CN102577806B - 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 the application with the probiotics that extracts in producing fertilizer from refuse in daily life.Say more specifically a kind of employing bacillus thuringiensis raising lawn plant salt-resistance method.
Background technology
Composting (Composting) refers to having 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 Composting is referred to as compost (Compost).Composting process can reduce 40%-50% effectively with the volume of mixture, and can rely on heat that metabolism in hot stage produces and kill cause of disease material in rubbish.Compost is not a new technology, but processes in this way urban solid garbage, meets the interests of environment, because remove in composting process or reduced the toxicity in the urban solid garbage and made final product can be used to recycle.The program of compost comprises pretreatment, fermenting raw materials and the post processing of raw material, and composting process is subjected to the impact of the factors such as moisture, oxygen content, C/N ratio, temperature, pH and ventilation situation.
The essence of Composting Process is microorganism metabolism under optimum conditions, in Composting Process, solubilized organic substance in house refuse sees through the cell wall of microorganism and cell membrane and is absorbed by Institute of Micro-biology, and microorganism changes into inorganic matter to the organic matter that absorbs by 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, in compost, the research of microorganism to exploitation compost microbe resource, is accelerated composting process, 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 be identified, 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 compost.The environmental problem that may 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, in solution compost application process, issuable environmental problem is the focus that the related scientific research personnel study concern always.For the existing many reports of research of the composition of the microorganism in compost and the variation of the microorganism in composting process, but for microorganism in compost being separated, is applied to the rare report of research of other matrix.
Consumer garbage compost as lawn matrix, not only can be avoided food chain, solved again the problem of outlet of rubbish simultaneously.but contain the materials such as heavy metal in garbage compost and may cause adverse influence to environment, this is the major issue during compost is used always, the useful microorganism fungus kind of separation and extraction from garbage compost, being mixed with different microbial bacterial agents is inoculated in the turf establishment system, both can improve the utilization ratio of compost, simultaneously solved again the environmental threat that compost heavy metal etc. may consist of, embody the superiority of biologic product, meet the requirement of sustainable development, by studying different compost microbe microbial inoculums to the impact of lawn plant salt-resistance, purpose is in order to screen suitable microbial bacterial agent, improve quality and the resistance of lawn plant, the quality of improving lawn soil matrix provides theoretical foundation.
Bacillus thuringiensis is the present maximum of output in the world and the microorganism insecticide that is successfully used to prevent and treat agriculture, woods, pest of stored grain and some sanitary insect pests, bacillus thuringiensis is when forming gemma, can form a kind of parasporal crystal that is comprised of insecticidal crystal protein, its main active insecticidal components is parasporal crystal protein.The research discovery, this albumen has specific killing action to multiple agriculture and forestry injurious insect.After insect's food-taking gemma mixed crystal; crystal in insect bodies middle intestines alkaline environment and the effect of protease under be degraded and form insecticidal activity albumen; result of study shows between these toxalbumin independent roles or different albumen that synergy has in various degree toxic action to insect; further strengthen the research of bacillus thuringiensis aspect control and Control pests harm, realize that for the to protect mankind living environment 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 the bacillus thuringiensis ferment filtrate to be used for improving the method for 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% is subject to the impact of high salt.The soil salt damage almost affects plant physiology and biochemical movable various aspects, and its main manifestations is: the plant physiology lack of water, nutrient absorption is unbalance and the toxic action etc. that is subject to ion.Research is found, microorganism can be alleviated salt stress, Promoting plant growth, microorganism can be improved the root structure of plant, reduce the Permeability of plant leaf blade, improve the activity of superoxide dismutase in its improving activity of root system and blade (SOD), 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, improves gas exchange capacity, the raising plant Net Photosynthetic Rate of plant leaf blade, reduces salt stress to the extent of injury of 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 on improving the impact 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 acute LD of Oral Administration in Rats
50852.7-856.7 mg/kg, 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 by studying a kind of employing bacillus thuringiensis, for the saline-alkali soil planting of lawn plant provides foundation.
For achieving the above object, the 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; Also can separate the consumer garbage compost from Tianjin Xiao Dian garbage compost treatment plant; 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 as follows:
1) fresh compost sample is taken 10g, put into the 90ml sterile water triangular flask that is added with 20 beades, vibration, make in sample microorganism fully and be uniformly distributed in liquid, get the lml mother liquor with liquid-transfering gun in triangular flask, 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 respectively l0
-2, l0
-3, l0
-4, l0
-5, l0
-6The compost dilution of several concentration; Drawing 0.1 ml concn with liquid-transfering gun respectively is l0
-2, l0
-3, l0
-4, l0
-5, l0
-6Dilution is injected on 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 whole beef extract-peptone solid culture medium, microorganism is evenly distributed, the flat-plate inverted that will contain the beef extract-peptone solid culture medium is placed in 37 ℃ of constant incubators and cultivated 1 day;
2) preparation of bacillus thuringiensis zymotic fluid:
A. isolated bacillus thuringiensis purifying agaric was cultivated for 2 generations; Then access is equipped with in 250 mL triangular flasks of 50mL beef extract-peptone liquid nutrient medium, 180 r/min37 ℃ cultivation; Select the 600nm wavelength to carry out turbidimetric assay, take the OD value of bacteria suspension as ordinate, incubation time is abscissa, draws the microbial growth curve; Obtain growth of microorganism to time of stationary phase according to growth curve, the bacterial classification of getting stationary phase adopts the ascites method to calculate viable count in every mL bacterium liquid;
B. get purifying bacillus thuringiensis bacterial classification, access is equipped with in 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 ascites method to calculate viable count in every mL bacterium liquid, the bacterium liquid of stationary phase is carried out suction filtration, be the miillpore filter of 0.22um by the aperture with bacterium liquid after suction filtration, the filtered fluid of acquisition is the filtered fluid after microbial fermentation; The clump count of bacillus thuringiensis (individual) wherein
Conclusion: the clump count situation by the bacillus thuringiensis that grows on beef-protein medium can find out, the bacillus thuringiensis clump count in compost contrasts higher than soil, due to l0
-4In soil under times concentration and l0
-5, l0
-6Concentration under bacterium colony number average in compost and 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 respectively 4 times of filtrates and 8 times of filtrates, standby;
4) a kind of employing bacillus thuringiensis is improved the lawn plant salt-resistance:
Method for potted is adopted in experiment, in being the disposal plastic cup of 7cm, diameter adds 200g, the saline-alkali soil of 0.4-0.8% concentration, in each cup, sow respectively perennial ryegrass, Festuca Arundinacea grass seeds 0.5g, experiment is 3 repetitions, until lawn plant growth approximately during 5cm, add the 1ml zymotic fluid respectively in soil, control group adds aseptic liquid nutrient medium, plastic cup is moved to sample plot buried in soil, to keep plant growth environment with experimental field soil environment is consistent, every day, quantitative water supply, guaranteed that each is consistent between processing; Measure indices until its growth after 2 months; Emerge stable after (10 d) to be transplanted to the field buried in soil, to keep plant growth environment with experimental field soil environment is consistent;
Purifying is wherein cultivated 2 cultures and is referred to: directly provoke bacterium colony to be purified with connecing collarium, be inoculated in 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 be two generations of purifying to picking colony again from the bacterial classification of a purifying generation.
The prepared bacillus thuringiensis of the present invention with from the prepared bacillus thuringiensis Physiology and biochemistry character of consumer garbage compost and commercially available identical, therefore not preservation.
Qualification result about bacillus thuringiensis
The picking bacterium colony similar to bacillus thuringiensis on beef-protein medium, the bacterium colony that discovery forms on medium is circle, white, the smooth of the edge, opaque, Gram’s staining is positive, and it is shaft-like that thalline is examined under a microscope ovalize, and gemma is oval, carry out parasporal crystal dyeing with bromophenol blue and sarranine dye liquor, Microscopic observation is to there being red thalline, colourless gemma and blue parasporal crystal, and it is bacillus thuringiensis (with commercially available identical) for Preliminary Identification.
The Physiology and biochemistry experimental result of bacterium
The bacterial strain that is initially identified as 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 result is as above shown.Bacterial strain to be measured is Gram-positive, the hydrogen peroxide enzyme positive, and the V.P reacting positive, glucose fermentation produces acid, and hydrolyzed starch, gelatin can 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 PEARLITOL 25C azymic.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 as follows:
1 materials and methods
1.1 experiment material
The experiment be the bacillus thuringiensis that separates in compost with microbial inoculum, lawn plant select the more common English ryegrass of northern China (
Lolium perenne L.) and Festuca Arundinacea (
Festuca arundinacea L.) be experiment material.Matrix saline-alkali soil for examination is taken from seashore, Huanghua City, Hebei province.Experiment is respectively 0.4% and 0.8% with the saline-alkali soil salt content, is moderate and severe water stress.
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 being the disposal plastic cup of 7cm, diameter adds 200g, the saline-alkali soil of 0.4-0.8% concentration, in each cup, sow respectively perennial ryegrass, Festuca Arundinacea grass seeds 0.5g, experiment is 3 repetitions, until lawn plant growth approximately during 5cm, add the 1ml zymotic fluid respectively in soil, control group adds aseptic liquid nutrient medium, plastic cup is moved to sample plot buried in soil, to keep plant growth environment with experimental field soil environment is consistent, every day, quantitative water supply, guaranteed that each is consistent between processing; Measure indices until its growth after 2 months; Emerge stable after (10 d) to be transplanted to the field buried in soil, to keep plant growth environment with experimental field soil environment is consistent;
Purifying is wherein cultivated 2 cultures and is referred to: directly provoke bacterium colony to be purified with connecing collarium, be inoculated in 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 be two generations of purifying to picking colony again from the bacterial classification of a purifying generation.
Consistent in order to guarantee each processing in experimentation, the plastic greenhouse waterproof is built in employing, the height of canopy is about 1 m, 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 affects in 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, malonaldehyde, proline content
Crude enzyme liquid extracts: accurately take 0.5 g sample leaf, with phosphate buffer (PBS, pH7.8) ice bath milling and extracting, constant volume 25 ml get 10 ml in centrifuge tube, 10000 rmin
-1Centrifugal 20 min of EPPENDOFF centrifuge, supernatant is thick zyme extract.
The POD determination of activity: adopt guaiacol method, get the 3 mixed liquid of ml reaction in cuvette, contrast replaces with the pH=7.8 phosphate buffer, add the 0.1 thick zyme extract of mL in cuvette, open immediately manual time-keeping, measure light absorption value under 470 nm, the number of reading 1 time per minute is read 3 times altogether.Change 0.01 as a peroxidase activity unit take △ A470 per minute.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 3 mixed liquid of ml reaction comprise: 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.Then measure light absorption value rapidly under 560 nm.Take suppress the NBT photochemical reduction 50% as a unit of enzyme activity.
CAT determination of activity: adopt ultraviolet spectrophotometry.Add 1.5 ml pH=7.8 phosphate buffers, 1 ml distilled water, 0.2 ml zyme extract in test tube, contrast replaces enzyme liquid with buffer solution.Then adding 0.3 ml concentration in measuring pipe is 0.1 molL
-1Hydrogen peroxide, timing is immediately simultaneously measured light absorption value rapidly, the number of reading 1 time per minute under 240 nm.With △ A per minute
240Changing 0. 1 is a catalase activity unit.
Malonaldehyde (MDA) assay
Take 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 boiling water, and centrifugal 10 min get supernatant and survey light absorption value under 532 nm and 450 nm.
Proline:
Taking 0.5 g blade shreds, be placed in respectively Boiling tube, then the yellow basic salicylic acid that adds respectively 10 ml 3%, extracting in boiling water 10 min, cooling rear absorption 2 ml add 2 ml glacial acetic acid and 2 ml acid ninhydrines in another clean test tube with ground stopper, heating 30 min in boiling water, cooling use 4 ml toluene extract, 3000 rmin
-1Centrifugal 5 min get upper strata liquid and survey light absorption value under 520 nm.
2.3. the mensuration of photosynthetic index of correlation
Measure its Photosynthetic Index in lawn plant growth after 60 days, under natural lighting, adopt the LI-6400 photosynthesis measurement system from the morning 8:00 to afternoon 18:00 measure that each group is processed and intercellular CO of contrast every 2 h are random
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 processes each 3 data of measuring, and 3 repetitions are established in experiment altogether.When measuring These parameters, automatically record photosynthetic active radiation, 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 the impact of different compost microbe microbial inoculums on the 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, then descends gradually, and afternoon, 17:00 was 26.01 ℃.Lawn surface air temperature and humidity also changes along with the variation of Intensity of the sunlight.After measured, photosynthetic active radiation from the excursion of 7:00-17:00 at 79-1272 μ molm-
2Between g, 11:00 reaches maximum (1272 μ molm-
2G), descend gradually afterwards, become 79 μ molm-to 17:00
2G, air CO
2Concentration is always on a declining curve 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 the amplitude that descends from 7:00 to 11:00 has reached 34.7%.
3.2 the impact of compost microbe microbial inoculum on the lawn plant 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.During 7:00, the Festuca Arundinacea blade Net Photosynthetic Rate that adds the bacillus thuringiensis microbial inoculum is 39.8 μ mol (m in the morning
2G
-1), exceed 5.0% than contrast, As time goes on, the net photosynthetic rate difference of experimental group and control group reduces gradually, and after 15:00, the net photosynthetic rate of experimental group and control group does not almost have difference.When 7:00, experimental group ryegrass leaves Net Photosynthetic Rate is respectively 40.0%, 41.8% and 39.4%, and the experimental group that bacillus thuringiensis is processed has exceeded .6%. than contrast.
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.During 7:00, add the lawn plant net photosynthetic rate of bacillus thuringiensis microbial inoculum to be respectively 40.1,37.8,39.5,38.9,38.5 and 38.1 μ mol (m in the morning
2G
-1), from 7:00 to 11:00 between, the net photosynthetic rate ascensional range of lawn plant is larger, during 11:00, the lawn plant 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 respectively 39.7%, 49.5%, 42.5%, 48.3%, 51.4% and 54.9%, the rising of the net photosynthetic rate of lawn plant tends towards stability after 11:00.In addition, under saline-alkali soil stress conditions in various degree, the net photosynthetic rate of lawn plant there is no obvious difference.
3.3 the impact of compost microbe microbial inoculum on lawn plant blade intercellular gas concentration lwevel
Under the moderate condition of salt stress, compared with the control, different compost microbe microbial inoculums all have facilitation to lawn plant blade intercellular gas concentration lwevel, when 11:00, the intercellular gas concentration lwevel of the experimental group Festuca Arundinacea that different compost microbe microbial inoculums are processed reaches maximum, be respectively 56.0,56.5,56.3 μ mol/mol, improved 1.1%, 1.4%, 1.1% than contrast, the blade intercellular gas concentration lwevel of perennial ryegrass experimental group is respectively 57.7,58.3,59.0 μ mol/mol, has improved 1.2%, 1.0%, 2.3% than contrast.After 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 impact of compost microbe microbial inoculum on lawn plant growth physical signs
3.4.1 the impact of compost microbe microbial inoculum on lawn plant protective enzyme activity
The impact of different compost microbe microbial inoculums on lawn plant protective enzyme activity under table 1 severe salt stress
Annotate: in table, * represents
p＜0.05, * * represents
p＜0.01, lower same
Under the severe condition of salt stress; different compost microbe microbial inoculums are as shown in table 1 on the active impact of lawn plant protective enzyme (CAT, POD, SOD); add Festuca Arundinacea experimental group POD and the SOD activity of bacillus thuringiensis microbial inoculum to reduce by 35.4% and 68.8% than contrast respectively; add the bacillus thuringiensis microbial inoculum to Festuca Arundinacea CAT activity, obvious reduction to be arranged also, but do not reach significance degree (
p0.05).
Add the perennial ryegrass CAT activity of bacillus thuringiensis microbial inoculum to reduce compared with the control 179.5%, add perennial ryegrass CAT after actinomycetes active, add the active not appreciable impact of POD, SOD after bacillus thuringiensis (
p0.05).
The impact of different compost microbe microbial inoculums on lawn plant protective enzyme activity under table 2 moderate salt stress
Annotate: in table, * represents
p＜0.05, * * represents
p＜0.01, lower same
Under the moderate condition of salt stress, different compost microbe microbial inoculums are as shown in table 2 for the active impact of lawn plant protective enzyme (CAT, POD, SOD), add the bacillus thuringiensis microbial inoculum there is no facilitation significantly to Festuca Arundinacea CAT activity.Add POD, the contrast of SOD specific activity of the perennial ryegrass experimental group of bacillus thuringiensis to reduce by 40.6%, but significantly do not act on for perennial ryegrass CAT is active (
p0.05).
3.4.2 the impact of compost microbe microbial inoculum on lawn plant malonaldehyde, proline content
The impact of different compost microbe microbial inoculums on lawn plant malonaldehyde, proline content under table 3 severe salt stress
Annotate: in table, * represents
p＜0.05, * * represents
p＜0.01, lower same
Different compost microbe microbial inoculums are as shown in table 3 for the impact of lawn plant malonaldehyde, proline content under the severe condition of salt stress, add the bacillus thuringiensis microbial inoculum to the Festuca Arundinacea mda content without obvious reducing effect, add the proline content comparison of Festuca Arundinacea after bacillus subtilis microbial agent to take a picture and reduced by 88.4%.
The impact of different compost microbe microbial inoculums on lawn plant malonaldehyde, proline content under table 4 moderate salt stress
Annotate: in table, * represents
p＜0.05, * * represents
p＜0.01, lower same
Under the moderate condition of salt stress, different compost microbe microbial inoculums are as shown in table 4 for the impact of the malonaldehyde 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 there is no remarkable effect to the Festuca Arundinacea proline content, the experimental group perennial ryegrass mda content that adds the bacillus thuringiensis microbial inoculum has reduced by 14.7% than contrast, add bacillus thuringiensis to the proline content of perennial ryegrass without significantly effect (
p0.05).
3.4.3 the impact of compost microbe microbial inoculum on lawn plant biomass, plant height
The impact of different compost microbe microbial inoculums on lawn plant biomass, plant height under table 5 severe salt stress
Annotate: in table, * represents
p＜0.05, * * represents
p＜0.01, lower same
As shown in table 5, under the severe condition of salt stress, add the bacillus thuringiensis microbial inoculum there is no facilitation significantly for ground fresh weight and dry weight, the fresh and dried ratio of Festuca Arundinacea, add bacillus thuringiensis there is no obvious facilitation to the Festuca Arundinacea plant height.Add different compost microbe microbial inoculums to perennial ryegrass on the ground fresh weight and on the ground dry weight without facilitation significantly, reduced on the contrary the fresh and dried ratio of lawn plant perennial ryegrass ground biomass, on the also impact significantly of plant height of perennial ryegrass.
The impact of different compost microbe microbial inoculums on lawn plant biomass, plant height under table 6 moderate salt stress
Annotate: in table, * represents
p＜0.05, * * represents
p＜0.01, lower same
As shown in table 6, under the moderate condition of salt stress, add the bacillus thuringiensis microbial inoculum for the ground fresh weight of Festuca Arundinacea, obvious facilitation to be arranged, improved respectively compared with the control 66.8% and 55.3%, dry weight, fresh and dried ratio, plant height do not have facilitation significantly on the ground to Festuca Arundinacea to add different compost microbe microbial inoculums.The perennial ryegrass that adds the bacillus thuringiensis microbial inoculum on the ground fresh weight exceeds respectively contrast 33.5%, and the perennial ryegrass that adds bacillus thuringiensis on the ground dry weight on the contrary lower than contrast 31.8%.
4. development conclusion
If salinity has exceeded the tolerance limit of plant, thereby will destroy so plant cell membrane permeability and ionic equilibrium, plant is produced injury, suppress the growth of plant, in addition, Salt Strees Condition can affect the chlorophyll content of plant, inhibited 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 may be owing to containing the nutritive elements such as nitrogen, phosphorus, potassium in microbial metabolic products, improved the photosynthetic capacity of lawn plant, studies show that, in plant corpus, the photosynthetic rate of the raising of nitrogen concentration and plant is proportional, in addition, the increase of available phosphorus can improve chlorophyll and the protein content of plant, thereby improves photosynthetic rate.The 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 and injure; under stress conditions; plant cell is obstructed and produces a large amount of active oxygens, superoxide radical etc. due 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 have no significant effect the proline content of lawn plant.The compost microbe microbial inoculum also increases to a certain extent to the ground biomass of lawn plant Festuca Arundinacea and perennial ryegrass, 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 bacillus thuringiensis bacterium colony photo;
Fig. 3 is that the compost microbe microbial inoculum affects figure to the lawn plant photosynthesis characteristics;
The affect figure of compost bacterium on the lawn plant net photosynthetic rate under Fig. 4 severe salt stress;
The affect figure of compost bacterium on the lawn plant net photosynthetic rate under Fig. 5 moderate salt stress;
The affect figure of compost bacterium on lawn plant intercellular gas concentration lwevel under Fig. 6 severe salt stress;
The affect figure of compost bacterium on lawn plant intercellular gas concentration lwevel under Fig. 7 moderate salt stress.
Embodiment
In order to explain more fully enforcement of the present invention, provide following preparation method's embodiment.These embodiments are only to explain rather than limit the scope of the invention.Wherein bacillus thuringiensis (Bacillusthuringiensis) has commercially availablely, also can be obtained 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 adopts 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 10g, put into the 90ml sterile water triangular flask that is added with 20 beades, vibration, make in sample microorganism fully and be uniformly distributed in liquid, get the lml mother liquor with liquid-transfering gun in triangular flask, 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 respectively l0
-2, l0
-3, l0
-4, l0
-5, l0
-6The compost dilution of several concentration; Drawing 0.1 ml concn with liquid-transfering gun respectively is l0
-2, l0
-3, l0
-4, l0
-5, l0
-6Dilution is injected on 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 whole beef extract-peptone solid culture medium, microorganism is evenly distributed, the flat-plate inverted that will contain the beef extract-peptone solid culture medium is placed in 37 ℃ of constant incubators and cultivated 1 day;
2) preparation of bacillus thuringiensis zymotic fluid:
A. isolated bacillus thuringiensis purifying agaric was cultivated for 2 generations; Then access is equipped with in 250 mL triangular flasks of 50mL beef extract-peptone liquid nutrient medium, 180 r/min37 ℃ cultivation, select the 600nm wavelength to carry out turbidimetric assay, take the OD value of bacteria suspension as ordinate, incubation time is abscissa, draw the microbial growth curve, obtain according to growth curve the time that bacillus thuringiensis grows to stationary phase;
The clump count of bacillus thuringiensis (individual) wherein
Conclusion: the clump count situation by the bacillus thuringiensis that grows on beef-protein medium can find out, the bacillus thuringiensis clump count in compost contrasts higher than soil, due to l0
-4In soil under times concentration and l0
-5, l0
-6Concentration under bacterium colony number average in compost and soil be less than 30, therefore can't carry out statistical counting.
B. get purifying bacillus thuringiensis bacterial classification, access is equipped with in 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 ascites method to calculate viable count in every mL bacterium liquid, the bacterium liquid of stationary phase is carried out suction filtration, be the miillpore filter of 0.22um by the aperture with bacterium liquid after suction filtration, the filtered fluid of acquisition is the filtered fluid after microbial fermentation;
3) dilution bacillus thuringiensis zymotic fluid:
Get a part of filtered fluid and be diluted to respectively 4 times of filtrates and 8 times of filtrates, standby;
Get a part of filtered fluid and be diluted to respectively 4 times of filtrates and 8 times of filtrates, standby;
4) adopt bacillus thuringiensis to improve the lawn plant salt-resistance:
Method for potted is adopted in experiment, adds 200g, the saline-alkali soil of 0.4-% concentration in diameter is the disposal plastic cup of 7cm, in each cup, sow respectively perennial ryegrass, Festuca Arundinacea grass seeds 0.5g, experiment is to repeat for 3 times, until the lawn plant growth approximately during 5cm, add the 1ml zymotic fluid respectively in soil, control group adds aseptic liquid nutrient medium, plastic cup is moved to sample plot buried in soil, to keep plant growth environment with experimental field soil environment is consistent, every day, quantitative water supply, guaranteed that each is consistent between processing; Measure indices until its growth after 2 months; Emerge stable after (10 d) to be transplanted to the field buried in soil, to keep plant growth environment with experimental field soil environment is consistent;
Purifying is wherein cultivated 2 cultures and is referred to: directly provoke bacterium colony to be purified with connecing collarium, be inoculated in 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 be two generations of purifying to picking colony again from the bacterial classification of a purifying generation.
1) preparation of bacillus thuringiensis zymotic fluid:
A. the bacillus thuringiensis purifying agaric of buying was cultivated for 2 generations; Then access is equipped with in 250 mL triangular flasks of 50mL beef extract-peptone liquid nutrient medium, 180 r/min37 ℃ cultivation, select the 600nm wavelength to carry out turbidimetric assay, take the OD value of bacteria suspension as ordinate, incubation time is abscissa, draw the microbial growth curve, obtain according to growth curve the time that bacillus thuringiensis grows to stationary phase; Purifying is wherein cultivated 2 cultures and is referred to: directly provoke bacterium colony to be purified with connecing collarium, be inoculated in 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 be two generations of purifying to picking colony again from the bacterial classification of a purifying generation.
B. get purifying bacillus thuringiensis bacterial classification, access is equipped with in 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 ascites method to calculate viable count in every mL bacterium liquid, the bacterium liquid of stationary phase is carried out suction filtration, be the miillpore filter of 0.22um by the aperture with bacterium liquid after suction filtration, the filtered fluid of acquisition is the filtered fluid after microbial fermentation;
3) dilution bacillus thuringiensis zymotic fluid:
Get a part of filtered fluid and be diluted to respectively 4 times of filtrates and 8 times of filtrates, standby;
Get a part of filtered fluid and be diluted to respectively 4 times of filtrates and 8 times of filtrates, standby;
4) adopt bacillus thuringiensis to improve the lawn plant salt-resistance:
Method for potted is adopted in experiment, adds 200g, the saline-alkali soil of 0.8% concentration in diameter is the disposal plastic cup of 7cm, in each cup, sow respectively perennial ryegrass, Festuca Arundinacea grass seeds 0.5g, experiment is to repeat for 3 times, until the lawn plant growth approximately during 5cm, add the 1ml zymotic fluid respectively in soil, control group adds aseptic liquid nutrient medium, plastic cup is moved to sample plot buried in soil, to keep plant growth environment with experimental field soil environment is consistent, every day, quantitative water supply, guaranteed that each is consistent between processing; Measure indices until its growth after 2 months; Emerge stable after (10 d) to be transplanted to the field buried in soil, to keep plant growth environment with experimental field soil environment is consistent;
Purifying is wherein cultivated 2 cultures and is referred to: directly provoke bacterium colony to be purified with connecing collarium, be inoculated in 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 be two generations of purifying to picking colony again from the bacterial classification of a purifying generation.
The present invention is after the preferred embodiment that describes in detail, being familiar with this technology personage can be well understood to, can carry out various variations and modification not breaking away under above-mentioned claim and spirit, all foundations technical spirit of the present invention all belongs to the scope of technical solution of the present invention to any simple modification, equivalent variations and modification that above embodiment does.And the present invention also is not subject to the embodiment of example in specification.
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 10g, put into the 90ml sterile water triangular flask that is added with 20 beades, vibration, make in sample microorganism fully and be uniformly distributed in liquid, get the lml mother liquor with liquid-transfering gun in triangular flask, 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 respectively l0
-2, l0
-3, l0
-4, l0
-5, l0
-6The compost dilution of several concentration; Drawing 0.1 ml concn with liquid-transfering gun respectively is l0
-2, l0
-3, l0
-4, l0
-5, l0
-6Dilution is injected on 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 beef extract-peptone solid culture medium, microorganism is evenly distributed, the flat-plate inverted that will contain the beef extract-peptone solid culture medium is placed in 37 ℃ of constant incubators and cultivated 1 day;
2) preparation of bacillus thuringiensis zymotic fluid:
A. isolated bacillus thuringiensis purifying agaric was cultivated for 2 generations; Then access is equipped with in 250 mL triangular flasks of 50mL beef extract-peptone liquid nutrient medium, 180 r/min37 ℃ cultivation, select the 600nm wavelength to carry out turbidimetric assay, take the OD value of bacteria suspension as ordinate, incubation time is abscissa, draw the microbial growth curve, obtain according to growth curve the time that bacillus thuringiensis grows to stationary phase;
B. get purifying bacillus thuringiensis bacterial classification, access is equipped with in 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 ascites method to calculate viable count in every mL bacterium liquid, the bacterium liquid of stationary phase is carried out suction filtration, be the miillpore filter of 0.22um by the aperture with bacterium liquid after suction filtration, the filtered fluid of acquisition is the filtered fluid after microbial fermentation;
3) dilution bacillus thuringiensis zymotic fluid:
Get a part of filtered fluid and be diluted to respectively 4 times of filtrates and 8 times of filtrates, standby;
4) adopt bacillus thuringiensis to improve the lawn plant salt-resistance:
Method for potted is adopted in experiment, in being the disposal plastic cup of 7cm, diameter adds 200g, the saline-alkali soil of 0.4-0.8% concentration, in each cup, sow respectively perennial ryegrass, Festuca Arundinacea grass seeds 0.5g, experiment is 3 repetitions, until lawn plant growth approximately during 5cm, add the 1ml zymotic fluid respectively in soil, control group adds aseptic liquid nutrient medium, plastic cup is moved to sample plot buried in soil, to keep plant growth environment with experimental field soil environment is consistent, every day, quantitative water supply, guaranteed that each is consistent between processing; Measure indices until its growth after 2 months; Emerge stablize 10 d after, be transplanted to the field buried in soil, to keep plant growth environment with experimental field soil environment is consistent;
Purifying is wherein cultivated 2 cultures and is referred to: directly provoke bacterium colony to be purified with connecing collarium, be inoculated in 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 be two generations of purifying to picking colony again from the bacterial classification of a purifying generation.
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| 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 |
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