CN110396485A - Generate class Brevibacillus brevis and its application of auxin - Google Patents

Generate class Brevibacillus brevis and its application of auxin Download PDF

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CN110396485A
CN110396485A CN201910507692.1A CN201910507692A CN110396485A CN 110396485 A CN110396485 A CN 110396485A CN 201910507692 A CN201910507692 A CN 201910507692A CN 110396485 A CN110396485 A CN 110396485A
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auxin
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魏士平
刘贝贝
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China University of Geosciences Beijing
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Abstract

Generate class Brevibacillus brevis and its application of auxin.The invention discloses one plant of class Brevibacillus brevis Brevibacillus parabrevis bacterial strain and its applications.The class Brevibacillus brevis Brevibacillus parabrevis is class Brevibacillus brevis Brevibacillus parabrevis BIO7 CGMCC NO.17202.Such Brevibacillus brevis Brevibacillus parabrevis BIO7 can generate auximone, in the culture medium of Tryptone for being added to 0.05% tryptophan, its auxin yield can be used for fermentation industry up to 36.1 μ g/ml, produce and prepare auximone.Also it can be used as a kind of microorganism formulation to directly apply to agriculturally, for promoting the growth of crop.

Description

Generate class Brevibacillus brevis and its application of auxin
Technical field
The present invention relates to the class Brevibacillus brevis for generating auxin and its applications.
Background technique
Auximone is the important hormone of coordinate plant growth.Its chemical property is heteroauxin, this is to send out earliest The hormone of existing promotion plant growth.It influences the division of plant cell, elongation and differentiation, controls taking root and sprouting for plant, And the growth of plant organ is influenced, mature and aging.Most important natural auxin is β-heteroauxin.Although plant endogenous swash Cellulose content is very low, but they play very important effect in the growth and development of plant.Due to plant endogenous hormones content It is low, therefore can not largely extract them and be used for agricultural production.Therefore it generates or synthesizes by other approach and plant The development trend that there is object hormone the substance of identical physiological effect to have become Modern Agricultural.
Barazani and Friedman (1999) discovery has the bacterium classification that much can produce auxin in the soil [16]；Finnie and Van Staden (1985) has found that a variety of edaphons include that bacterium, fungi etc. can generate plant life Long element, this may establish certain contact with the growth and development of plant；Existing research shows that bacteriogenic auxin for The growth of plant has certain facilitation, and Mohite (2013) research discovery is isolated from plant rhizosphere soil Bacterium can produce auximone, and can dramatically increase plant height, stem length, root long and the chlorophyll content of wheat seedling； Sun et al. (2014) discovery can increase the quantity of arabidopsis lateral root by the auxin that saccharomycete generates and reduce root elongation, And promote the formation of root hair；The bacterium bacterial strain of production auxin Shahab et al. (2009) isolated from soil can promote Rhizome into soybean extends and promotes the formation of root hair.Barbieri et al. (1986) discovery is vaccinated with the wheat seedling of bacterium The quantity and length of lateral root have apparent increase；Glick et al. (1986) discovery bacterial treatment rape seed, so that rape The length of the root of seedling increases 2 to 3 times；Caron et al. (1995) has found the bacterium separated from rhizosphere soil, nothing By under the premise of existing or physiology precursor substance tryptophan (Trp) is not present, all there is the energy of synthetic auxin in vitro Power.
Ocean is a huge resource treasure-house, its biology and chemical resource rich in.It is mentioned compared to by fertilising High soil nutrient is sprayed insecticide to promote crop growth, is improved for the original method of yield, by the production in Yu Haiyang The bacterium of auxin as formulation application in agricultural, it is not only pollution-free, but also sustainable use, meet green development view, will not The pollution of soil environment and water environment is caused, this is advocating green living, and protect environment is particularly important, its gesture instantly The following key breakthrough point for improving agriculture value must be become, there is high development prospect, or the hair that the future of agriculture will be become Spread gesture.
Summary of the invention
The object of the present invention is to provide one plant of class Brevibacillus brevis and its generating the application in auxin.
Class Brevibacillus brevis provided by the present invention is class Brevibacillus brevis Brevibacillus parabrevis BIO7CGMCC No.17202。
The class Brevibacillus brevis Brevibacillus parabrevis BIO7 was protected on 01 16th, 2019 It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address are as follows: court, city, BeiJing, China The institute 3 of positive area's North Star West Road 1) deposit number be CGMCC NO. 17202.
The class Brevibacillus brevis Brevibacillus parabrevis BIO7 is isolated from the new river mouth ocean in Bei Dai River Deposit is gram-positive bacterium.The bacterium can form milky bacterium colony, bacterium colony circle on beef-protein medium Shape, regular edges, circular colonies center projections, surface wettability.Lipase, cellulase, protease and siderophore can be generated (siderophore)。
The class Brevibacillus brevis Brevibacillus parabrevis BIO7CGMCC No.17202 gives birth in production Application in long element also belongs to protection scope of the present invention.
The class Brevibacillus brevis Brevibacillus parabrevis BIO7CGMCC No.17202 is planted in preparation Application in object growth regulating preparation also belongs to protection scope of the present invention.
Class Brevibacillus brevis Brevibacillus parabrevis BIO7CGMCC No.17202 of the invention may be used also Using as preparation promote plant growth microbial-bacterial fertilizer in application.
The present invention also provides a kind of bacteria agents for promoting plant growth, which is characterized in that its bioactive ingredients is class Brevibacillus brevis Brevibacillus parabrevis BIO7CGMCC No.17202.
The present invention also provides a kind of microbial-bacterial fertilizers for promoting plant growth, including the bacteria agent.
Preferably, the microbial-bacterial fertilizer further includes tryptophan.
The present invention also provides a kind of plant growth regulating compositions, including class Brevibacillus brevis Brevibacillus parabrevis BIO7CGMCC No.17202。
Preferably, the plant growth regulating composition further includes tryptophan.
In an embodiment of the invention, the tryptophan is the tryptophan solution that mass percentage concentration is 5%.
The present invention also provides class Brevibacillus brevis Brevibacillus parabrevis BIO7CGMCC No.17202 Cultural method, which is characterized in that class Brevibacillus brevis Brevibacillus parabrevis BIO7 is inoculated in containing color In the Tryptone culture medium of propylhomoserin, it is to cultivate in 30 DEG C of concussion and cultivate cases；The Tryptone culture medium be containing Tryptone (tryptone, Britain OXOID, article No. LP0042) 10g/L, the fluid nutrient medium of NaCl 5g/L；The color ammonia The whole mass percentage concentration of acid is preferably 0.05%；The time of the culture is preferably 4 days.
The revolution of the shake culture is preferably 150rpm/min.
This research, by the separation of bacterium, screening, can get using the bacterium in ocean as research object and generate plant growth The bacterium of element produces auximone with bacterial fermentation, auxin compound can be prepared, for cuttage Plants and Seeds It takes root；Also can be used as a kind of microorganism formulation and directly apply to agricultural, this by be the following green agriculture sustainable development new shape Formula.Taken root, germinateed to seed directly as microbial inoculum by auxin caused by the bacterium and the bacterium respectively, potting etc. it is real It tests, it is inquired into the facilitation of plant growth, is made into plant growth regulator products application in agricultural Production has great development prospect.
Detailed description of the invention
Fig. 1 is auxin standard sample color reaction.
Fig. 2 is auxin standard curve.
Influence (the A:YMD culture medium of Fig. 3 culture medium and tryptophan to color reaction；B:Tryptone culture medium；C:LB Culture medium；D:CM culture medium.1: original culture medium；2: BIO7 is vaccinated in original culture medium；3: being added in original culture medium 0.05% tryptophan；4: being added in the original culture medium of 0.05% tryptophan and be vaccinated with BIO7.
Fig. 4 is that (A: supporting from earth culture for wheat is separately added into for observation of the bacteriogenic auxin to wheat root long, plant height Auxin standard solution or the bacteriogenic auxin crude product of BIO7；B: the wheat young plant of auxin standard items processing；C: The wheat young plant of the bacteriogenic auxin crude product processing of BIO7).
Fig. 5 is observation of the bacterium BIO7 to wheat root long, plant height.
Specific embodiment
Method in following embodiments is unless otherwise instructed conventional method.
Percentage composition in following embodiments is unless otherwise instructed mass percentage.
Embodiment 1, the bacterial strain class Brevibacillus brevis Brevibacillus parabrevis BIO7 for producing auxin The screening and identification of CGMCC No.17202
One, the acquisition and dilution of sample
1, using the new river mouth marine sediment in Bei Dai River as sample；
2, sediment sample dilute solution is prepared, the step is carried out in super-clean bench using aseptic technique: weighing 10g Deposit is put into the conical flask for filling 90ml sterile water, is shaken 10 min using oscillator machinery, is kept deposit breakup uniform, Obtaining diluted concentration is 10-1Sample；
3, deposit suspension is serially diluted with constant gradient dilution method, obtaining dilution is 10-2, 10-3, 10-4, 10-5Sediment sample solution.
Two, in sediment sample bacterium isolation and purification
1, the configuration of culture medium: configuration beef extract-peptone solid medium (beef extract 3g/L, peptone 10g/L, NaCl 5g/L, agar 20g/L), CM fluid nutrient medium (casein 7.5g/L, yeast 10 g/L, sodium citrate 3g/L, MgSO4·7H2O 5g/L, KCl 2g/L, FeSO4·7H2O 0.05g/L, NaCl 5g/L) high steam under the conditions of 121 DEG C Sterilize 20min, and inverted plate is spare after cooling；
2, the separation of bacterium: taking cooling solid beef-protein medium plate, is 10 by dilution-2, 10-3, 10-4, 10-5Sediment sample solution respectively draw 100 μ L drops in plate center, be coated with using the spreader after sterilizing uniform.It will Culture medium is sealed and is inverted, and the culture medium of inoculated bacteria is placed in constant incubator, is cultivated 2 days at 30 DEG C.It is long to bacterium colony After out, the picking that appropriate dilution carries out bacterium colony counting and single colonie is selected；
3, the purifying of bacterium: to prevent, picking single colonie from mixing, and the single colonie of above-mentioned institute's picking is carried out plate and is drawn Line purifying, cultivates 2 days at 30 DEG C, obtains the pure culture of bacterium.Finally obtain 61 plants of pure culture bacterium.
Three, the preservation of pure culture bacterium
Bacterium is saved using the method for liquid freezing, specific operating process is as follows:
1, liquid CM fluid nutrient medium is prepared, it is stand-by after 121 DEG C of high pressure steam sterilization 20min；
2, the liquid CM culture medium 1mL of sterilizing is added in 1.5mL sterile centrifugation tube, scrapes the training of beef extract-peptone solid Each bacterium pure culture lawn on base is supported, is put into above-mentioned centrifuge tube；Then final concentration of 20% sterile glycerol is added, be vortexed shake It swings uniformly, is placed in -70 DEG C of refrigerators and saves.
Four, auxin generates the screening and identification of bacterium
1, the measurement of auxin
With 35%HClO4Solution and 0.5mol/L FeCl3Solution is mixed to get Salkowski reagent with the ratio of 50:1, The liquid culture and Salkowski reagent of bacterium are uniformly mixed with the ratio that volume ratio is 1: 2.At room temperature, it is kept in dark place Color reaction can occur for 30min, Salkowski reagent and auxin (heteroauxin IAA), if pink occurs in mixed liquor, i.e., It indicates to contain auxin in bacterium solution.Bacterium can be trained a length of 530 nanometers according to the maximum absorption wave of generated pink material It supports generated auxin in object and carries out quantitative analysis.
2, the screening of auxin producing strains
(1) the qualitative primary dcreening operation of auxin producing strains: the bacterium solution saved in 50 μ L step 3 is respectively drawn with liquid-transfering gun and is inoculated in Equipped with 5ml YMD culture medium, (yeast 4g/L, malt root soak powder 10g/L, glucose 4 g/L, K2HPO42g/L, tryptophan 0.5g/ L in test tube), after cultivating 2 days in 30 DEG C of concussion and cultivate case, take 50 μ L bacteria suspensions and 100 μ L Salkowski reagents mixed It closes uniformly (the YMD culture medium of bacterial strain and 1: 2 mixed solution of Salkowski reagent is not added as blank control), in room temperature Under, observation liquid color variation after 30min is kept in dark place, pink occur indicates that auxin IAA can be generated.
(2) the quantitative secondary screening of auxin producing strains:
The production of auxin standard curve.10mg IAA standard sample is weighed, is dissolved with a small amount of ethyl alcohol, 100mL is added and steams Distilled water is diluted to the IAA standard solution of 0,0.5,1.0,5.0,10.0,15.0,20.0,25.0 μ g/ mL after mixing, with Salkowski reagent is mixed with the volume ratio of 1:2, and avoid light place 30 minutes, measure the OD530 value of each gradient respectively at room temperature (as the 1:2 mixed solution of distilled water and Salkowski reagent as blank control).Finally using IAA concentration as abscissa, OD530 is that ordinate is mapped to get IAA standard curve is arrived.It observes color change (Fig. 1), and measures the OD530 of each gradient respectively It is worth (as the 1:2 mixed solution of distilled water and Salkowski reagent as blank control).Finally with IAA concentration for horizontal seat Mark, OD530 are that ordinate is mapped to get IAA standard curve (Fig. 2) is arrived.Obtaining IAA calibration curve equation is Y=0.0287X, Wherein R2=0.9942.
The measurement of auxin concentration in bacterium solution.The bacterial strain that step 3 kind saves is activated, the correspondence bacterium of preservation is drawn Strain 2 μ L of solution is inoculated on beef extract-peptone plate, after 30 DEG C are cultivated 2 days, with one ring of aseptic inoculation ring picking, is inoculated in In 5mL YMD liquid tube, in being cultivated on 30 DEG C, 150rpm/min shaking table 3 days, then it is equipped with by 1% inoculum concentration access In 100mL YMD fluid nutrient medium, be added filtration sterilization tryptophan solution make its final concentration of 0.1%.At 30 DEG C, Training 2-4 days is shaken in the concussion and cultivate case of 150rpm/min.2mL bacterium solution is taken, under conditions of centrifugal speed is 10000rpm/min 5 min are centrifuged, then takes 1.9mL supernatant to be uniformly mixed with 3.8mL Salkowski reagent, at room temperature, is kept in dark place 30min (the YMD culture medium of bacterial strain and 1: 2 mixed solution of Salkowski reagent is not added as blank control), measures light respectively Density, and corresponding IAA concentration is calculated from the standard curve of IAA concentration and OD530.Three repetitions of every group of experimental setup.
As a result, it has been found that the bacterium separated in marine sediment has sub-fraction to can produce auxin, each bacterium is produced The capacity of water of raw auxin have by force have it is weak.It wherein obtains the bacterial strain that one plant of number is BIO7 and generates auxin ability highest.
3, the physiological and biochemical property of bacterial strain and identification
1) bacterial strain physiological and biochemical property
Colony colour and form.It goes bail for the BIO7 bacterial strain deposited, dips bacterium solution with the oese that calcination sterilizes, use plate streaking For method in the flat lining out of beef extract-peptone, which is inverted culture in 30 DEG C of incubators, until there is single colonie.
Strain enzyme-producing detection.Lipase detects culture medium (1L): beef extract 3g, peptone 10g, tributyrin 6ml, NaCl 10g.Agar 20g.Cellulose detects culture medium (1L)；Carboxymethyl cellulose 5g, NaNO31g, K2HPO42g, KCl 1g, MgSO4.7H2O 0.5g, 2 g of yeast powder, glucose 1g, NaCl 10g, agar 20g.Protease detects culture medium (1L): de- Rouge milk powder 15g, yeast powder 2g, NaCl 10g, agar 20g.Amylase detects culture medium (1L): starch 5g, yeast powder 2g, (NH4)2SO41.4g, K2KPO42g, MgSO4·7H2O 0.2g, NaCl 10g, agar 20g.The BIO7 bacterial strain of activation is distinguished It is inoculated into the center of lipase detection, cellulase detection, protease detection and amylase detection plate, is fallen in 30 DEG C of incubators Set culture 5 days.Such as bacterial strain detects in lipase and forms macroscopic transparent circle on culture medium and protease detection culture medium, then It indicates to generate lipase and protease；Cellulase detects 0.1% Congo red flushing of plate, as periphery of bacterial colonies appearance is transparent Circle then shows that the bacterium generates cellulase；Amylase detects plate and is rinsed with 0.3% iodine solution, as periphery of bacterial colonies appearance is transparent Circle then shows that the bacterium generates amylase.
Bacterial strain produces siderophore detection.The detection of siderophore has become an important indicator of screening plant-growth promoting rhizobacteria.CAS Culture medium (1L): the MgSO of 1mM4The CaCl of 10mL, 1mM2 10mL,MM9 (K2HPO46g, KH2PO40.3g, NaCl 0.5g, NH4Cl 1g, deionized water 100mL) 5 mL, 20% C6H12O6Solution 10mL, 10% caseinhydrolysate 30mL, fine jade Rouge 16g.After sterilizing, when culture medium is cooled to 50-60 DEG C, CAS solution is added, and (0.06g chromazurine is dissolved in 50mL water and obtains solution 1；0.073g HDTMA is dissolved in 40mL water and obtains solution 2；10mL FeCl is added into solution 23Obtain solution 3；Solution 1 is slowly added Enter solution 3 and obtain CAS solution) 50mL.BIO7 bacterial strain after activation is connected to CAS solid medium tablets center, is trained in 30 DEG C It supports and is inverted culture 5 days in case, which yellow halo occurs, then illustrates that the bacterial strain can produce siderophore.
Physiology and biochemistry testing result is as follows:
The bacterial strain forms milky bacterium colony on beef-protein medium, and bacterium colony is round, regular edges, round bacterium Fall center projections, surface wettability.
The bacterial strain can produce lipase, cellulase, protease and siderophore, not generate amylase；The result is shown in tables 1.
The producing enzyme of 1 BIO7 of table detects
Enzyme class Producing enzyme Transparent loop diameter (mm)
Lipase + 19.5±0.35
Cellulase + 5.0±0.03
Protease + 18.8±1.06
Amylase - -
Siderophore + 0.5±0.05
Note: "+" indicates to generate；"-" expression does not generate；Experiment sets three repetitions.
2) strain classification is identified
Extract bacterium BIO7 genomic DNA, using this DNA as template, using the general 16S rRNA primer of bacterium come Carry out PCR amplification:
Upstream primer 27f:5 '-GTTTGATCCTGGCTCAG-3 '.
Downstream primer 1492r:5 '-CTACGGCTACCTTGTT-3 '.
Pcr amplification product after purification, is carried out with 27f and 1492r primer respectively through agarose gel electrophoresis and gel extraction Double sequencings, sequence are corrected through Chromas, and are spliced by DANMAN software, and the sequence of 1420bp, sequence such as sequence are obtained In table shown in sequence 1.The sequence is compared in ncbi database (https: //www.ncbi.nlm.nih.gov/) It analyzes, as the result is shown the 16S rRNA sequence of the bacterial strain and Brevibacillus parabrevis NAP3 in database (KJ872854) and the homology of Brevibacillus parabrevis IFO 12334 (NR_040981) reaches 99.86%.
Such Brevibacillus brevis Brevibacillus parabrevis BIO7, in preservation on the 16th in 01 month in 2019 In China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address are as follows: city, BeiJing, China southern exposure The institute 3 of area North Star West Road 1) deposit number be CGMCC NO.17202.
Embodiment 2, class Brevibacillus brevis Brevibacillus parabrevis BIO7 generate the condition choosing of auxin It selects
One, culture medium and tryptophan generate the influence of auxin to bacterium
The nutritional ingredient as contained by various culture mediums is different, and intrinsic color is also different, in order to reduce culture medium face Interference of the color to auxin color reaction, convenient for the screening to auxin bacterium is generated, we respectively carry out 4 kinds of culture mediums Comparison: CM culture medium, YMD culture medium, LB culture medium (NaCl 5g/L, peptone 10g/L, yeast powder 5g/L), Tryptone training It supports base (Tryptone (tryptone, Britain OXOID, article No. LP0042) 10g/L, NaCl 5g/L).In addition, due to auxin The direct precursor substance of synthesis is tryptophan (Trp), we compared being separately added into 0.05% in above 4 kinds of culture mediums simultaneously (mass percentage concentration) tryptophan and be added without tryptophan to generate auxin influence.
Take 50 μ L to save bacterium solution to be inoculated in the test tube equipped with the above-mentioned each culture medium of 5ml, using do not connect the culture medium of bacterium as Control.It is cultivated 2 days in 30 DEG C of concussion and cultivate cases.1.9ml supernatant is taken after bacterium solution centrifugation, do not connect bacterium then directly takes 1.9ml Culture medium is uniformly mixed with 3.8ml Salkowski reagent respectively, at room temperature, observes liquid color after 30min is kept in dark place Variation, pink occur indicates that IAA can be generated.Then its OD is measured530Value, obtains its corresponding IAA content.
Above 4 kinds of culture mediums are in addition tryptophan and do not add tryptophan and be inoculated with Brevibacillus Parabrevis BIO7 be not inoculated with Brevibacillus parabrevis BIO7 and combine for totally 16 kinds, after cultivation, carry out The color reaction of auxin and quantitative determination.Color reaction result is shown in Fig. 3.From figure 3, it can be seen that adding when in 4 kinds of culture mediums Add (A3, A4, B3, B4, C3, C4, D3, D4) after tryptophan, all than not adding the color of corresponding culture medium of tryptophan It is deep.In addition it can find out, after having accessed Brevibacillus parabrevis BIO7 in 4 kinds of culture mediums (A2, A4, B2, B4, C2, C4, D2, D4), all than no corresponding culture medium face for accessing Brevibacillus parabrevis BIO7 Color is deep, and the apparent macroscopic pink colour reaction (Fig. 3 B2,3B4) of the appearance having.By contrast, we can see that YMD culture medium color is relatively deep (Fig. 3 A), masks pink colour reaction, it is not easy to observe color change；Tryptone culture medium face Color is shallower (Fig. 3 B), it is easily observed that color change；Although LB culture medium dyes shallower (Fig. 3 C), access Brevibacillus parabrevis BIO7 does not occur apparent pink colour reaction；CM culture medium color is shallower (Fig. 3 D), relatively holds Easily observe color change.By contrast, Tryptone culture medium access Brevibacillus parabrevis BIO7 (figure 3B2,3B4), pink colour even deeper color reaction is all produced, illustrates the bacterium that the culture medium is suitable for being used to generate auxin Screening, and after tryptophan is added in the culture medium, pink colour color is further deepened, and illustrates that tryptophan generates auxin to bacterium There is apparent influence.
Further to quantify Brevibacillus parabrevis BIO7 generated auxin in various culture mediums Content, determine 4 kinds of culture mediums and tryptophans be added and are added without the contents of auxin caused by tryptophan bacterium, as a result see Table 2, in YMD, Tryptone and CM culture medium after tryptophan is added, the yield of auxin is obviously increased, in fermentation liquid The yield of auxin is respectively 11.8 μ g/ml, 24.1 μ g/ml, 13.8 μ g/ml (table 2)；And tryptophan is being added in LB culture medium Afterwards, auxin yield reduces instead compared with the control.Therefore, it is to generate that 0.05% tryptophan, which is added, in Tryptone culture medium The better suited culture medium of auxin, can be used for the fermenting and producing of auxin.
The influence of 2 culture medium of table and tryptophan to auxin yield
Two, incubation time generates the influence of auxin to bacterium
It selects Tryptone culture medium that 0.05% tryptophan is added, accesses BIO7 bacterial strain, cultivated, shaken in 30 DEG C In incubator (150rpm/min), when cultivating 2 days, auxin yield is up to 24.1 μ g/ml；When cultivating 4 days, auxin yield can Up to 36.1 μ g/ml；But with the further extension of incubation time, auxin yield was declined slightly, therefore using fermentation 4 days It is preferable that mode prepares auxin.
Influence experiment of the bacteriogenic auxin of embodiment 3, BIO7 to wheat growth
One, the bacteriogenic auxin of Brevibacillus parabrevis BIO7 is to the wheat plant supported from earth culture The influence of growth
1, culture BIO7 bacterium generates the fermentation liquid containing auxin
Configuration beef extract-peptone solid medium and the Tryptone culture medium for being added to 0.05% tryptophan. BIO7 Bacterial strain activation and the preparation of seed culture medium are equipped with 100mL by 1% inoculum concentration access with the measurement of auxin concentration in bacterium solution It is added in the Tryptone fluid nutrient medium of 0.05% tryptophan, training 4 days is shaken under conditions of 30 DEG C, is in revolving speed by bacterium solution 5min is centrifuged under conditions of 8000rpm/min, supernatant is the fermentation liquid containing auxin.Above-mentioned supernatant is carried out 10 times Constant gradient dilution, be diluted to 10-1-10-5；Auxin IAA standard solution, which is also done constant gradient, is diluted to 10 simultaneously-1-10-9。
2, influence of the fermentation liquid containing auxin to being grown from native cultivated wheat
Wheat seed is put in the conical flask for filling a certain amount of distilled water, soaked overnight after washing 5 to 10 times.It will impregnate Overnight wheat seed vernalization selects that germination degree is close, the similar wheat of size after showing money or valuables one carries unintentionally at seed embryo, small to guarantee Wheat vigor is roughly the same, excludes bring to influence in subsequent processing caused by wheat oneself factor.It will urge Wheat seed dibbling after bud in the test tube for filling water agar, after wheat malt it is long to a certain extent afterwards be transferred to normal illumination item Under part.And it is separately added into above-mentioned corresponding bacterial fermentation dilution and the diluted auxin mark of 10 times of constant gradients every three days Quasi- product solution, with each 100 μ l of original culture medium solution, every group of experiment and control three repetitions of each setting.
After wheat is supported 10 days from earth culture (Fig. 4 A), the water agar in test tube is taken out together with whole wheat, carefully peels off fine jade Rouge obtains complete wheat plant (Fig. 4 B, 4C).Then overall merit is carried out to the wheat of each dilution gradient, measures wheat Root long and plant height.From Fig. 4, it is apparent that being shown with the wheat that the auxin standard items of different gradient dilution degree are handled bright Aobvious effect has apparent inhibiting effect to wheat root that is, in Seedling height element concentration, with the dilution of auxin standard items, The overall length of wheat root gradually increases, when auxin standard items are diluted to 1.6 × 10-2When μ g/ml (table 3), it can promote tomorrow The elongation of wheat root；And the plant height for corresponding to the wheat of each gradient processing is not distinguished significantly then.This and auxin for The intrinsic propesties of plant growth are related, i.e., inhibit the growth of root under high concentration, promote the elongation of root under low concentration；And stem of plant Increase the reaction to auxin, then promote the production of stem in higher concentrations, but we do not observe increasing for stem in experiment, Illustrate that the concentration that wheat stalk can be promoted to extend has not been reached yet in the concentration of auxin standard items.Equally, the root of wheat plant, stem pair The sensibility of auxin concentration is different, so resulting in the small of the processing of the auxin standard items using different dilution gradients There is certain difference between wheat plant.It compares, joined not with control when with fermentation liquor treatment wheat containing auxin Wheat with the fermentation liquid of the bacterium of gradient dilution degree has at certain promotion or inhibiting effect and auxin standard items root The wheat of reason has high similitude, i.e., has apparent inhibiting effect to root at high concentrations, with the dilution of fermentation liquid, root Overall length had apparent increase before this, when dilution be 10-3When, auxin concentration is 3.61 × 10-2μ g/ml (table 3) the root totality longest of the wheat, handled, increases 25.0% compared with the control；It is small with the further dilution of fermentation liquid Wheat root long with compare no significant difference.Fermentation liquid is low and high dilution is all to observe that the promotion high to wheat plant is made With.
The influence of 3 auxin fermentation liquid of table and auxin standard items to wheat root long and plant height
Two, it is real that influence of the Brevibacillus parabrevis BIO7 bacterium to wheat growth is directly inoculated in soil It tests
1, the preparation of wheat is tested
It takes soil several, is sieved after grinding, makes uniform soil quality.Using the iron wire after heating in disposable plastic bottom of a cup Portion uniformly drills out 5 comparable apertures of diameter, and soil after cooling packing equivalent is weighed to these disposable plastics In cup.These disposal plastic cups are encased with newspaper later, for guaranteeing the dark of the soil bottom during plant strain growth Environment.Treated, these disposal plastic cups are put in plate.Later will under the conditions of 121 DEG C high pressure steam sterilization 20min sterile water after cooling pours into plate, soaks soil in entire plastic cup using the osmosis of water.It saves stand-by.
Select that germination degree is close, the similar wheat of size is dispensed by vernalization after wheat seed soaked overnight, after vernalization In the above-mentioned disposal plastic cup for filling sterile soil, to guarantee that wheat vigor is roughly the same, substantially eliminate due to wheat certainly It bring may be influenced in subsequent processing caused by body factor.The sterile soil of 4g is weighed after packing to be uniformly spread to mould Material cup soil surface covers wheat seed completely, be placed under dark condition and cultivate, after wheat malt is long to a certain extent It is transferred under normal lighting conditions.
2, the preparation of Brevibacillus parabrevis BIO7 bacteria preparation
BIO7 bacterial strain carries out scribing line culture on beef extract-peptone plate, is then washed the bacterium colony grown with sterile water Under, it is transferred in sterile centrifugation tube, with sterile water washing 2-3 times, is finally suspended in sterile water, adjusts its cell concentration to 109 Uniform bacterium solution is put in the conical flask equipped with a certain amount of sterile water and is diluted to bacterial cell number 10 by a/ml7A/ml, it is standby With.
3, influence of the Brevibacillus parabrevis BIO7 bacteria preparation to wheat growth
Experiment wheat is taken, 3 groups of experiments, three repetitions of every group of experiment are set.Experiment process is as follows: growing to 1- to wheat young plant When 2cm, respectively by the sterile water of 50ml, 50ml BIO7 bacteria preparation and be added to 0.05% Trp 50ml BIO7 it is thin Bacteria preparation is injected separately into the plastic cup of experiment wheat.Then it checks daily, according to the moisture in plastic cup bottom culture plate How much, the sterile water of each processing supplement equivalent, to keep ground moistening.
After experiment wheat cultivates 14 days at room temperature, by soil from being taken out in plastic cup and rinse out the soil on wheat root The wheat of each experiment of correspondence is carried out overall merit, measures wheat root long and plant height by earth.From fig. 5, it can be seen that joined The wheat growing way of BIO7 bacterium solution is preferable, especially in terms of root long, than control group and has added wheat in the experimental group of tryptophan Root long have apparent increase.The statistical result of wheat and plant height is shown in Table 4, and the wheat root that joined BIO7 bacteria preparation is up to To 26.2 ± 4.1cm, than the root long balanced growth 1.2% of wheat in control group；And it joined tryptophan and be inoculated with BIO7 Experimental group, there is inhibiting effect compared with the control group in the root long of wheat, this may be the presence due to there is tryptophan, The auxin concentration of its bacterium synthesis is slightly higher, plays certain inhibiting effect to the elongation of wheat root.It can be seen that BIO7 bacterium The growth that can promote wheat in more soil can be directly added as microorganism formulation, without supplementing tryptophan.In addition, three groups Plant Height in Wheat does not have apparent difference in experiment, and due to the experiment culture 14 days, the time was shorter, it is believed that with the extension of time, The microorganism BIO7 preparation has more obvious influence to the root of wheat and plant height.
Influence of the 4 bacterium BIO7 of table to wheat growth
Processing Total root long/the strain (cm) of wheat Total plant height/the strain (cm) of wheat
Sterile water 23.4±4.2 23.9±4.2
BIO7 26.2±4.1 24.9±2.8
BIO7+Trp 21.8±3.1 23.9±3.8
Conclusion
This experiment is screened from the bacterium separated in marine sediment, and it is relatively strong to obtain one plant of generation auxin ability Bacterial strain, 16S rRNA sequence and the similitude of the 16S rRNA sequence of Brevibacillus parabrevis are 99.86%, it is named as Brevibacillus parabrevis BIO7.Using the bacterial strain as experimental subjects, culture has been probed into respectively Base and tryptophan, incubation time and auxin crude product is prepared, has rated the physiological action of the bacteriogenic auxin, And biological agent is made in the bacterial strain, is applied to potted plant experiment, observes and records the facilitation to plant growth.As a result table Bright: Tryptone fluid nutrient medium is conducive to generate the screening of auxin bacterium, and is adding 0.05% tryptophan In Tryptone fluid nutrient medium, the yield of auxin is higher than other culture mediums, cultivates two days, auxin yield reaches 24.1 μ g/ml can reach 36.1 μ g/ml when cultivating 4 days.
Auxin crude product prepared by Brevibacillus parabrevis BIO7 bacterial fermentation carries out 10 times of ladder Degree dilution, observes their influences to the root long and plant height of wheat under the conditions of from soil.BIO7 ferment product, which is worked as, to be diluted to 10-3When dilution, auxin concentration is 3.61 × 10-2μ g/ml can be obviously promoted the elongation of wheat root；In low dilution, i.e., It is when auxin concentration is high, then inhibited to the elongation of wheat root.No matter auxin standard items or BIO7 bacterial growth Plain crude product does not all influence Plant Height in Wheat significantly.It under normal conditions, is to promote root to the auxin concentration that stem of plant promotes 100 times of growth concentration, illustrate bacteriogenic auxin concentration have not been reached yet can be obviously promoted it is dense required for Stem nematode Degree.
Brevibacillus parabrevis BIO7 bacterium is applied in soil directly as microorganism formulation, as a result Show: BIO7 bacterium is directly appended in soil as biological agent, can promote the elongation of wheat root；Add the processing of additive color propylhomoserin Wheat with compare no significant difference, and add tryptophan and access in the processing of bacterium BIO7, the root of wheat is by certain The inhibition of degree, this may be that tryptophan transfer has been turned to auxin by bacterium, so that the increase of auxin concentration is caused, certain The elongation of root is inhibited in degree.
In conclusion Brevibacillus parabrevis BIO7 is one plant of bacterial strain that can generate auxin, fermentation Product and microbial inoculum itself can promote the elongation of wheat root, can be used for industrial fermentation and prepare auxin compound being answered With, can also directly as biological agent be applied to agricultural production.
Described above to be merely exemplary for the purpose of the present invention, and not restrictive, those of ordinary skill in the art understand, In the case where not departing from spirit and scope as defined in the appended claims, many modifications, variation or equivalent can be made, but all It will fall within the scope of protection of the present invention.
Sequence table
<110>The Chinese Geology Univ. (Beijing)
<120>class Brevibacillus brevis and its application of auxin are generated
<130>WHOI190073
<170> Patent-In 3.5
<160> 1
<210> 1
<211> 1420
<212> DNA
<213>class Brevibacillus brevis (Brevibacillus parabrevis)
<400> 1
atgcagtcga gcgagggttt tcggacccta gcggcggacg ggtgagtaac acgtaggcaa 60
cctgcctctc agaccgggat aacataggga aacttatgct aataccggat aggtttttgg 120
attgcatgat ccgaaaagaa aagatggctt cggctatcac tgggagatgg gcctgcggcg 180
cattagctag ttggtggggt aacggcctac caaggcgacg atgcgtagcc gacctgagag 240
ggtgaccggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg 300
gaattttcca caatggacga aagtctgatg gagcaacgcc gcgtgaacga tgaaggtctt 360
cggattgtaa agttctgttg tcagggacga acacgtgccg ttcgaatagg gcggtacctt 420
gacggtacct gacgagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 480
gtggcaagcg ttgtccggat ttattgggcg taaagcgcgc gcaggcggct atgtaagtct 540
ggtgttaaag cccggagctc aactccggtt cgcatcggaa actgtgtagc ttgagtgcag 600
aagaggaaag cggtattcca cgtgtagcgg tgaaatgcgt agagatgtgg aggaacacca 660
gtggcgaagg cggctttctg gtctgtaact gacgctgagg cgcgaaagcg tggggagcaa 720
acaggattag ataccctggt agtccacgcc gtaaacgatg agtgctaggt gttgggggtt 780
tcaataccct cagtgccgca gctaacgcaa taagcactcc gcctggggag tacgctcgca 840
agagtgaaac tcaaaagaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 900
tcgaagcaac gcgaagaacc ttaccaggtc ttgacatccc gctgaccgct ctggagacag 960
agcttccctt cggggcagcg gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga 1020
gatgttgggt taagtcccgc aacgagcgca acccttatct ttagttgcca gcattcagtt 1080
gggcactcta gagagactgc cgtcgacaag acggaggaag gcggggatga cgtcaaatca 1140
tcatgcccct tatgacctgg gctacacacg tgctacaatg gttggtacaa cgggatgcta 1200
cctcgcgaga ggacgccaat ctctgaaaac caatctcagt tcggattgta ggctgcaact 1260
cgcctacatg aagtcggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt 1320
cccgggcctt gtacacaccg cccgtcacac cacgggagtt tgcaacaccc gaagtcggtg 1380
aggtaaccgc aaggagccag ccgccgaagg tgggtgcaaa 1420

Claims (10)

1. one plant of class Brevibacillus brevis Brevibacilus parabrevis, which is characterized in that the entitled short gemma bar of class Bacterium Brevibacilus parabrevis BIO7, in China Committee for Culture Collection of Microorganisms's common micro-organisms center Deposit number be CGMCC No.17202.
2. class Brevibacillus brevis Brevibacilus parabrevis BIO7 CGMCC No.17202 is in production auxin Application.
3. class Brevibacillus brevis Brevibacilus parabrevis BIO7 CGMCC No.17202 is preparing plant growth Adjust the application in preparation.
4. class Brevibacillus brevis Brevibacilus parabrevis BIO7 CGMCC No.17202 promotes plant in preparation Application in the microbial-bacterial fertilizer of growth.
5. a kind of bacteria agent for promoting plant growth, which is characterized in that its bioactive ingredients is class Brevibacillus brevis Brevibacilus parabrevis BIO7 CGMCC No.17202。
6. a kind of microbial-bacterial fertilizer for promoting plant growth, including the bacteria agent described in claim 5.
7. microbial-bacterial fertilizer according to claim 6, it is characterised in that: the microbial-bacterial fertilizer further includes tryptophan.
8. a kind of plant growth regulating composition, including class Brevibacillus brevis Brevibacilus parabrevis BIO7 CGMCC No.17202。
9. plant growth regulating composition according to claim 8, it is characterised in that: the plant growth regulating composition also wraps Include tryptophan.
10. the cultural method of class Brevibacillus brevis Brevibacillus parabrevis BIO7 CGMCC No.17202, It is characterized by: being inoculated in class Brevibacillus brevis Brevibacillus parabrevis BIO7 containing tryptophan In Tryptone culture medium, it is to cultivate in 30 DEG C of concussion and cultivate cases；The Tryptone culture medium is to contain tryptone The fluid nutrient medium of 10g/L, NaCl 5g/L；The whole mass percentage concentration of the tryptophan is preferably 0.05%；The culture Time is preferably 4 days.
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CN112322518A (en) * 2020-10-15 2021-02-05 华南农业大学 Bacillus alpinus with potassium-dissolving effect and application thereof
CN115109710A (en) * 2021-03-18 2022-09-27 中国农业大学 Bacillus 1603IPR-02 with siderophore production capacity and application thereof

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CN112322518A (en) * 2020-10-15 2021-02-05 华南农业大学 Bacillus alpinus with potassium-dissolving effect and application thereof
CN115109710A (en) * 2021-03-18 2022-09-27 中国农业大学 Bacillus 1603IPR-02 with siderophore production capacity and application thereof
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