CN106085925A - A kind of salt tolerant antibacterial T5S2 and the application in microbial manure thereof - Google Patents
A kind of salt tolerant antibacterial T5S2 and the application in microbial manure thereof Download PDFInfo
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Abstract
nullThe present invention discloses a kind of salt tolerant antibacterial T5S2 and the application in microbial manure thereof，By salt radix unguis border, Junggar Basin, Xinjiang, china varieties in saline-alkali areas pedotheque separates a strain salt tolerant antibacterial novel species T5S2，It is resistant to the NaCl of 1 15% concentration，At 30 DEG C of 37 DEG C of well-growns，Growth course need not be induced and can produce acc deaminase and IAA heteroauxing，Utilize strain to prepare microbial manure and be applied to alkaline land cotton、In the industrial crops such as Semen Maydis，Ensure that the germination percentage of alkaline land cotton reaches 77.8%，Survival rate is to 65.4%，Plant height、Strain number and single bell strain number will be apparently higher than conventional treatment Cotton Gossypiis，Salt-soda soil corn emergence rate all increases to more than 80% from 64.5%，Per mu yield Semen Maydis reaches 600kg/ mu，0 30cm soil layer soil total salt content is made to have dropped 60 70%，There is obvious salt reduction production-increasing function，It is with a wide range of applications.
Description
Technical field
The present invention relates to microorganism fungus kind applied technical field, be specifically related to a kind of salt tolerant antibacterial and preparing micro-life
The technical field of application in thing fertilizer.
Background technology
Halomonas (Halomonas) is the antibacterial that a class salt tolerance is the strongest, mostly can be in 0.1%-20%NaCl concentration
Lower growth, have also can grow under 0.1%-32.5% concentration, is typically distributed about salt lake, saltern etc., belongs to moderate addicted to salt
Bacterium (moderatehalophiles).They and extremely halophilic archaea (extrem eha lophiles) broadly fall into the most micro-life
Thing, but more extensive than halophilic archaea distribution, low to nutritional requirement, it is easier to adapt to environment.The biological characteristics that Halomonas is unique
Property, physiological mechanism and genetic mechanism gradually cause the interest of research worker.
Fairly common owing to irrigating improper and caused by other unreasonable cultivation soil secondary salinization at present, it is to cause
The Main Barrier Factors of the crop failure reduction of income, is that " drought, flood, salt, alkali, thin " five evils are various, the growth promoter shadow to crop
Ringing greatly, under salt stress, cotton leaf feels like jelly, color is dark, functional period is short, comes off early, and side root occurs less, generating region yellowish-brown,
Dry-matter accumulation reduces, growth retardation, even dead Seedling.The method improving the soil salinization at present is mainly chemical method, but
The excess of chemical fertilizer and pesticide is used and is brought the most serious environmental problem and food-safety problem, along with people are to protection
The pay attention to day by day of environment, in order to improve ecological environment, improve people's life, it is current for using biological method to solve these problems
Development trend.
Microbial manure has low input, high production, high benefit, high-quality, raw material is sufficient, processing technology is simple, easily
The advantages such as popularization, microbial manure will give full play to its due economic benefit and ecological benefits in agricultural production, therefore, raw
The technological development of thing fertilizer and application, be the inexorable trend of agricultural production and Fertilizer development, and the exploitation of microbial manure is non-
Often meet the requirement of modern material reserve and agricultural sustainable development.
The patent of invention " a kind of Salt And Alkali Tolerance microorganism species and application thereof " of Application No. 201410623410.1 discloses
A kind of Salt And Alkali Tolerance compounds microbial activity flora, discloses existing common different Shewanella, pseudomonas and Halomonas, but
It is that this Salt And Alkali Tolerance compounds microbial activity flora mainly for the treatment of the waste drilling mud of high salinity content and oily sludge.Shen
Please number be 201210572369.0 patent of invention " the Yellow River Halomonas BJGMM-B45 and application thereof " disclose a strain and have excellent
Antibacterial the Yellow River Halomonas (Halomonas huanghegensis) BJGMM-B45 of good salt toleration, discloses in literary composition
This bacterial strain has antibiotic resistance, as the genetic donor of new genetically modified organism, but can record other spies the most in detail
Different effect.The patent of invention " method of a kind of microorganism improvement salt affected soil " of Application No. 201310466328.8 discloses
A kind of method of the microbial inoculum improvement salt affected soil utilizing elongation Halomonas, lignin bacillus cereus and alkaline bacillus mixing,
But three kinds of strains that this technical scheme comprises are existing common strain, and only disclose this microorganism formulation mycopowder and can improve
Semen sojae atricolor, the germination percentage of Fructus Lycopersici esculenti, do not record more beneficial effect.
Due to the territorial environment that Xinjiang is wide, be richly stored with microorganism.Research Xinjiang extreme microorganism not only helps
In expanding extreme microorganism resources bank, it is also beneficial to the development and utilization of Xinjiang special environment microorganism.Prior art research table
Bright, study the most rare in terms of Biological control and microbial manure about Halomonas, therefore, research and utilization extreme microorganism
Novel bacterial characteristic and application thereof have the meaning of reality.
Summary of the invention
The less state of the art is recorded about Halomonas research in terms of microbial manure in prior art, this
Invention aims to provide a kind of novel bacterial salt tolerant antibacterial T5S2 and the application in microbial manure thereof.The present invention is by accurate in Xinjiang
Varieties in saline-alkali areas, basin, Gaer foliated kalidium rhizosphere soil sample separates a strain Halomonas novel species (Halomonas sp.)
T5S2CGMCC No.12642, utilizes isolated strain as microbial manure, is applied to the economy such as alkaline land cotton, Semen Maydis
In crop, the growth characteristics such as crops emergence rate, survival rate can be significantly improved so that soil layer soil total salt content have dropped 60-
70%, there is obvious salt reduction production-increasing function, be with a wide range of applications in microbial manure technical field.
The main technical schemes that the present invention uses:
The present invention is by isolated from the foliated kalidium rhizosphere soil sample of Junggar Basin, Xinjiang, china varieties in saline-alkali areas.Sample is adopted
Take gradient dilution method, weigh 1g pedotheque and be suspended in the triangular flask containing PAF culture fluid, 30 DEG C, 150r/min shaken cultivation
Through DF culture fluid, twice switching of ADF culture fluid after 24h, carry out gradient dilution, coat ADF solid plate constant temperature culture.Therefrom
Filter out the Halomonas novel species T5S2 in terms of a strain can be applicable to microbial manure.Utilize the numbering that the present invention separates
Bacterial isolates for T5S2 prepares microbial manure, is applied in the industrial crops such as alkaline land cotton, Semen Maydis, can significantly improve salt
The growth characteristics such as alkali ground crop emergence rate, survival rate, effectively reduce soil salt content, the microorganism fertilizer present invention provided simultaneously
Material is applied in the industrial crops such as Herba Apocyni veneti, Helianthi and Radix Betae have obvious salt reduction production-increasing function, it is seen then that utilize the present invention
The Halomonas novel species T5S2 provided prepares the exploitation application of microbial manure and has the biggest application prospect.
The present invention specifically provides a kind of Halomonas novel species (Halomonas sp.) T5S2CGMCC No.12642.Pass through
From the foliated kalidium rhizosphere soil sample of Junggar Basin, Xinjiang, china varieties in saline-alkali areas, isolated sampling, filters out a strain salt tolerant antibacterial salt
Zymomonas mobilis (Halomonas sp.) T5S2, this bacterium has higher salt-tolerant trait, need not induce and can produce in growth course
Acc deaminase and IAA heteroauxing, this bacterial strain has good growth-promoting by the microbial manure that conventional fermentation techniques obtains
Long, effect of salt reduction.
The present invention is according to the enrichment ground feature of the particularity of Xinjiang geographical environment and Xinjiang Situation of Microorganism Under Extremity Environment resource, logical
Cross and from the foliated kalidium rhizosphere soil sample of Junggar Basin, Xinjiang, china varieties in saline-alkali areas, carry out the cultivation of microorganism fungus kind, separate and screen
A collection of excellent species, and therefrom separate the Halomonas (Halomonas sp.) of a numbered T5S2 of strain, through microorganism
Credit class and qualification, belong to Halomonas, is resistant to the NaCl of 1-15% concentration, in 30 DEG C of-37 DEG C of well-growns, 45 DEG C of lifes
Long the most suppressed, can be at the LB cultured on solid medium containing 1.0%NaCl, bacterial growth is circular, microprotrusion, butter
Color, mucoid colony, it is seen then that Halomonas (Halomonas sp.) T5S2 is resistant to the NaCl of 1-15% concentration, 30 DEG C-
37 DEG C of well-growns, have need not induce in growth course and can produce the spy that acc deaminase and IAA heteroauxing are distinct
Point, carries out morphology, Physiology and biochemistry survey with reference to " primary Jie Shi Bacteria Identification handbook " and " common bacteria identification handbook " etc. to bacterial strain
Fixed, determine that T5S2 bacterial strain is member in Halomonas；Sequence sees the SEQUENCE LISTING of attached offer, through 16SRNA
Homogeneous assays, Phylogenetic Analysis, by BLAST homology comparison, determine the kind nearest with experimental strain sibship.From number
Obtain the sequence of related species according to storehouse, and combine bacterium living beings Study on Diversity central database, carry out cluster analysis and system
Cladogram builds.T5S2 bacterial strain belongs to Halomonas (Halomonas sp.), bacterial strain T5S2 and this genus type strain sequence ratio
Homology is respectively 96.7% (Halomonas ventosae Al12T), 96.4% (Halomonas jebtebsis
AIR-2T), 96.4% (Halomonas desisderata FB2T), 96.2% (Halomonas rifensis HK31T), temporarily
Named Halomonas (Halomonas sp.) T5S2.This bacterial strain was preserved in budapest treaty microorganism before the applying date
International Depository Authority: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)；Address: court of Beijing
North Star West Road 1 institute of sun district 3, Institute of Microorganism, Academia Sinica, postcode: 100101；Preservation date is 2016 06 month
20, culture presevation number was CGMCC No.12642, identified temporary named Halomonas (Halomonas sp.) through microbiology
T5S2。
The physio-biochemical characteristics of Halomonas (Halomonas sp.) T5S2: Gram’s staining is negative, aerobic, bacillus,
Individually or in pairs occurring, being positive in catalase and oxidase are tested, sugar fermentation does not produce acid；Indole, clark and Lubsreaction
Negative；Measuring card with the GN 2 of Biolog Microstation system to test, bacterial strain T5S2 can utilize acetic acid, Fructus Citri Limoniae
Acid, D-galactose, D-Glucose, D-Glucose, lactic acid, malonic acid, maltose, propanoic acid, sorbitol, succinic acid, L-are Arabic
Sugar, cellobiose, ethanol.
Meanwhile, the present invention further provides one and utilize Halomonas (Halomonas sp.) T5S2CGMCC
No.12642 prepares the method for microbial manure, specifically comprises the following steps that
(1) Halomonas (Halomonas sp.) T5S2CGMCC No.12642 is inoculated in LB solid slant culture base,
Cultivate 3d, i.e. obtain slant strains for 33 DEG C；Slant tube adds after 5mL sterilized water acutely vibrates, use dual-layer sterilization gauze mistake
Filter, filtrate is prepared as containing 10 through dilution6Individual spore suspension；Draw above-mentioned spore suspension 1mL, be inoculated in LB liquid culture
In base, 33 DEG C, 150rpm shaken cultivation 3d, obtain seed liquor.
(2) preparation method of liquid microbe fertilizer: draw above-mentioned seed liquor, is inoculated in fermentation culture by 3% inoculum concentration
Base, 33 DEG C, 150rpm cultivate 6d, results thalline and fermentation liquid, prepare corresponding liquid microbe fertilizer；The fermentation used
Culture medium is: according to peptone 5g in every 100ml fermentation medium, yeast extract powder 2g, glucose 20g, dipotassium hydrogen phosphate 1g,
Magnesium sulfate 0.5g, distilled water 1000mL, pH 6.6.
(3) preparation method of solid microbe fertilizer: draw above-mentioned seed liquor, is inoculated in solid fermentation by 3% inoculum concentration
Culture medium, 33 DEG C of solid fermentation 6d, cover with solid medium to mycelia, obtain solid fermentation microbial inoculum, prepare corresponding solid
Body microbial manure；The solid fermentation culture medium used according to weight ratio proportioning is: pulverize cotton stalk 65%, wheat bran 20%, Semen Gossypii
Shell 13%, sucrose 1%, CaCO31%, then add tap water and fully stir evenly, controlling final water content is 60%.
There is provided above-mentioned concrete technical scheme by implementing the present invention, implement present invention, following useful effect can be reached
Really:
(1) Halomonas (Halomonas sp.) the T5S2CGMCC No.12642 that the present invention provides is a kind of typical
Novel bacterial, is resistant to the NaCl of 1-15% concentration, at 30 DEG C of-37 DEG C of well-growns, need not induce and can produce in growth course
Raw acc deaminase and IAA heteroauxing, have condition of culture simple, breed fast feature.
(2) by Halomonas (Halomonas sp.) the T5S2CGMCC No.12642 of separation screening of the present invention by often
The microbial manure that rule fermentation technique obtains, is applied in the industrial crops such as alkaline land cotton, Semen Maydis, it is possible to ensure that salt-soda soil is cotton
The germination percentage of flower reaches 77.8%, survival rate to 65.4%, and plant height, strain number and single bell strain number are cotton apparently higher than conventional treatment
Flower, salt-soda soil corn emergence rate all increases to more than 80% from 64.5%, and per mu yield Semen Maydis reaches 600kg/ mu so that 0-30cm soil layer
Soil total salt content have dropped 60-70%, has obvious salt reduction production-increasing function, the microbial manure simultaneously present invention provided
It is applied in the industrial crops such as Herba Apocyni veneti, Helianthi and Radix Betae, all there is obvious salt reduction production-increasing function, it is seen then that utilize this
The Halomonas novel species T5S2 of bright offer prepares the exploitation application of preparation and has the biggest application prospect.Meanwhile, the present invention produces
Product can be administered alone, and has good growth promoting function, it is possible to carries out joining executing with fertilizer, reduces soil while improving soil nutrient
The salinity of earth, application process is various, and Field information is easy, it is possible to take into account environmental friendliness and yield promotes.
Accompanying drawing explanation
Fig. 1 show bacterial strain Halomonas (Halomonas sp.) T5S2CGMCC No.12642 Phylogenetic dendrogram.
Fig. 2 show bacterium Halomonas (Halomonas sp.) T5S2CGMCC No.12642 and produces in different culture media
Acc deaminase situation.
Fig. 3 show bacterium Halomonas (Halomonas sp.) T5S2CGMCC No.12642 and produces in different culture media
IAA situation.
Detailed description of the invention
Below, for embodiment, the present invention is described, but, the present invention is not limited to following embodiment.The present invention selects
All raw and auxiliary materials, and select spawn culture method be all to it is well known that selection, the % related in the present invention
All be weight percentage, unless otherwise indicated except.
The embodiment of the present invention uses following basal medium:
PAF culture medium: peptone 10g, casein hydrolysate 10g, MgSO41.5g, K2HPO41.5g, glycerol 10mL, NaCl
10g, pH7.0, distilled water 1000mL.
DF culture medium: glucose 2g, gluconic acid 2g, citric acid 2g, NaCl10g, K2HPO44.0g, Na2HPO46.0g,
MgSO40.2g, (NH4)2SO42.0g, 0.1mL trace element solution, pH7.2, agar 15g.
Trace element: FeSO41mg, H3BO310mg, MnSO411.19mg, ZnSO4124.6mg, CuSO478.22mg,
MoO310mg, is dissolved in 100mL distilled water.
ADF culture medium: the DF culture medium that sterilizing prepares, takes out, and after culture medium is cooled to about 50 DEG C, adds and filters
After the ACC (1-amino-cyclopropane-1-carboxylic acid) to final concentration 3umol/L of degerming 0.3mol/L, it is down flat plate standby.
Product heteroauxing (lAA) culture fluid: glucose 1g, L-Trp 0.05g, K2HPO48.709g,
KH2PO46.805g, MgS041.232g, (NH4)2SO43.303g, NaCl 10g, distilled water 1000mL.
TSB culture medium: tryptone 5.0g, soya peptone 1.0g, NaCl 10g, distilled water 1000mL, pH value=7.2.
LB culture medium: peptone 10.0g, yeast extract 5.0g, NaCl 10g, distilled water 1000mL, pH value=7.2.
Embodiment one: the separation of Halomonas (Halomonas sp.) T5S2, screen and identify
1, separate: isolated from the foliated kalidium rhizosphere soil sample of Junggar Basin, Xinjiang, china varieties in saline-alkali areas.Weigh 1g soil
Earth sample is suspended in the triangular flask containing PAF culture fluid, 30 DEG C, 150r/min shaken cultivation 24h, draws 1ml bacteria suspension extremely
In 50ml DF culture fluid, 30 DEG C, 150r/min shaken cultivation 24h, connect bacteria suspension in ADF culture fluid with inoculating loop, 30 DEG C,
150r/min shaken cultivation 24h, repeats to transfer after 2 times, and gradient dilution is coated ADF solid plate, 28 DEG C of constant temperature culture, treated
Flat board grows single bacterium colony.
2, purification: the bacterium colony that picking shape, size, color etc. are different after growing bacterial clump streak inoculation respectively is in phase
The flat board answered, 30 DEG C of 37 DEG C of purification are cultivated, it is preferred to use 33 DEG C of cultivations, until falling without miscellaneous bacteria, the most preferably go out bacterial strain.
3, classify, identify: be special by observing Halomonas (Halomonas sp.) T5S2 colony shape and Physiology and biochemistry
Point, extract bacterial strain STb gene, use antibacterial ITS PCR expand universal primer, carry out PCR amplification, PCR primer through cutting glue purification,
Order-checking, carries out ITS BLAST and compares, and carries out cluster analysis and systematic evolution tree builds.
Halomonas used in the present invention (Halomonas sp.) T5S2 is from Junggar Basin, Xinjiang, china varieties in saline-alkali areas salt pawl
Isolated in the pedotheque of radix unguis border.Weigh 1g pedotheque to be suspended in the triangular flask containing 50ml PAF culture fluid, 30 DEG C,
150r/min shaken cultivation 24h, repeats to transfer 1 time.Draw 1ml bacteria suspension in 50ml DF culture fluid, 30 DEG C, 150r/min
Shaken cultivation 24h.Bacteria suspension is connect in 50ml ADF culture fluid, 30 DEG C, 150r/min shaken cultivation 24h with inoculating loop.Repeat
Transfer after 2 times, gradient dilution, coat ADF solid plate, 28 DEG C of constant temperature culture, picking shape, big after growing bacterial clump
The bacterium colony streak inoculation respectively that little, color etc. are different, in corresponding flat board, until falling without miscellaneous bacteria, the most preferably goes out a strain numbered
The bacterial strain of T5S2.
Halomonas (Halomonas sp.) T5S2 can be at 30 DEG C of-37 DEG C of well-growns, and 45 DEG C of growths are the most suppressed,
Can be at the LB cultured on solid medium containing 1.0%NaCl, bacterial growth is circular, microprotrusion, cream-colored, mucoid colony,
With reference to " primary Jie Shi Bacteria Identification handbook " and " common bacteria identification handbook ", bacterial strain is carried out taxonomic identification, tentatively predicate salt list
Born of the same parents bacterium (Halomonas sp.).
By extracting the STb gene of Halomonas (Halomonas sp.) T5S2, antibacterial ITS PCR is used to expand general drawing
Thing, carries out PCR amplification, and PCR primer is through cutting glue purification, order-checking, and its sequence sees the SEQUENCE LISTING of attached offer.
Gained T5S2 region sequence carries out BLAST with the known array in GenBank data base and compares, and determines and experimental strain
The kind that sibship is nearest.Obtain the sequence of related species from data base, and combine calculation in bacterium living beings Study on Diversity
According to the mode standard bacterium sequence in storehouse, MEGA 5.0 software kit is utilized to use adjacent method (Neighbor-Joining method) to enter
Row cluster analysis and systematic evolution tree build, and systematic evolution tree sees accompanying drawing 1, and from systematic evolution tree, bacterial strain belongs to salt
Zymomonas mobilis (Halomonas sp.), bacterial strain T5S2 and this genus type strain sequence alignment homology are respectively 96.7%
(Halomonas ventosae Al12T), 96.4% (Halomonas jebtebsis AIR-2T), 96.4% (Halomonas
desisderataFB2T), 96.2% (Halomonas rifensisHK31T), tentatively predicate the potential novel species of this genus and name temporarily
For Halomonas (Halomonas sp.) T5S2.
The physio-biochemical characteristics of Halomonas (Halomonas sp.) T5S2: Gram’s staining is negative, aerobic, bacillus,
Individually or in pairs occurring, being positive in catalase and oxidase are tested, sugar fermentation does not produce acid；Indole, clark and Lubsreaction
Negative；Measuring card with the GN 2 of Biolog Microstation system to test, bacterial strain T5S2 can utilize acetic acid, Fructus Citri Limoniae
Acid, D-galactose, D-Glucose, D-Glucose, lactic acid, malonic acid, maltose, propanoic acid, sorbitol, succinic acid, L-are Arabic
Sugar, cellobiose, ethanol.
Bacterial strain Halomonas (Halomonas sp.) T5S2 was preserved in budapest treaty microorganism state before the applying date
Border depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC).Address: Beijing is exposed to the sun
North Star West Road, district 1 institute 3, Institute of Microorganism, Academia Sinica, postcode: 100101.Preservation date on 06 20th, 2016,
Culture presevation number is CGMCC No.12642.Temporary named Halomonas (Halomonas sp.) T5S2 is identified through microbiology.
By the above-mentioned thalli morphology for strain Halomonas (Halomonas sp.) T5S2CGMCC No.12642,
Cultural characteristic observation, Determination of Physiological And Biochemical Indices and molecular level are identified, are i.e. seen by thalli morphology observation, strain culturing feature
Examine, growth temperature mensuration, resistance test etc. are tested, compared with common Halomonas, with reference to " primary Jie Shi Bacteria Identification handbook "
And the method for " common bacteria identification handbook " is carried out, although the strain of numbered T5S2 and common Halomonas bacteria culture
Compare, there are some attributes of general character, but have obvious physio-biochemical characteristics poor with common Halomonas bacteria culture
The different diversity with molecular level, shows that T5S2 bacterial strain is a kind of typical novel bacterial, from bacterium classification angle by bacterium numbering
Bacterial strain comprehensive identification for T5S2 is Halomonas (Halomonas sp.).
Halomonas (Halomonas sp.) the T5S2CGMCC No.12642 that the present invention provides can be suitable for its propagation
Solid medium or fluid medium in carry out enrichment culture, utilize Conventional solid slant culture cultivate, the side of low-temperature preservation
Method, pass on every time can preservation more than 3 months, the long term storage strain manufactured with the method for being frozen and dried, can preservation more than 1 year, or
Long term storage is carried out with glycerol pipe.
Embodiment two: Halomonas (Halomonas sp.) T5S2CGMCC No.12642 produces ACC at different culture media and takes off
Ammonia enzyme situation alive
Bacterial strain bacterial strain T5S2 is inoculated in LB, TSB and ADF culture fluid containing 1%NaCl respectively, and 30 DEG C, 150rpm shakes
After swinging cultivation 2 days, 4 DEG C of centrifugal thalline of collecting, after washing 3 times with 0.1mol/L Tris-HCl buffer (pH=7.6), resuspended
Float in 600 μ l 0.1mol/L Tris-HCl buffer (pH=8.0), add 30 μ l toluene rapid oscillation 30s with broken
Cell.Draw above-mentioned cell extract 200 μ L, add 20 μ l 0.5mol/L ACC mixings, do the blank without ACC simultaneously
Test, 30 DEG C of water bath with thermostatic control 15min.Adding 1ml 0.56mol/L HCl, 16000g is centrifuged 5min, takes this suspension of 1ml, adds
Enter 800 μ l 0.56mol/L HCl and 300 μ l 2,4 dinitrophenyl hydrazines, 30 DEG C of water bath with thermostatic control 30min, add 2ml 2mol/L
NaOH, 540nm survey absorbance.
Result sees accompanying drawing 2, and Halomonas (Halomonas sp.) T5S2 all can produce in three kinds of culture medium of experiment
Acc deaminase, wherein in TSB culture medium, single enzymatic productivity is better than LB, ADF culture medium, and maximum enzyme work, up to 0.25U/mg, shows
This bacterium can produce acc deaminase in the simple culture medium being suitable for strain growth, and without induction.
Embodiment three: Halomonas (Halomonas sp.) T5S2CGMCC No.12642 produces IAA feelings at different culture media
Condition
Bacterial strain T5S2 is inoculated in respectively in LB, the TSB containing 1%NaCl and product heteroauxing (lAA) culture medium, 30
DEG C, 150rpm shaken cultivation is after 2 days, centrifuging and taking 1ml supernatant, adds the orthophosphoric acid of 50 μ l 10mmol/L, then adds 2ml
Salkowski ' s developer, dark lower 25 DEG C of colour developing 30min, measure the light absorption value under 530nm wavelength.Training is replaced with distilled water
Nutrient solution is reacted equally as comparison zeroing, makees standard curve with the IAA titer of variable concentrations with method, calculates IAA in fermentation liquid
Concentration.
Result sees accompanying drawing 3, and t bacteria 5S2 all can produce IAA in three kinds of culture medium of experiment, wherein produces IAA culture medium
Yield is higher than LB, TSB culture medium containing 1%NaCl, and maximum production can reach 259mg/L, shows that this bacterium can be simply
It is suitable in the culture medium of strain growth producing IAA, utilizes for later development and lay a good foundation.
Embodiment four: Halomonas (Halomonas sp.) T5S2CGMCC No.12642 prepares the side of microbial manure
Method
Halomonas (Halomonas sp.) T5S2CGMCC No.12642 is utilized to prepare microbial manure concrete steps such as
Under:
Halomonas (Halomonas sp.) T5S2CGMCC No.12642 is inoculated in LB solid slant culture base, 33
DEG C cultivate 3d, i.e. obtain slant strains.Slant tube adds after 5mL sterilized water acutely vibrates, uses dual-layer sterilization filtered through gauze,
Filtrate is prepared as containing 10 through suitably dilution6Individual spore suspension.Draw above-mentioned spore suspension 1mL, be inoculated in the training of LB liquid
Support in base, 33 DEG C, 150rpm shaken cultivation 3d, obtain seed liquor.
(1) preparation method of liquid microbe fertilizer: draw above-mentioned seed liquor, is inoculated in fermentation culture by 3% inoculum concentration
Base, 33 DEG C, 150rpm cultivate 6d, results thalline and fermentation liquid；According to fermentation medium in every 100ml fermentation medium it is, egg
White peptone 5g, yeast extract powder 2g, glucose 20g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, distilled water 1000mL, pH 6.6.
(2) preparation method of solid microbe fertilizer: draw above-mentioned seed liquor, is inoculated in solid fermentation by 3% inoculum concentration
Culture medium, 33 DEG C of solid fermentation 6d, cover with solid medium to mycelia, i.e. obtain solid fermentation microbial inoculum；Solid fermentation culture medium
For, pulverize cotton stalk 65%, wheat bran 20%, cotton seed hulls 13%, sucrose 1%, CaCO31%, then add tap water and fully stir evenly,
Controlling final water content is 60%.
In the present invention, when the liquid microbe fertilizer using above-mentioned offer to prepare, the basic bacterium present invention can prepared
Body fermentation liquid is directly as liquid microbe Fertilizer application, it is also possible to use after mixing with other nutritional solutions.
In the present invention, when the solid microbe fertilizer using above-mentioned offer to prepare, can be by the basis of the preparation of the present invention
Solid fermentation microbial inoculum is directly as liquid microbe Fertilizer application, it is also possible to use after mixing with other fertilizers.
Embodiment five: Halomonas (Halomonas sp.) T5S2CGMCC No.12642 prepares microbial manure at salt
Application in alkali ground cotton planting
In the Different Crop of salt-soda soil plantation, design matched group, compare the Halomonas that the present invention provides
(Halomonas sp.) T5S2CGMCC No.12642 prepares microbial manure effect in terms of improving plant growth characteristic.
(1) application in alkaline land cotton plantation:
Test arrangement is 3 mu in farmland, Manas County, area, Changji 16, testing ground area, and cotton variety selects " middle cotton
Institute 41 ".Manas County 16 agricultural land soil salt content is 1.9%, and pH is 8.5, and soil salt type is sulfate-chlorination
Species type, soil organism 11.5g/kg, alkali-hydrolyzable nitrogen 72mg/kg, rapid available phosphorus 12mg/kg, available potassium 185mg/kg, soil fertility
Medium.
EXPERIMENTAL DESIGN standing grain health group, test group and comparison blank group totally three groups, test group uses above-described embodiment four offer
The microbial manure prepared of Halomonas (Halomonas sp.) T5S2CGMCC No.12642 process, standing grain health group is adopted
Processing with standing grain health fertilizer, comparison blank group is not used any fertilizer and is used conventional cultivation processing mode.Test group and standing grain health group
Apply fertilizer with cotton growing stage the most prior to seeding.Deep placement solid fertilizer 30-40cm before cotton culture is sowed, dose 50kg/
Mu, the whole trophophase of Cotton Gossypii use drip irrigation apply Liquid Fertilizer, Irrigation Frequency be 30 days once, irrigation volume is 40m3/ mu, test
If 3 times are repeated.
10 days statistics germination percentages of cotton seed and survival rate after Cotton Gossypii emerges, in the cotton boll blowing phase, every community diagonal 3 point
Sampling, strain number, Bolls per plant and the plant height of statistical unit area, result of the test sees table 1.
Table 1: different fertilization index each on Cotton Gossypii affect result
| Germination percentage (%) | Survival rate (%) | Plant height (cm) | Strain number (ten thousand strains/hm<sup>2</sup>) | Single basal munure (individual) | |
| Test group | 77.8 | 65.4 | 62.3 | 24.8 | 5.9 |
| Standing grain health group | 66.1 | 58.5 | 59.5 | 23.2 | 5.4 |
| Matched group | 50.3 | 47.6 | 58.6 | 20.85 | 4.8 |
As shown in Table 1, the process of test group and standing grain health group all can promote the growth of Cotton Gossypii, especially applies above-described embodiment
The test group of four microbial manures provided, effect of increasing production is obvious, and its germination percentage, survival rate, plant height, strain number and single bell strain number are equal
It is higher than and standing grain health group.By the microbial manure using above-described embodiment four to provide, it is possible to ensure that the germination percentage of Cotton Gossypii reaches
77.8%, survival rate to 65.4%, plant height, strain number and single bell strain number will be apparently higher than conventional treatment Cotton Gossypiis.
The biggest obstacle of general planting Cotton Gossypii is exactly that its survival rate is the highest, and along with irrigation, its survival rate declines, and irrigates one
Secondary, Cotton Gossypii death 2-5%, and the application utilizes Halomonas (Halomonas sp.) the T5S2CGMCC No.12642 of offer
Preparing microbial manure and can improve Cotton Gossypii emergence rate, reduce this risk, the plantation for alkaline land cotton has extensively
Practicality.
Application in the corn planting of salt-soda soil:
Test arrangement tests farmland in Xinjiang Agricultural Univ, and testing ground area is 2 mu, corn variety selection " new beautiful No. 62,
This agricultural land soil salt content is 1.7%, and pH is 8.0, and soil salt type is sulfate-chloride type, the soil organism
12.1g/kg, alkali-hydrolyzable nitrogen 85mg/kg, rapid available phosphorus 15mg/kg, available potassium 136mg/kg, soil fertility is medium.
EXPERIMENTAL DESIGN test group and matched group, test group uses the Halomonas (Halomonas that above-described embodiment four provides
Sp.) microbial manure prepared by T5S2CGMCC No.12642 processes, and matched group uses conventional treatment mode.Test group
Deep placement microbial solid fertilizer 30-40cm before corn seeding, dose 25kg/ mu, use drip irrigation to execute at the whole trophophase of Semen Maydis
Add Liquid Fertilizer, Irrigation Frequency be 7 days once, irrigation volume is 30m3/ mu, test sets 3 repetitions, and result of the test sees table 2.
Table 2: different fertilization index each on Semen Maydis affect result
| Emergence rate (%) | Survival rate (%) | |
| Test group | 80.8 | 78 |
| Matched group | 64.35 | 58.5 |
As seen from the experiment, during the microbial manure using above-described embodiment four to provide, alkaline land soil is no longer
Period of duration earth's surface accumulation of salt in the surface soil problem occur, in period of duration, 0-30cm soil layer soil total salt content have dropped 60-70%, salt-soda soil Semen Maydis
Emergence rate all increases to more than 80% from 64.5%, and per mu yield Semen Maydis reaches 600kg/ mu.
From above-mentioned alkaline land cotton and Semen Maydis application effect test it can be seen that the application of the invention provide salt
Zymomonas mobilis (Halomonas sp.) T5S2CGMCC No.12642 prepares corresponding microbial manure, not only significantly improves saline and alkaline
The growth characteristics such as ground crop crop emergence rate, survival rate, and ensure that alkaline land soil no longer occurs that the period of duration earth's surface accumulation of salt in the surface soil is asked
Topic.By selecting other to apply the present invention to carry such as the industrial crops such as Herba Apocyni veneti, Helianthi and Radix Betae, fruit tree such as Fructus Mali pumilae, fragrant pear etc.
The microbial manure of confession all has obvious salt reduction production-increasing function, can effectively drop soil salt content simultaneously.
Halomonas (Halomonas sp.) the T5S2CGMCC No.12642 provided by above-mentioned series embodiment is one
Plant typical novel bacterial, be resistant to the NaCl of 1-15% concentration, at 30 DEG C of-37 DEG C of well-growns, need not lure in growth course
Lead and can produce acc deaminase and IAA heteroauxing, there is condition of culture simple, breed fast feature.By Halomonas
The microbial manure that (Halomonas sp.) T5S2CGMCC No.12642 is obtained by conventional fermentation techniques, is applied to saline and alkaline
In the ground industrial crops such as Cotton Gossypii, Semen Maydis, it is possible to ensureing that the germination percentage of alkaline land cotton reaches 77.8%, survival rate arrives
65.4%, plant height, strain number and single bell strain number will be apparently higher than conventional treatment Cotton Gossypiis, and salt-soda soil corn emergence rate all increases from 64.5%
Being added to more than 80%, per mu yield Semen Maydis reaches 600kg/ mu so that 0-30cm soil layer soil total salt content have dropped 60-70%, has
Significantly salt reduction production-increasing function, is applied to the warps such as Herba Apocyni veneti, Helianthi and Radix Betae by the microbial manure that the present invention provides simultaneously
In Ji crop, all there is obvious salt reduction production-increasing function, it is seen then that utilize the Halomonas novel species Halomonas that the present invention provides
(Halomonas sp.) T5S2CGMCC No.12642 prepares the exploitation application of preparation and has the biggest application prospect.Meanwhile,
Product of the present invention can be administered alone, and has good growth promoting function, it is possible to carries out joining executing with fertilizer, is improving the same of soil nutrient
Time reduce the salinity of soil, application process is various, and Field information is easy, it is possible to take into account environmental friendliness and yield promotes.
Above-described embodiment is only for clearly demonstrating example of the present invention, and not restriction to embodiment.
For those of ordinary skill in the field, the change of other multi-form can also be made on the basis of the above description
Or variation.Here without also cannot all of embodiment be given exhaustive.And the obvious change thus extended
Or change among still in protection scope of the present invention.
SEQUENCE LISTING
<110>Xinjiang Shanchuanxiuli Biology Co., Ltd
<120>a kind of salt tolerant antibacterial T5S2 and the application in microbial manure thereof
<130> T5S2
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1421
<212> DNA
<213> T5S2
<220>
<221> T5S2
<222> (1)..(1421)
<400> 1
gctaccatgc agtcgagcgg taacaggggg agcttgctcc ctgctgacga gcggcggacg 60
ggtgagtaat gcataggaat ctgcccggta gtgggggata acctggggaa acccaggcta 120
ataccgcata cgtcctacgg gagaaagcag gggaccttcg ggccttgcgc tatcggatga 180
gcctatgtcg gattagctgg ttggtgaggt aatggctcac caaggcaacg atccgtagct 240
ggtctgagag gatgatcagc cacatcggga ctgagacacg gcccgaactc ctacgggagg 300
cagcagtggg gaatattgga caatgggggc aaccctgatc cagccatgcc gcgtgtgtga 360
agaaggcctt cgggttgtaa agcactttca gtgaggaaga aggccttggg gctaataccc 420
tcgaggaagg acatcactca cagaagaagc accggctaac tccgtgccag cagccgcggt 480
aatacggagg gtgcgagcgt taatcggaat tactgggcgt aaagcgcgcg taggtggctt 540
gataagccgg ttgtgaaagc cccgggctca acctgggaac ggcatccgga actgtcaggc 600
tagagtgcag gagaggaagg tagaattccc ggtgtagcgg tgaaatgcgt agagatcggg 660
aggaatacca gtggcgaagg cggccttctg gactgacact gacactgagg tgcgaaagcg 720
tgggtagcaa acaggattag ataccctggt agtccacgcc gtaaacgatg tcgactagcc 780
gttgggtccc tcgcggactt tgtggcgcag ttaacgcgat aagtcgaccg cctggggagt 840
acggccgcaa ggttaaaact caaatgaatt gacgggggcc cgcacaagcg gtggagcatg 900
tggtttaatt cgatgcaacg cgaagaacct tacctaccct tgacatcctc ggaatctgtc 960
ggagacggcg gagtgccttc gggaaccgag tgacaggtgc tgcatggctg tcgtcagctc 1020
gtgttgtgaa atgttgggtt aagtcccgta acgagcgcaa cccttgtccc tatttgccag 1080
cgattcggtc gggaactcta gggagactgc cggtgacaaa ccggaggaag gtggggacga 1140
cgtcaagtca tcatggccct tacgggtagg gctacacacg tgctacaatg gccggtacaa 1200
agggttgcga agccgcgagg tggagctaat cccgaaaagc tggtctcagt ccggatcgga 1260
gtctgcaact cgactccgtg aagtcggaat cgctagtaat cgtgaatcag aatgtcacgg 1320
tgaatacgtt cccgggcctt gtacacaccg cccgtcacac catgggagtg gactgcacca 1380
gaagtggtta gcctaacttc ggagggcgat caccacgagg t 1421
Claims (4)
1. a Halomonas (Halomonas sp.) T5S2, it is characterised in that described Halomonas (Halomonas sp.) culture presevation number of T5S2 is CGMCC No.12642.
2. one kind utilize Halomonas (Halomonas sp.) T5S2 CGMCC No.12642 prepares liquid microbe fertilizer
Method, concrete grammar is as follows:
(1) by Halomonas (Halomonas sp.) T5S2 CGMCC No.12642 is inoculated in LB solid slant culture base, 33
DEG C cultivate 3d, i.e. obtain slant strains；Slant tube adds after 5 mL sterilized water acutely vibrate, use dual-layer sterilization gauze mistake
Filter, filtrate is prepared as containing 10 through dilution6Individual spore suspension；Draw above-mentioned spore suspension 1mL, be inoculated in LB liquid
In culture medium, 33 DEG C, 150rpm shaken cultivation 3d, obtain seed liquor；
(2) preparation method of liquid microbe fertilizer: draw above-mentioned seed liquor, is inoculated in fermentation medium by 3% inoculum concentration, and 33
DEG C, 150rpm cultivate 6d, results thalline and fermentation liquid, prepare corresponding liquid microbe fertilizer；The fermentation culture used
Base is: according to peptone 5g in every 100ml fermentation medium, yeast extract powder 2g, glucose 20g, dipotassium hydrogen phosphate 1g,
Magnesium sulfate 0.5g, distilled water 1000mL, pH 6.6.
3. one kind utilize Halomonas (Halomonas sp.) T5S2 CGMCC No.12642 prepares solid microbe fertilizer
Method, concrete grammar is as follows:
(1) by Halomonas (Halomonas sp.) T5S2 CGMCC No.12642 is inoculated in LB solid slant culture base, 33
DEG C cultivate 3d, i.e. obtain slant strains, slant tube adds after 5 mL sterilized water acutely vibrate, use dual-layer sterilization gauze mistake
Filter, filtrate is prepared as containing 10 through suitably dilution6Individual spore suspension, draws above-mentioned spore suspension 1mL, is inoculated in LB
In fluid medium, 33 DEG C, 150rpm shaken cultivation 3d, obtain seed liquor；
(2) drawing above-mentioned seed liquor, be inoculated in solid fermentation culture medium by 3% inoculum concentration, 33 DEG C of solid fermentation 6d, to mycelia length
Full solid medium, obtains solid fermentation microbial inoculum, prepares corresponding solid microbe fertilizer；The solid fermentation used is cultivated
Base according to weight ratio proportioning is: pulverize cotton stalk 65%, wheat bran 20%, cotton seed hulls 13%, sucrose 1%, CaCO31%, then add from the beginning
Water fully stirs evenly, and controlling final water content is 60%.
4. the microbial manure prepared such as claim 1,2 or 3 any one.
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| CN106857674A (en) * | 2017-02-09 | 2017-06-20 | 河北省科学院生物研究所 | Application of bacillus methylotrophicus BH21, microbial fertilizer and preparation method thereof |
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| CN113897305A (en) * | 2021-09-10 | 2022-01-07 | 中国烟草总公司贵州省公司 | Shewanella subsp and application thereof |
| CN114686391A (en) * | 2020-12-31 | 2022-07-01 | 中国石油化工股份有限公司 | High-salinity-tolerance bacterium and application thereof |
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| CN106857674B (en) * | 2017-02-09 | 2019-02-26 | 河北省科学院生物研究所 | Application of bacillus methylotrophicus BH21, microbial fertilizer and preparation method thereof |
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| WO2020107760A1 (en) * | 2018-11-30 | 2020-06-04 | 江苏大学 | Moderately halophilic bacteria and method for fermenting fish meat sauce by using same |
| CN109355232B (en) * | 2018-11-30 | 2021-06-29 | 乌鲁木齐永丰天农农业科技发展有限公司 | Termite fungus IS7 and application thereof in plant growth promotion |
| CN109355232A (en) * | 2018-11-30 | 2019-02-19 | 乌鲁木齐永丰天农农业科技发展有限公司 | A kind of termite bacterium IS7 and its application in plant growth-promoting |
| US11974590B2 (en) | 2018-11-30 | 2024-05-07 | Jiangsu University | Method of fermentation of fish paste by medium halophilic bacteria |
| CN109486709B (en) * | 2018-12-03 | 2019-07-02 | 山东农业大学 | One plant of Fan Shi Halomonas bacterial strain JPT10-1 for improving corn salt resistance ability and its microbial inoculum and application |
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| CN114686391A (en) * | 2020-12-31 | 2022-07-01 | 中国石油化工股份有限公司 | High-salinity-tolerance bacterium and application thereof |
| CN114686391B (en) * | 2020-12-31 | 2023-07-04 | 中国石油化工股份有限公司 | High-salt-tolerance bacterium and application thereof |
| CN113897305A (en) * | 2021-09-10 | 2022-01-07 | 中国烟草总公司贵州省公司 | Shewanella subsp and application thereof |
| CN113897305B (en) * | 2021-09-10 | 2024-04-09 | 中国烟草总公司贵州省公司 | Isowa and application thereof |
| CN115161217A (en) * | 2022-04-19 | 2022-10-11 | 中农新科(苏州)有机循环研究院有限公司 | Salt-tolerant degradable organic macromolecular substance waste salt field branch bacillus H83 and preparation and application of microbial inoculum thereof |
| CN115161217B (en) * | 2022-04-19 | 2024-02-09 | 中农新科(苏州)有机循环研究院有限公司 | Waste salt Tian Zhi bacillus H83 of salt-resistant degradable organic macromolecular substance and preparation and application of microbial inoculum thereof |
| CN117363523A (en) * | 2023-10-11 | 2024-01-09 | 石河子大学 | Salmonella ZH-1 and application thereof |
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Effective date of registration: 20230109 Address after: 830011 the Xinjiang Uygur Autonomous Region Urumqi city Beijing Road No. 11 Diamond City JETCO building 12 floor Patentee after: XINJIANG SHANCHUANXIULI BIOLOGICAL Co.,Ltd. Address before: 271600 No. 338, Mazhuang village, Wangguadian Town, Feicheng City, Tai'an City, Shandong Province Patentee before: Hu Xuyan Patentee before: Chen Yuanfen Patentee before: Li Tianquan Patentee before: Zhou Weihong |