CN106978347A - The screening and its application in improvement booth vegetable soil hardening of one plant of dissolving phosphor and dissolving potassium bacillus - Google Patents
The screening and its application in improvement booth vegetable soil hardening of one plant of dissolving phosphor and dissolving potassium bacillus Download PDFInfo
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- CN106978347A CN106978347A CN201710226896.9A CN201710226896A CN106978347A CN 106978347 A CN106978347 A CN 106978347A CN 201710226896 A CN201710226896 A CN 201710226896A CN 106978347 A CN106978347 A CN 106978347A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
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- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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Abstract
The present invention relates to one plant of dissolving phosphor and dissolving potassium bacillus JJMSH1 screening and its application in improvement booth vegetable soil hardening, the bacterial strain has good dissolving phosphor and dissolving potassium function.The bacterial screening is from green house of vegetables plant rhizosphere soil, it is enriched with using organophosphor culture medium, Phos and silicic acid salt culture medium are screened successively, and available phosphorus and quick-acting potassium content are detected successively using molybdenum antimony resistance colorimetric method and flame spectrometry, and finally selected JJMSH1 is desirable strain.Gram-positive Bacillus is identified as by Gram's staining and spore staining.Handle available phosphorus and quick-acting potassium content in the hardened soil of greenhouse, soil using the invention bacterial strain has more obvious raising relatively, and OD600 reaches that 1.5 microbial inoculums act on soil after diluting 100 times, and available phosphorus content adds 35.7%, and quick-acting potassium content adds 26.1%.
Description
Technical field
Present invention design agricultural and microorganism field, particularly this bacterial strain have dissolving phosphor and dissolving potassium effect can be to a certain extent
Improve the hardened phenomenon of plastic shed soil.
Background technology
Phosphorus and potassium are all nutritional factors necessary to plant growth, the photosynthesis of phosphorus wide participation plant and internal life
Change reaction, the metabolism and growth of potassium and plant are closely related.Phosphorus in soil it is main with organophosphor and Phos form
In the presence of the most of form with phytic acid, nucleic acid, phosphatide of organophosphor is present, it is impossible to directly absorbed, it is necessary under microbial action
The form for being transformed into Phos could be absorbed.More than 95% phosphorus can not directly be absorbed by plant in soil.In soil
Content of soluble potassium contains substantial amounts of potassium-bearing mineral than relatively low in soil, simply they are main with stable alumino-silicates
Form is present, and more than 90% potassium can not absorb for crop.Mineral containing potassium silicate in soil are only in chemical factors
In the presence of microorganism, available potassium is progressively discharged by differentiation and decomposition and utilized for plant growth.Soluble potassium in soil
Content also constantly decline with very high speed, cause the shortage of soil effective K, cause soil fertility to decline.We
The supplement of phosphorus and potassium is agriculturally generally carried out by applying the method for chemical fertilizer.But apply chemical fertilizer and easily cause the broken of soil texture
It is bad, and cause unnecessary pollution.And administration chemical fertilizer phosphorus and potassium can not make full use of, especially phosphate fertilizer utilization efficiency is less than
30%, most phosphorus, which is solidificated in soil, to be utilized.
Booth vegetable be it is a kind of can create the agricultural production method of high-quality and efficient industrial crops, be Current Farmers cause
The very important approach that richness is striven for a relatively comfortable life.But with the accumulation of planting time, fertilizer practice it is improper, it is easy to there is soil
The hardened phenomenon of earth, has severely impacted the sustainable development of greenhouse vegetable.A soil hardening very big reason is exactly nitrogen
Fertilizer applies excessive, and available phosphorus, available potassium and other trace elements are few.Although phosphorus and potassium content content are abundant in soil,
It can not directly be absorbed, microorganism promotes available phosphorus and Potassium release in soil to improved soil rhizospheric environment, improve soil fertilizer
Power has great role, so can effectively to alleviate soil element unbalance for dissolving phosphor and dissolving potassium bacterial strain, soil element is fully used.
The content of the invention
First purpose of the present invention is to obtain the bacterial strain with good dissolving phosphor and dissolving potassium function.
Second object of the present invention is that institute's bacterium can play certain improvement result to the hardened soil of greenhouse, is increased
The growing power of thing is pretended, technical support is provided for the related microbial inoculum of exploitation.It is corresponding to reduce related economic loss, income is improved, is had
Develop beneficial to environmental health and agriculture sustainable development.
Embodiment one, bacterial screening
Bacillus JJMSH1 is isolated from green house of vegetables soil, and to realize the invention, primary dcreening operation is rich using organophosphor culture medium
Collection, reuses Phos culture medium secondary screening, the bacterial strain point with phosphorus decomposing function is connected into silicate bacteria culture medium afterwards.Therefore can
The bacterial strain may with dissolving phosphor and dissolving potassium effect is obtained simultaneously.
It is enriched with first by organophosphor culture medium, takes 1g booth vegetables Rhizosphere sampling in 100ml enriched mediums,
28 DEG C of 200r/min are enriched with 2d.Sterile saline dilution gradient is taken to be coated with to 10-8 and then by -6, -7, -8 three gradients afterwards
In Phos culture medium, growing way, observation Phos culture medium hydrolysis circle are observed in 30 DEG C of cultures daily, and record its Soluble phosphorus circle with
The diameter ratio (JJMSH1 diameters ratio is 4.1) of colony diameter, is connected to silicic acid salt culture medium by the bacterium colony point with hydrolysis circle and notes
Meaning observation colonial morphology, is chosen at the more vigorous bacterium colony of growing way on culture medium, the bacterial strain of acquisition is stored in into LB inclined-planes, final choosing
Take 5 plants of larger bacterial strains grown fine on potassium ore culture medium of hydrolysis circle
Screening, culture, preservation culture medium
1. organophosphor culture medium, Meng Jinna basal mediums add egg yolk liquid (1000ml)：Glucose 10g, NH4SO40.5g,
NaCl0.3g, KCl0.3g, 7H2O.MgSO40.3g, 7H2O.FeSO40.03g, 4H2O.MnSO 0.03g, CaCO3 5g,
(NH4)3PO40.5g, yeast extract 0.4g, adjust pH7,121 DEG C of 20min, and egg yolk liquid 10ml (sterile saline and egg are added afterwards
Huang 1:1) (solid adds 20g agar)
2. Phos culture medium, Meng Jinna basal mediums add Ca3(PO3)2：Glucose 10g, NH4SO40.5g,
NaCl0.3g, KCl0.3g, 7H2O.MgSO40.3g, 7H2O.FeSO40.03g, 4H2O.MnSO 0.03g, CaCO35g, Ca3
(PO3)22g, (solid adds 20g agar) adjusts pH7,121 DEG C of sterilizing 20min
3. silicate bacteria culture medium：Sucrose 5g, MgS047H20 0.5g, FeCl30.005g, Na2HPO4 2g, potassium
Feldspar powder (800 mesh, containing K2O：9.8%) 2g, water 1000mL, (agar 20g) adjusts pH7,121 DEG C of sterilizing 20min
4.LB：Tryptone 10g/L, yeast extract 5g/L, NaCl10g/L, agar (Gu) regulation PH7,121 DEG C of sterilizings
20min, is placed in room temperature and tilts 15 ° or so natural cooling solidifications, be placed in 4 DEG C of refrigerators standby afterwards.
The bacterial strain being separated to is activated using LB culture mediums, three kinds of fluid nutrient mediums are respectively connected to afterwards, is respectively
Phos, organophosphor, silicate bacteria culture medium, inoculum concentration are 2%, 200r/min, and culture 5d determines its phosphorus potassium content.
Embodiment two, available phosphorus available potassium are determined
The present invention surveys available phosphorus content using molybdenum antimony resistance colorimetric method, and flame spectrometry surveys quick-acting potassium content
Molybdenum antimony resistance colorimetric method surveys available phosphorus
Molybdenum antimony resistance colorimetric method principle：With 2,4- dinitrophenol dinitrophenolates as indicator, soluble phosphate can be with the anti-colour developing of molybdenum antimony
Agent is reacted, and generates P-Mo blue, and colorimetric estimation is carried out at 660nm wavelength, the content of available phosphorus is calculated according to standard curve, is led to
The concentration for crossing available phosphorus represents the height of microorganism phosphate solubilization.
Molybdenum antimony resistance colorimetric method common agents
1. the anti-storage liquid of molybdenum antimony：98% concentrated sulfuric acid 153ml is taken gently to be poured into about 400ml distilled water, stirring, cooling.
Then l0.0g ammonium molybdates are weighed to be dissolved in about 60 DEG C of 300m1 water, are cooled down.Then above-mentioned sulfuric acid solution is slowly added into this molybdenum
In acid ammonium solution, 5g/L antimony tartrate potassium solution 100ml are added, lL is finally diluted with water to, is contained in brown bottle, be put in the moon
Dark place is preserved
2. the anti-developer of molybdenum antimony：Ascorbic acid 1.5g, is dissolved in the anti-storage liquid of l00ml molybdenum antimony, now with the current
3. phosphorus standard liquid (10mg/L)：Potassium dihydrogen phosphate takes 0.439g, is dissolved in 100 DEG C of oven for drying 2h, cooling
In 100ml distilled water, the 5ml98% concentrated sulfuric acids are added, 1L is settled to, 10 times of dilution is 10mg/L phosphorus standard liquids
Phosphorus Specification Curve of Increasing：5mg/L phosphorus standard liquid 0,2,4,6,8,10,12ml are drawn respectively, are diluted with water respectively
To 26.3m1, add the anti-developer of 5ml molybdenum antimony, shake up rear constant volume, produce 0,0.2,0.4,0.6,0.8,1.0,1.2mg/L phosphorus it is molten
Liquid, is placed after 4h at 20 DEG C, using 0mg/L phosphorus titer as contrast solution, remaining phosphorus standard liquid and prepare liquid colorimetric simultaneously, and
Record the absorbance under 660nm.Using the absorbance that measures as ordinate, phosphorus concentration (mg/L) is abscissa, is depicted as standard
Phosphorus curve
Zymotic fluid 50ml, 4000r/min centrifugation 30min is taken, supernatant is taken, is fitted with distilled water diluting after 50 times, take 25ml,
It is added dropwise 2~3 and drips 2,4- dinitrophenol dinitrophenolate indicator solutions, it is firm to solution to adjust pH with 1mol/L sodium hydroxide solutions or sulfuric acid solution
Micro- Huang is presented, adds the anti-developer 5m1 of molybdenum antimony and shakes up, be settled to scale.Blank test zymotic fluid is cooked identical processing.660nm
Locate colorimetric, determine absorbance, using blank test zymotic fluid as comparison liquid zeroising, read absorbance, looked on working curve
Go out the concentration of phosphorus titer, calculating is obtained.
Bacterial strain JJMSH1 bacterial strain Phos culture mediums available phosphorus content is obtained for 59.81mg/L culture mediums by detection, its
He four plants of bacterium JJMSH2,3,4,5 is respectively, 23.10,41.32,39.28,65.45mg/L
Bacterial strain JJMSH1 bacterial strain organophosphor culture mediums available phosphorus content is 41.23mg/L culture mediums, other four plants of bacterium
JJMSH2,3,4,5 are respectively, 29.88,40.51,39.11,17.82mg/L
Flame spectrometry surveys available potassium
1.1mol/L cation exchange resin membrane solution：Weigh pure ammonium acetate 77.09g to be dissolved in water, be settled to nearly 1L, detect pH,
Adjusted with acetic acid and ammoniacal liquor to ph7
2. the standard liquid of potassium is prepared：KCl100 DEG C degree Celsius of drying 2h, weighing 0.1907g, to be dissolved in 1mol/L ammonium acetates molten
In liquid, 1L is settled to, as K containing 100ug/mL titers 0,2.5,5,7.5,10,15,20mL are put into 50mL volumetric flasks, used
1mol/L ammonium acetate solution constant volumes, as 0,5,10,15,20,30,40ug/mL K standard serial solutions
Take 25ml to be all poured into evaporating dish bacterium solution, be concentrated into 5ml or so on water-bath, plus 2ml hydrogen peroxide after
Continuous evaporation, constantly agitation, repeatedly plus several times, untill mucous substance digests completely.3500r/min centrifuges 10min, by supernatant
Liquid is collected in 50ml volumetric flasks, uses distilled water constant volume.Control medium does same treatment.The titer of filtrate and potassium exists together
Determined on flame photometer
Detection obtains bacterial strain JJMSH1 bacterial strain Phos culture mediums quick-acting potassium content for 28.56mg/L culture mediums, and other four
Strain bacterium JJMSH2,3,4,5 are respectively, 23.11,7.32,19.18,22.37mg/L
Selected bacterial strain JJMSH1 is ideal bacterial strain
Embodiment three, bacterial strain JJMSH1 strain idenfications
Bacterium colony configuration of surface is coarse, opaque, color milky is a little partially yellow, similar to some bacterial strain bacterium of bacillus
Fall form
Gram's staining and spore staining
Gram's staining：Smear is fixed, and is contaminated 1 minute at the beginning of crystal violet, and running water is rinsed, and is careful not to thalline to wash out, iodine
Liquid mordant dyeing about 1 minute, washing, moisture is sucked with blotting paper, 95% alcohol one drips, and is washed after 15~20 seconds, is sucked moisture, Huang red
Dyeing liquor was redyed after 1 minute, and running water is rinsed, oil mirror observation.
Bacterial strain of the present invention passes through Gram's staining, is accredited as positive bacteria, and thalline is elongated rod shape, accidental to have similar uncolored bud
Spore is produced.
Spore staining
Smear, drying, fixation, are added dropwise 3-5 and drip peacock green dyes, slide heats on flame, make dye liquor emit steam but
Do not seethe with excitement, be not evaporated dye liquor, about 4-5 minutes, be washed to untill peacock green no longer fades, sarranine is redyed 1 minute, is washed, and is done
After dry, oil mirror observation
It was observed that having a little green gemma and a large amount of red thalline
The present invention is accredited as bacillus
Example IV, fermentation of bacillus
Bacterial strain JJMSH1 is activated in 100mlLB fluid nutrient mediums, 35 DEG C of 200r/min activation, is then inoculated in 10L hairs
Fermentation tank, culture medium includes tryptone:5-10g/L, yeast extract:5-10g/L, sodium chloride:2-10g/L, rotating speed 300rpm,
25-35 DEG C of temperature, cultivates 12-24h, and it is 10,000,000,000/mL then to take suitable gradient to carry out colony counting.
Example IV, plastic shed soil application test
Bacterium solution dilutes to 100 respectively, 200,400 times.
The hardened mesh sieve of soil 4000 sieving of greenhouse
Take 12 1L beakers, respectively 1~12,121 DEG C of sterilizing 20min of label, be respectively charged into identical and sieved big refractory slab
Soil 0.5kg is tied, 1~3 is control group, and 4~12 be experimental group, and experimental group respectively sprays 50ml bacterium solutions, and every 3 are a dilution times
Number, control group each sprays the sterilized water of same volume
25 DEG C of culture 15d
Soil available phosphorus assay
Each sample difference soil sampling 2g
Add 50ml0.5mol/L NaHCO3Solution, 25 DEG C of 180r/min shake 30min, and 8000r/min takes supernatant
25ml, identical method do one group of blank be used for mark zero, be specifically shown in embodiment two
Control group available phosphorus content is 9.75mg/kg, and experimental group sprinkling 100 times of microbial inoculum soil sample available phosphorus contents of dilution are about
13.23mg/kg, sprinkling 200 times of microbial inoculum soil sample available phosphorus contents of dilution are about 12.11mg/kg, sprinkling 400 times of microbial inoculum soil of dilution
Sample available phosphorus content is about 10.87mg/kg
Dilution 100,200,400 times of available phosphorus contents add 35.7%, 24.2%, 11.9% respectively
Soil available nitrogen assay
Each sample difference soil sampling 2g
50ml1mol/L ammonium acetate solutions are added, 25 DEG C of 180r/min shake 30min, and 8000r/min takes supernatant 25ml, phase
With method do one group of blank be used for mark zero, be specifically shown in embodiment two
Control group quick-acting potassium content is 5.01mg/kg, and experimental group sprinkling 100 times of microbial inoculum soil sample available phosphorus contents of dilution are about
6.32mg/kg, sprinkling 200 times of microbial inoculum soil sample available phosphorus contents of dilution are about 5.77mg/kg, sprinkling 400 times of microbial inoculum soil samples of dilution
Available phosphorus content is about 5.27mg/kg
Dilution 100,200,400 times of quick-acting potassium contents add 26.1%, 15.2%, 5.2% respectively
<110>Chinese Academy Of Sciences Process Engineering Research Institute
<120>The screening and its application in improvement booth vegetable soil hardening of one plant of dissolving phosphor and dissolving potassium bacillus
<130> 2017
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1442
<212> DNA
<213> Bacillus subtilis JJMSH1
<400> 1
ggggcgggtg ctatacatgc agtcgagccc acagatggga gcttgctccc tgatgttagc 60
ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag actgggataa ctccgggaaa 120
ccggggctaa taccggatgg ttgtttgaac cgcatggttc agacataaaa ggtggcttcg 180
gctaccactt acagatggac ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca 240
aggcgacgat gcgtagccga cctgagaggg tgatcggcca cactgggact gagacaccgc 300
ccagactcct acgggagcca gcagtaggga atcttccgca atggacgaaa gtctgacgga 360
gcaacgccgc gtgagtgatg aaggttttcg gatcgtattg ctctgttgtt agggaagaac 420
aagtgccgtt caaatagggc ggcaccttga cggtacctaa ccagaaagcc acggctaact 480
acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggaatt attgggcgta 540
aagggctcgc aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg 600
tcattggaaa ctggggaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt 660
gaaatgcgta gagatgtgga ggaacaccag tggcgttggc gactctctcc tctgtaactg 720
acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg 780
taaacgatga gtgctaagtg ttagggggta accgcccctt agtgctgcag ctaacgcatt 840
aagcactccg cctggggaga acggtcgcaa gactgaaact caaacgaatt gacgggcccc 900
cgcacaagcg gtggagcatg acctttaatt cgaagcaacg cgaagaacct taccaggtct 960
tgacatcctc tgacaatcct agagatagga cgtccccttc gggggcagag tgacaggtgg 1020
tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1080
cccttgatct tagttgccag cattcagttg ggcactctaa ggtgactgcc ggtgacaaac 1140
cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt 1200
gctacaatgg acagaacaaa gggcagcgaa accgcgaggt taagccaatc ccacaaatct 1260
gttctcagtt cggatcgcag tctgcaactc gactgcgtga agctggaatc gctagtaatc 1320
gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc 1380
acgagagttt gtaacacccg aagtcggtga ggtaacctaa aggagccagc cgccgaagaa 1440
gt 1442
Claims (6)
1. the screening of one plant of dissolving phosphor and dissolving potassium bacillus, fermentation and its application in improvement booth vegetable soil hardening.
2. the screening technique of dissolving phosphor and dissolving potassium bacillus, first exists the soil from booth vegetable according to claim 1
Enrichment 2-6 days is carried out in organophosphor culture medium, then Phos culture medium and silicic acid salt culture medium are screened, and are chosen at training
Growing way is vigorous on foster base, contain the larger bacterium colony for hydrolyzing circle.
3. bacterial strain is Gram-positive bacillus according to claim 1.
4. the 16SRNA gene orders of bacterial strain such as SEQ ID NO according to claim 1:Shown in 1.
5. the fermentation condition of bacterial strain is according to claim 1：Culture medium：Tryptone:5-10g/L, yeast extract:5-
10g/L, sodium chloride:2-10g/L, rotating speed 200-400rpm, 25-35 DEG C of temperature cultivate 12-24h.
6. the application conditions in improvement booth vegetable soil hardening are according to claim 1：Microbial inoculum is diluted into 100-400
Apply again, drip irrigation to plastic shed soil.
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| CN112980728A (en) * | 2021-03-08 | 2021-06-18 | 领先生物农业股份有限公司 | Bacillus flexus endophytic strain for salt-tolerant alkaline hydrolysis of silicon, phosphorus and potassium and application thereof |
| CN113150788A (en) * | 2021-04-22 | 2021-07-23 | 浙江树人学院（浙江树人大学） | Preparation method and application of seaweed oligosaccharide composite microbial material for preventing soil hardening |
| CN113293116A (en) * | 2021-07-10 | 2021-08-24 | 西南林业大学 | Bacillus bacteria with phosphate solubilizing capability and application thereof |
| CN114105409A (en) * | 2021-11-19 | 2022-03-01 | 苏州中晟环境修复有限公司 | Method for treating antimony in underground water polluted by printing and dyeing wastewater |
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| CN112980728A (en) * | 2021-03-08 | 2021-06-18 | 领先生物农业股份有限公司 | Bacillus flexus endophytic strain for salt-tolerant alkaline hydrolysis of silicon, phosphorus and potassium and application thereof |
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| CN113150788A (en) * | 2021-04-22 | 2021-07-23 | 浙江树人学院（浙江树人大学） | Preparation method and application of seaweed oligosaccharide composite microbial material for preventing soil hardening |
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| CN114105409A (en) * | 2021-11-19 | 2022-03-01 | 苏州中晟环境修复有限公司 | Method for treating antimony in underground water polluted by printing and dyeing wastewater |
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