterial strain to be measured, to its detection of carrying out the Physiology and biochemistry experiment, obtains result such as following table:
The Physiology and biochemistry experimental result of bacterium
Bacterial strain to be measured has been carried out the Physiology and biochemistry test, shown in the result as above shows.Bacterial strain to be measured is a Gram-positive, the hydrogen peroxide enzyme positive, and the V.P reacting positive, glucose fermentation produces acid, and hydrolyzed starch, gelatin can both utilize citrate.
The distinguishing feature of bacterial strain to be measured is: bacterial strain to be measured produces acid to the azymic of D-wood sugar, and acid, not aerogenesis are produced in the azymic of D-mannitol.According to above experimental result, identify that further bacterial strain to be measured is bacillus thuringiensis (with commercially available identical).With the bacterial strain purification storage, use in order to subsequent experimental.
The more detailed test method of the present invention is following:
1 materials and methods
1.1 experiment material
The experiment use microbial inoculum to be the bacillus thuringiensis that separates in the compost, lawn plant select for use the more common English ryegrass of northern China (
Lolium perenne L.) and Festuca Arundinacea (
Festuca arundinacea L.) be experiment material.Supply the matrix saline-alkali soil of examination to take from seashore, Huanghua City, Hebei province.Experiment is respectively 0.4% and 0.8% with the saline-alkali soil salt content, for moderate and severe are coerced.
1.2 experimental technique
1.2.1. the preparation of microbial inoculum
1.2.2. turf establishment
Method for potted is adopted in experiment, in diameter is the disposal plastic cup of 7cm, adds 200g, the saline-alkali soil of 0.4-0.8% concentration; In each cup, sow perennial ryegrass, Festuca Arundinacea grass seeds 0.5g respectively, experiment is 3 repetitions; When treating the about 5cm of lawn plant growth, in soil, add the 1ml zymotic fluid respectively, control group adding aseptic liquid nutrient medium; It is buried in soil that plastic cup is moved to sample plot; To keep plant growth environment with experimental field soil environment is consistent, every day, quantitative water supply guaranteed that each is consistent between handling; Treat to measure each item index after it was grown 2 months; Emerging, to be transplanted to the field buried in soil in stable back (10 d), to keep plant growth environment with experimental field soil environment is consistent;
Purifying cultivation 2 wherein is commissioned to train to support and is referred to: directly provoke bacterium colony to be purified with connecing collarium; Be inoculated in the preprepared Gause I flat-plate solid medium; Line gently from left to right on flat board; Culture dish is inverted in 37 ℃ of constant incubators cultivated 1 day, this process is a purifying generation, and to repeat this process again be two generations of purifying to picking colony from the bacterial classification of a purifying generation.
Consistent in the experimentation in order to guarantee each processing; The plastic greenhouse waterproof is built in employing, and the height of canopy is about 1 m, and canopy covers the movable transparent plastic film; Fine day is removed plastic film; So that plant can fully be accepted sunlight, the rainy day cover film prevents the excessive salt content that influences in the soil matrix of rainwater.
1.2.3. sample plot overview
Sample plot is located in the Tianjin Normal University campus, the geographical position be 39 ° 13 of north latitude ', 117 ° 2 of east longitude ', belong to warm temperate zone half moistening monsoon type weather, 12.3 ℃ of average temperatures of the whole year, average precipitation 550-680 mm.Experiment field soil belongs to damp cinnamon soil, and 24.5 ℃ of experimental session mean temperature of air, medial humidity are 35%, average precipitation 380-560 mm.
The mensuration of 2 indexs of correlation
2.1. the mensuration of lawn plant biomass, plant height
2.2. the mensuration of lawn plant protective enzyme, MDA, proline content
Crude enzyme liquid extracts: accurately take by weighing 0.5 g appearance leaf, (constant volume 25 ml get 10 ml in centrifuge tube, 10000 rmin for PBS, pH7.8) ice bath milling and extracting with phosphate buffer
-1Centrifugal 20 min of EPPENDOFF centrifuge, supernatant is thick zyme extract.
The POD determination of activity: adopt guaiacol method, get the mixed liquid of 3 ml reaction in cuvette, contrast replaces with the pH=7.8 phosphate buffer; In cuvette, add the thick zyme extract of 0.1 mL, open manual time-keeping immediately, measure light absorption value down in 470 nm; Per minute is read number 1 time, reads altogether 3 times.Changing 0.01 with per minute △ A470 is a peroxidase activity unit.The phosphate buffer that the mixed liquid composition of reaction is 50 mL pH=6.0 adds 28 μ L guaiacol and 19 μ L hydrogen peroxide.
SOD determination of activity: adopt the NBT method.The mixed liquid of 3 ml reaction comprises: the phosphate buffer of pH=7.8, and 0.1 mMEDTA, 13 mM methionine, 75 μ MNBT, the zyme extract of 2 μ M vitamin b3 and 0.1mL, enzyme-added liquid is not contrast.In climatic cabinate the irradiation 15 min, not enzyme-added liquid and unglazed photograph as blank.Under 560 nm, measure light absorption value then rapidly.To suppress 50% of NBT photochemical reduction is a unit of enzyme activity.
CAT determination of activity: adopt ultraviolet spectrophotometry.In test tube, add 1.5 ml pH=7.8 phosphate buffers, 1 ml distilled water, 0.2 ml zyme extract, contrast replaces enzyme liquid with buffer solution.In measuring pipe, adding 0.3 ml concentration then is 0.1 molL
-1Hydrogen peroxide, light absorption value is measured in timing immediately simultaneously rapidly under 240 nm, and per minute is read number 1 time.With per minute △ A
240Changing 0. 1 is a catalase activity unit.
MDA (MDA) assay
Take by weighing 0.5 g blade, extract constant volume 10 ml, 4000 rmin with 10%TCA
-1Centrifugal 10 min get the 0.6%TBA that liquid 2 ml in upper strata add 2 ml again, boil 15 min in the boiling water, and centrifugal 10 min get supernatant and survey light absorption value down in 532 nm and 450 nm.
Proline:
Take by weighing 0.5 g blade and shred, place Boiling tube respectively, add the yellow basic salicylic acid of 10 ml 3% then respectively; Extracting in boiling water 10 min; 2 ml are drawn in another clean test tube with ground stopper in the cooling back, add 2 ml glacial acetic acid and 2 ml acid ninhydrines, heating 30 min in the boiling water; Cooling is extracted with 4 ml toluene, 3000 rmin
-1Centrifugal 5 min get upper strata liquid and survey light absorption value down in 520 nm.
2.3. the mensuration of photosynthetic index of correlation
Measure its photosynthetic index after 60 days in lawn plant growth, under natural lighting, adopt the photosynthetic mensuration of LI-6400 system from the morning 8:00 to afternoon 18:00 whenever measure the intercellular CO that each group is handled and contrasted at random at a distance from 2 h
2The variation of concentration (Ci), Net Photosynthetic Rate (Pn).Relative air humidity between test period is between 9.86%-38.21%, and morning, 7:00 was the highest, and afternoon 15:00 is minimum.The air temperature variations scope is between 19.51-31.15 ℃.Each handles each 3 data of measuring, and 3 repetitions are established in experiment altogether.When measuring These parameters, write down photosynthetic active radiation automatically, air themperature, leaf temperature, air humidity, air CO
2Diurnal variation.
2.4 statistical analysis
Data analysis adopts Excel 2003 and SPSS 17.0 statistical analysis softwares to analyze.
3 development results
3.1 different compost microbe microbial inoculums are to the influence of lawn plant photosynthesis characteristics
Relative air humidity between test period between 9.86%-38.21%, 7:00 in morning the highest (38.21%), afternoon 15:00 minimum (9.86%).The air temperature variations scope is between 19.51-31.15 ℃, and 7:00 rises gradually since the morning, reaches the highest (31.15 ℃) at 15:00, descends gradually then, and afternoon, 17:00 was 26.01 ℃.Lawn surface air temperature and humidity also changes along with the variation of solar irradiation intensity.Through measuring, photosynthetic active radiation from the excursion of 7:00-17:00 at 79-1272 μ molm-
2Between the g, 11:00 reaches maximum (1272 μ molm-
2G), descend gradually afterwards, become 79 μ molm-to 17:00
2G, air CO
2Concentration is on a declining curve always on the same day, has dropped to 386.1 μ mol/mol from 421.7 μ mol/mol, and 11:00-17:00 descends comparatively slow, and has reached 34.7% from 7:00 to the 11:00 decline scope.
3.2 the compost microbe microbial inoculum is to the influence of lawn plant blade Net Photosynthetic Rate
Under the severe condition of salt stress, used the photosynthetic capacity that different compost microbes have obviously improved the lawn plant blade.In morning during 7:00, the Festuca Arundinacea blade Net Photosynthetic Rate that adds the bacillus thuringiensis microbial inoculum is 39.8 μ mol (m
2G
-1), comparison is according to exceeding 5.0%, and As time goes on, the blade Net Photosynthetic Rate difference of experimental group and control group reduces gradually, and after 15:00, the blade Net Photosynthetic Rate of experimental group and control group does not almost have difference.When 7:00, experimental group perennial ryegrass blade Net Photosynthetic Rate is respectively 40.0%, 41.8% and 39.4%, and the experimental group comparison that bacillus thuringiensis is handled is according to having exceeded .6%..
Under the moderate condition of salt stress, using different compost microbe microbial inoculums does not have obvious facilitation to the Net Photosynthetic Rate of lawn plant blade.In morning during 7:00, the lawn plant blade Net Photosynthetic Rate that adds the bacillus thuringiensis microbial inoculum is respectively 40.1,37.8,39.5,38.9,38.5 and 38.1 μ mol (m
2G
-1), from 7:00 to 11:00 between, the blade Net Photosynthetic Rate ascensional range of lawn plant is bigger, during 11:00, lawn plant blade Net Photosynthetic Rate is respectively 56.0,56.5,56.3,57.7,58.3 and 59.0 μ mol (m
2G
-1), having improved 39.7%, 49.5%, 42.5%, 48.3%, 51.4% and 54.9% respectively, the rising of the blade Net Photosynthetic Rate of lawn plant tends towards stability after 11:00.In addition, under saline-alkali soil stress conditions in various degree, the blade Net Photosynthetic Rate of lawn plant does not have evident difference.
3.3 the compost microbe microbial inoculum is to the influence of lawn plant blade intercellular gas concentration lwevel
Under the moderate condition of salt stress; Compare with contrast; Different compost microbe microbial inoculums all have facilitation to lawn plant blade intercellular gas concentration lwevel, and when 11:00, the intercellular gas concentration lwevel of the experimental group Festuca Arundinacea that different compost microbe microbial inoculums are handled reaches maximum; Be respectively 56.0,56.5,56.3 μ mol/mol; Comparison is according to having improved 1.1%, 1.4%, 1.1%, and the blade intercellular gas concentration lwevel of perennial ryegrass experimental group is respectively 57.7,58.3,59.0 μ mol/mol, and comparison is according to having improved 1.2%, 1.0%, 2.3%.After the 11:00, lawn plant blade intercellular gas concentration lwevel begins to descend.Compare with the severe salt stress, under the moderate condition of salt stress, lawn plant blade intercellular gas concentration lwevel is higher.
3.4 the compost microbe microbial inoculum is to the influence of lawn plant growth physical signs
3.4.1 the compost microbe microbial inoculum is to the active influence of lawn plant protective enzyme
Different compost microbe microbial inoculums are to the active influence of lawn plant protective enzyme under the table 1 severe salt stress
Annotate: * representes in the table
p<0.05 * * representes
p<0.01, down together
Under the severe condition of salt stress; Different compost microbe microbial inoculums are as shown in table 1 to the active influence of lawn plant protective enzyme (CAT, POD, SOD); The Festuca Arundinacea experimental group POD and the SOD activity that add the bacillus thuringiensis microbial inoculum are compared respectively according to having reduced by 35.4% and 68.8%; Adding the bacillus thuringiensis microbial inoculum also has tangible reduction to Festuca Arundinacea CAT activity, but do not reach significance degree (
p>0.05).
The perennial ryegrass CAT activity that adds the bacillus thuringiensis microbial inoculum is compared with contrast and has been reduced by 179.5%, add perennial ryegrass CAT behind the actinomycetes active, add the active not appreciable impact of POD, SOD behind the bacillus thuringiensis (
p>0.05).
Different compost microbe microbial inoculums are to the active influence of lawn plant protective enzyme under the table 2 moderate salt stress
Annotate: * representes in the table
p<0.05 * * representes
p<0.01, down together
Under the moderate condition of salt stress, different compost microbe microbial inoculums are as shown in table 2 for the active influence of lawn plant protective enzyme (CAT, POD, SOD), and adding the bacillus thuringiensis microbial inoculum does not have facilitation significantly to Festuca Arundinacea CAT activity.The POD of the perennial ryegrass experimental group of adding bacillus thuringiensis, the contrast of SOD specific activity have reduced by 40.6%, but significantly do not act on for perennial ryegrass CAT is active (
p>0.05).
3.4.2 the compost microbe microbial inoculum is to the influence of lawn plant MDA, proline content
Different compost microbe microbial inoculums are to the influence of lawn plant MDA, proline content under the table 3 severe salt stress
Annotate: * representes in the table
p<0.05 * * representes
p<0.01, down together
Different compost microbe microbial inoculums are as shown in table 3 for the influence of lawn plant MDA, proline content under the severe condition of salt stress; Adding the bacillus thuringiensis microbial inoculum does not have tangible reduction effect to the Festuca Arundinacea mda content, and the proline content comparison of Festuca Arundinacea is taken a picture and reduced by 88.4% behind the adding bacillus subtilis microbial agent.
Different compost microbe microbial inoculums are to the influence of lawn plant MDA, proline content under the table 4 moderate salt stress
Annotate: * representes in the table
p<0.05 * * representes
p<0.01, down together
Under the moderate condition of salt stress; Different compost microbe microbial inoculums are as shown in table 4 for the influence of the MDA of lawn plant Festuca Arundinacea and perennial ryegrass, proline content; Add bacillus thuringiensis to the not significantly effect of Festuca Arundinacea mda content; Add different compost microbe microbial inoculums to the not significantly effect of Festuca Arundinacea proline content; The experimental group perennial ryegrass mda content comparison that adds the bacillus thuringiensis microbial inoculum has been according to having reduced by 14.7%, add bacillus thuringiensis to the proline content of perennial ryegrass do not have tangible effect (
p>0.05).
3.4.3 the compost microbe microbial inoculum is to the influence of lawn plant biomass, plant height
Different compost microbe microbial inoculums are to the influence of lawn plant biomass, plant height under the table 5 severe salt stress
Annotate: * representes in the table
p<0.05 * * representes
p<0.01, down together
As shown in table 5, under the severe condition of salt stress, adding bacillus thuringiensis microbial inoculum does not have facilitation significantly for ground fresh weight and dry weight, the fresh and dried ratio of Festuca Arundinacea, and adding bacillus thuringiensis does not have obvious facilitation to the Festuca Arundinacea plant height.What add different compost microbe microbial inoculums does not have facilitation significantly to perennial ryegrass ground fresh weight and ground dry weight, has reduced the fresh and dried ratio of lawn plant perennial ryegrass ground biomass on the contrary, to the yet influence significantly of plant height of perennial ryegrass.
Different compost microbe microbial inoculums are to the influence of lawn plant biomass, plant height under the table 6 moderate salt stress
Annotate: * representes in the table
p<0.05 * * representes
p<0.01, down together
As shown in table 6; Under the moderate condition of salt stress; Adding bacillus thuringiensis microbial inoculum has obvious facilitation for the ground fresh weight of Festuca Arundinacea; Compare with contrast and to have improved 66.8% and 55.3% respectively, add different compost microbe microbial inoculums Festuca Arundinacea dry weight, fresh and dried ratio, plant height facilitation significantly not on the ground.The perennial ryegrass ground fresh weight that adds the bacillus thuringiensis microbial inoculum exceeds contrast 33.5% respectively, and the perennial ryegrass ground dry weight of adding bacillus thuringiensis is lower than contrast 31.8% on the contrary.
4. development conclusion
If salinity has exceeded the tolerance limit of plant, thereby will destroy plant cell membrane permeability and ionic equilibrium so plant is produced injury, suppress the growth of plant, in addition, salinity is coerced the chlorophyll content that can influence plant, suppresses photosynthesis.
Different compost microbe microbial inoculum is improved for Net Photosynthetic Rate and the intercellular gas concentration lwevel of lawn plant in this research; This possibly be owing to contain nutritive elements such as nitrogen, phosphorus, potassium in the microbial metabolic products, has improved the photosynthetic capacity of lawn plant, and research shows; The photosynthetic rate of the raising of nitrogen concentration and plant is proportional in the plant corpus; In addition, the increase of available phosphorus can improve chlorophyll and the protein content of plant, thereby improves photosynthetic rate.Protective enzymes such as SOD, CAT, POD act synergistically in plant corpus and remove excessive active oxygen; Keep metabolic balance, the diaphragm structure of active oxygen; Thereby make it restrain oneself to a certain extent, slow down or resist environment stress injury, under stress conditions, plant cell is obstructed and produces a large amount of active oxygens, superoxide radical etc. owing to metabolism; These active oxygens, superoxide radical can not get effective removing meeting cell membrane fat and carry out peroxidating, cause the cell membrane system damage.In this research; Under the condition of salt stress of severe; The compost microbe microbial inoculum all has the effect of reduction to the proline content of lawn plant Festuca Arundinacea and perennial ryegrass, but under the moderate condition of salt stress, different compost microbe microbial inoculums do not have obvious influence to the proline content of lawn plant.The compost microbe microbial inoculum also increases to the ground biomass of lawn plant Festuca Arundinacea and perennial ryegrass to a certain extent; Above result of study explanation compost microbe has been alleviated the degree that lawn plant is coerced to a certain extent, has promoted the growth of lawn plant.
Description of drawings:
Fig. 1 is the growth curve of bacillus thuringiensis;
Fig. 2 is a bacillus thuringiensis bacterium colony photo;
Fig. 3 is that the compost microbe microbial inoculum is to lawn plant photosynthesis characteristics influence figure;
Compost bacterium is to the figure that influences of lawn plant blade Net Photosynthetic Rate under Fig. 4 severe salt stress;
Compost bacterium is to the figure that influences of lawn plant blade Net Photosynthetic Rate under Fig. 5 moderate salt stress;
Compost bacterium is to the figure that influences of lawn plant intercellular gas concentration lwevel under Fig. 6 severe salt stress;
Compost bacterium is to the figure that influences of lawn plant intercellular gas concentration lwevel under Fig. 7 moderate salt stress.
Embodiment
In order to explain enforcement of the present invention more fully, following preparation method's embodiment is provided.These embodiments only are to explain rather than limit scope of the present invention.Wherein bacillus thuringiensis (Bacillusthuringiensis) has commercially availablely, also can obtain according to the method for embodiment 1, from the resulting bacillus thuringiensis Physiology and biochemistry of consumer garbage compost character with commercially available identical.The beef extract-peptone solid culture medium that is adopted has commercially available.Plating medium is: the medium of Gause I also has commercially available.
(1)The separation of bacillus thuringiensis:
1) fresh compost sample is taken by weighing 10g, put into the 90ml sterile water triangular flask that is added with 20 beades, vibration; Make in the sample microorganism fully and be uniformly distributed in the liquid; In triangular flask, get the lml mother liquor with liquid-transfering gun, add abundant mixing in the Boiling tube that fills the 9ml sterile water, this is l0
-1The dilute solution of concentration according to said method is diluted to l0 respectively
-2, l0
-3, l0
-4, l0
-5, l0
-6The compost dilution of several kinds of concentration; Use liquid-transfering gun to draw 0.1 ml concn respectively and be l0
-2, l0
-3, l0
-4, l0
-5, l0
-6Dilution is injected on the beef extract-peptone solid plate medium, and each dilution factor triplicate, sterile water are blank; With aseptic glass slicker bacteria suspension is evenly spread upon on the whole beef extract-peptone solid culture medium, microorganism is evenly distributed, the flat board that will contain the beef extract-peptone solid culture medium is inverted in 37 ℃ of constant incubators to be cultivated 1 day;
2) bacillus thuringiensis preparation of fermentation liquid:
A. isolated bacillus thuringiensis bacterial classification purifying was cultivated for 2 generations; Insert then and be equipped with in the 250 mL triangular flasks of 50mL beef extract-peptone liquid nutrient medium; 180 r/min37 ℃ cultivation; Selecting for use the 600nm wavelength to carry out turbidimetric assay, is ordinate with the OD value of bacteria suspension, and incubation time is an abscissa; Draw the microbial growth curve, obtain the time that bacillus thuringiensis grows to stationary phase according to growth curve;
The clump count of bacillus thuringiensis (individual) wherein
Conclusion: the clump count situation through the bacillus thuringiensis that on beef-protein medium, grows can find out that the bacillus thuringiensis clump count in the compost is higher than the soil contrast, because l0
-4In the soil under times concentration and l0
-5, l0
-6Concentration under bacterium colony number average in compost and the soil be less than 30, therefore can't carry out statistical counting.
B. get purifying bacillus thuringiensis bacterial classification; Access is equipped with in the 250 mL triangular flasks of 50mL Gause I liquid nutrient medium; Cultivate 8～12 h to stationary phase for 180 r/min37 ℃, the bacterial classification of getting stationary phase adopts the microscope direct counting method to calculate the viable count in every mL bacterium liquid, and the bacterium liquid of stationary phase is carried out suction filtration; Be the miillpore filter of 0.22um with bacterium liquid through the aperture behind the suction filtration, the filtered fluid of acquisition then is the filtered fluid behind the microbial fermentation;
3) dilution bacillus thuringiensis zymotic fluid:
Get a part of filtered fluid and be diluted to 4 times of filtratings and 8 times of filtratings respectively, subsequent use;
Get a part of filtered fluid and be diluted to 4 times of filtratings and 8 times of filtratings respectively, subsequent use;
4) adopt bacillus thuringiensis to improve the lawn plant salt-resistance:
Method for potted is adopted in experiment, in diameter is the disposal plastic cup of 7cm, adds 200g, and the saline-alkali soil of 0.4-% concentration is in each cup; Sow perennial ryegrass, Festuca Arundinacea grass seeds 0.5g respectively, experiment is to repeat for 3 times, when treating the about 5cm of lawn plant growth; In soil, add the 1ml zymotic fluid respectively, control group adds aseptic liquid nutrient medium, and it is buried in soil that plastic cup is moved to sample plot; To keep plant growth environment with experimental field soil environment is consistent, every day, quantitative water supply guaranteed that each is consistent between handling; Treat to measure each item index after it was grown 2 months; Emerging, to be transplanted to the field buried in soil in stable back (10 d), to keep plant growth environment with experimental field soil environment is consistent;
Purifying cultivation 2 wherein is commissioned to train to support and is referred to: directly provoke bacterium colony to be purified with connecing collarium; Be inoculated in the preprepared Gause I flat-plate solid medium; Line gently from left to right on flat board; Culture dish is inverted in 37 ℃ of constant incubators cultivated 1 day, this process is a purifying generation, and to repeat this process again be two generations of purifying to picking colony from the bacterial classification of a purifying generation.
1) bacillus thuringiensis preparation of fermentation liquid:
A. the bacillus thuringiensis bacterial classification purifying of buying was cultivated for 2 generations; Insert then and be equipped with in the 250 mL triangular flasks of 50mL beef extract-peptone liquid nutrient medium; 180 r/min37 ℃ cultivation; Selecting for use the 600nm wavelength to carry out turbidimetric assay, is ordinate with the OD value of bacteria suspension, and incubation time is an abscissa; Draw the microbial growth curve, obtain the time that bacillus thuringiensis grows to stationary phase according to growth curve; Purifying cultivation 2 wherein is commissioned to train to support and is referred to: directly provoke bacterium colony to be purified with connecing collarium; Be inoculated in the preprepared Gause I flat-plate solid medium; Line gently from left to right on flat board; Culture dish is inverted in 37 ℃ of constant incubators cultivated 1 day, this process is a purifying generation, and to repeat this process again be two generations of purifying to picking colony from the bacterial classification of a purifying generation.
B. get purifying bacillus thuringiensis bacterial classification; Access is equipped with in the 250 mL triangular flasks of 50mL Gause I liquid nutrient medium; Cultivate 12 h to stationary phase for 180 r/min37 ℃, the bacterial classification of getting stationary phase adopts the microscope direct counting method to calculate the viable count in every mL bacterium liquid, and the bacterium liquid of stationary phase is carried out suction filtration; Be the miillpore filter of 0.22um with bacterium liquid through the aperture behind the suction filtration, the filtered fluid of acquisition then is the filtered fluid behind the microbial fermentation;
3) dilution bacillus thuringiensis zymotic fluid:
Get a part of filtered fluid and be diluted to 4 times of filtratings and 8 times of filtratings respectively, subsequent use;
Get a part of filtered fluid and be diluted to 4 times of filtratings and 8 times of filtratings respectively, subsequent use;
4) adopt bacillus thuringiensis to improve the lawn plant salt-resistance:
Method for potted is adopted in experiment, in diameter is the disposal plastic cup of 7cm, adds 200g, and the saline-alkali soil of 0.8% concentration is in each cup; Sow perennial ryegrass, Festuca Arundinacea grass seeds 0.5g respectively, experiment is to repeat for 3 times, when treating the about 5cm of lawn plant growth; In soil, add the 1ml zymotic fluid respectively, control group adds aseptic liquid nutrient medium, and it is buried in soil that plastic cup is moved to sample plot; To keep plant growth environment with experimental field soil environment is consistent, every day, quantitative water supply guaranteed that each is consistent between handling; Treat to measure each item index after it was grown 2 months; Emerging, to be transplanted to the field buried in soil in stable back (10 d), to keep plant growth environment with experimental field soil environment is consistent;
Purifying cultivation 2 wherein is commissioned to train to support and is referred to: directly provoke bacterium colony to be purified with connecing collarium; Be inoculated in the preprepared Gause I flat-plate solid medium; Line gently from left to right on flat board; Culture dish is inverted in 37 ℃ of constant incubators cultivated 1 day, this process is a purifying generation, and to repeat this process again be two generations of purifying to picking colony from the bacterial classification of a purifying generation.
The present invention is after the preferred embodiment that specifies; Being familiar with this technological personage can be well understood to; Do not break away from above-mentioned claim with spirit under can carry out various variations and modification; All foundations technical spirit of the present invention all belongs to the scope of technical scheme of the present invention to any simple modification, equivalent variations and modification that above embodiment did.And the embodiment that the present invention also is not subject in the specification to be given an actual example.
Claims (1)
1. method that adopts the bacillus thuringiensis zymotic fluid to improve the lawn plant salt-resistance is characterized in that being undertaken by following step:
(1) separation of bacillus thuringiensis:
1) fresh compost sample is taken by weighing 10g, put into the 90ml sterile water triangular flask that is added with 20 beades, vibration; Make in the sample microorganism fully and be uniformly distributed in the liquid; In triangular flask, get the lml mother liquor with liquid-transfering gun, add abundant mixing in the Boiling tube that fills the 9ml sterile water, this is l0
-1The dilute solution of concentration according to said method is diluted to l0 respectively
-2, l0
-3, l0
-4, l0
-5, l0
-6The compost dilution of several kinds of concentration; Use liquid-transfering gun to draw 0.1 ml concn respectively and be l0
-2, l0
-3, l0
-4, l0
-5, l0
-6Dilution is injected on the beef extract-peptone solid plate medium, and each dilution factor triplicate, sterile water are blank; With aseptic glass slicker bacteria suspension is evenly spread upon on the whole beef extract-peptone solid culture medium, microorganism is evenly distributed, the flat board that will contain the beef extract-peptone solid culture medium is inverted in 37 ℃ of constant incubators to be cultivated 1 day;
2) bacillus thuringiensis preparation of fermentation liquid:
A. isolated bacillus thuringiensis bacterial classification purifying was cultivated for 2 generations; Insert then and be equipped with in the 250 mL triangular flasks of 50mL beef extract-peptone liquid nutrient medium; 180 r/min37 ℃ cultivation; Selecting for use the 600nm wavelength to carry out turbidimetric assay, is ordinate with the OD value of bacteria suspension, and incubation time is an abscissa; Draw the microbial growth curve, obtain the time that bacillus thuringiensis grows to stationary phase according to growth curve;
B. get purifying bacillus thuringiensis bacterial classification; Access is equipped with in the 250 mL triangular flasks of 50mL Gause I liquid nutrient medium; Cultivate 8～12 h to stationary phase for 180 r/min37 ℃, the bacterial classification of getting stationary phase adopts the microscope direct counting method to calculate the viable count in every mL bacterium liquid, and the bacterium liquid of stationary phase is carried out suction filtration; Be the miillpore filter of 0.22um with bacterium liquid through the aperture behind the suction filtration, the filtered fluid of acquisition then is the filtered fluid behind the microbial fermentation;
3) dilution bacillus thuringiensis zymotic fluid:
Get a part of filtered fluid and be diluted to 4 times of filtratings and 8 times of filtratings respectively, subsequent use;
4) adopt bacillus thuringiensis to improve the lawn plant salt-resistance:
Method for potted is adopted in experiment, in diameter is the disposal plastic cup of 7cm, adds 200g, the saline-alkali soil of 0.4-0.8% concentration; In each cup, sow perennial ryegrass, Festuca Arundinacea grass seeds 0.5g respectively, experiment is 3 repetitions; When treating the about 5cm of lawn plant growth, in soil, add the 1ml zymotic fluid respectively, control group adding aseptic liquid nutrient medium; It is buried in soil that plastic cup is moved to sample plot; To keep plant growth environment with experimental field soil environment is consistent, every day, quantitative water supply guaranteed that each is consistent between handling; Treat to measure each item index after it was grown 2 months; Emerging, to be transplanted to the field buried in soil in stable back (10 d), to keep plant growth environment with experimental field soil environment is consistent;
Purifying cultivation 2 wherein is commissioned to train to support and is referred to: directly provoke bacterium colony to be purified with connecing collarium; Be inoculated in the preprepared Gause I flat-plate solid medium; Line gently from left to right on flat board; Culture dish is inverted in 37 ℃ of constant incubators cultivated 1 day, this process is a purifying generation, and to repeat this process again be two generations of purifying to picking colony from the bacterial classification of a purifying generation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104789494A (en) * | 2015-03-30 | 2015-07-22 | 天津师范大学 | Method for improving salt resistance of turf by adopting reinforced garbage compost microbial agent |
| CN104838841A (en) * | 2015-04-20 | 2015-08-19 | 天津师范大学 | Method adopting salt-tolerant reinforcement nanometer garbage compost for reinforcing turf grass salt resistance |
| CN106431767A (en) * | 2016-10-18 | 2017-02-22 | 中国科学院武汉植物园 | Method for enhancing salt resistance of turf grass by using halophiles |
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| US20060123517A1 (en) * | 2004-12-06 | 2006-06-08 | Superak Theodore H | Inbred pumpkin line ZYPMB24 |
| CN101849011A (en) * | 2007-10-23 | 2010-09-29 | 先锋国际良种公司 | Regulatory elements associated with CBF transcription factor of rye |
| CN102172145A (en) * | 2011-02-14 | 2011-09-07 | 天津师范大学 | Application of waste compost microbial agent to improving salt resistance of lawn grass |
| CN102344812A (en) * | 2011-07-25 | 2012-02-08 | 北京平安福生物技术研究所有限公司 | Microbiological preparation for improving alkaline land, its preparation method and its application |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060123517A1 (en) * | 2004-12-06 | 2006-06-08 | Superak Theodore H | Inbred pumpkin line ZYPMB24 |
| CN101849011A (en) * | 2007-10-23 | 2010-09-29 | 先锋国际良种公司 | Regulatory elements associated with CBF transcription factor of rye |
| CN102172145A (en) * | 2011-02-14 | 2011-09-07 | 天津师范大学 | Application of waste compost microbial agent to improving salt resistance of lawn grass |
| CN102344812A (en) * | 2011-07-25 | 2012-02-08 | 北京平安福生物技术研究所有限公司 | Microbiological preparation for improving alkaline land, its preparation method and its application |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104789494A (en) * | 2015-03-30 | 2015-07-22 | 天津师范大学 | Method for improving salt resistance of turf by adopting reinforced garbage compost microbial agent |
| CN104789494B (en) * | 2015-03-30 | 2018-05-11 | 天津师范大学 | The method for improving turf salt-resistance using garbage compost microbial bacterial agent is strengthened |
| CN104838841A (en) * | 2015-04-20 | 2015-08-19 | 天津师范大学 | Method adopting salt-tolerant reinforcement nanometer garbage compost for reinforcing turf grass salt resistance |
| CN104838841B (en) * | 2015-04-20 | 2017-12-29 | 天津师范大学 | Strengthen the method for active nano garbage compost enhancing Salinity Tolerance of Turfgrass using salt |
| CN106431767A (en) * | 2016-10-18 | 2017-02-22 | 中国科学院武汉植物园 | Method for enhancing salt resistance of turf grass by using halophiles |
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