CN104195082B - A kind of travelling germ and its microbial inoculum and preparation and application - Google Patents
A kind of travelling germ and its microbial inoculum and preparation and application Download PDFInfo
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Abstract
The present invention relates to a kind of travelling germ and its microbial inoculum and preparation and application, the travelling germ of the bacterial strain belongs to bacterial strain G14 7（Planomicrobiumsp. G14‑7）It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No.9602.Microbial bacterial agent its active component of the present invention isPlanomicrobiumsp. G14‑7.What the present invention was providedPlanomicrobiumSp. G14 7 and its microbial inoculum can significantly improve the salt resistance ability of crop, can be used with the microbial inoculum that this technology is produced in salt affected soil, improve the growth of the salt resistance ability and crop of common crops in salt affected soil.
Description
Technical field
The present invention relates to a kind of travelling germ category (Planomicrobium) bacterium, further relate to utilize and be somebody's turn to do
The microbial bacterial agent of Planomicrobium bacteriums production, and the Planomicrobium bacteriums are improving crop with microbial inoculum
Application in salt resistance ability.
Background technology
Saline-alkali soil refers to the soil of soil salt content too high (more than 0.3%), and the salinization of soil of soil can increase agricultural production
Cost, even results in soil and falls into disuse.According to statistics, about 5.27 hundred million mu of the saline alkali land resource area of China, wherein Saline 0.88
Hundred million mu, it is mainly distributed on North China, northwest and the Northeast.The salinization of soil makes China cause substantial amounts of grain drop in production, agriculture every year
Grain drop in production not only causes serious economic loss caused by the salinization of soil of field, and the also grain security to country is constituted sternly
Threaten again.
Currently for this problem, people mainly first improve saline-alkali soil using the method for various physics, chemistry or biology
The physicochemical property of earth, reduction soil salt content etc., realize the utilization of wetland with saline-alkaline.Administer alkaline land improving measure have physics,
Chemistry and biological and agricultural tillage measure etc., including water conservancy ameliorative measure (irrigation, draining, warp, plant rice, antiseepage etc.)；Agriculture
Industry ameliorative measure (is leveled land, improves farming, applies soil moved in improve the original, fertilising, sowing, crop rotation, kind interplanting etc.)；Biological modification measure
(plantation salt-tolerant plant and herbage, green manure, afforestation etc.)；Chemical modifying measure (apply upgrade materials, such as gypsum, ardealite,
Calcium sulfite etc.), and improve soil property etc. using the fertilizer for the microbial strains formation screened from soil.
It was found that in the plant of nature or rhizosphere has substantial amounts of bacterium, fungi and actinomyces, these bacterial strains are not
But not cause the disease of host plant, the anti-adverse environmental factor of plant itself can be improved by the interaction with plant on the contrary
Including being resistant to saline and alkaline ability.These microbial resources are developed to have important practical significance to industrial and agricultural production.
The content of the invention
The salinization of soil of soil still lacks effective means to administer, and technical problem solved by the invention is by microorganism
To improve the salt resistance ability of common crops, the utilization of salinized soil is realized.
It is of the invention main out of, plant with relatively strong saline and alkaline tolerance, screen itself salt resistance ability by force and can carry
The bacterium of high crop salt tolerant alkali ability, to improve the salt resistance ability of common crops.The endophytic bacterium of this research can be
Plant rhizosphere forms mutualism relation with plant, and low using bacterium amount, easy to operate, cost is low.
One of inventive point of the present invention is to provide a kind of has better effects to improving salt tolerance of crops
Planomicrobium belongs to bacterium.
The two of the inventive point of the present invention are to provide a kind of microbial bacteria produced using above-mentioned Planomicrobium bacteriums
Agent.
The three of the inventive point of the present invention are to provide above-mentioned Planomicrobium category bacteriums and microbial inoculum is improving crop tolerance to salt
The application process of ability.
Specifically, the present invention is achieved through the following technical solutions：
First, a kind of travelling germ of present invention offer belongs to bacterial strain G14-7 (Planomicrobium sp.G14-7), preservation
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.9602.
In addition, the present invention, which also provides travelling germ, belongs to applications of the bacterial strain G14-7 in salt tolerance of crops is improved.
Wherein, described travelling germ belongs to applications of the bacterial strain G14-7 in salt tolerance of crops is improved, it is characterised in that
The salt affected soil refers to the soil that concentration containing soluble-salt is 0~2.0%.
The present invention also provides a kind of microbial bacterial agent, it is characterised in that it belongs to bacterial strain G14-7 using above-mentioned travelling germ
Production is obtained, and its active component is that the travelling germ described in claim 1 belongs to bacterial strain G14-7.
Wherein, in the microbial bacterial agent of the present invention, it is 1,000,000,000 that it, which contains described dynamic property Bacillus genus strain G14-7,
Individual/more than ml, the microbial inoculum belongs to bacterial strain G14-7 using described travelling germ and fermentation medium production is obtained, the fermentation training
Foster base contains following component in mass：Glucose 0.7~1.0%, (NH4)2SO40.8~1.2%, K2HPO40.1~0.3%,
MgSO40.03~0.07%, NaCl0.005~0.015%, CaCO30.2~0.4%, dusty yeast 0.01~0.03%, remaining is
Water, pH value 7.2~7.5.
In addition, the present invention also provides application of the described microbial bacterial agent in salt tolerance of crops is improved.
Wherein, the application of described microbial bacterial agent of the invention in the salt resistance ability for improving crop, it is characterised in that
The salt affected soil refers to the soil that concentration containing soluble-salt is 0~2.0%.
In addition, the present invention provides the preparation method of described microbial bacterial agent, it is characterised in that it passes through including following steps
Rapid preparation method is obtained：
1) inclined-plane kind culture：Test tube slant will be inoculated in after dynamic property Bacillus genus strain G14-7 activation described in claim 1
On；
2) shaking flask kind culture：Step 1) obtained test tube strains be inoculated in broth bouillon, constant-temperature shaking culture is extremely
Exponential phase；
3) seeding tank inoculated and cultured：By step 2) obtained shaking flask strain is inoculated in seeding tank according to 10% volume ratio and trains
Support to exponential phase；
4) tank culture is produced：By step 3) obtained seeding tank strain is inoculated in production tank by 10% volume ratio, temperature
Control is cultivated at 30-35 DEG C, obtains microbial inoculum.
Specifically, the Planomicrobium sp.G14-7 that the present invention is provided, are preserved on the 26th in August in 2014
China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.9602.
The Planomicrobium sp.G14-7 of present invention morphological feature：Thalline is spherical.
The Planomicrobium sp.G14-7 of present invention physiological and biochemical property：Gram positive；Contact
Enzyme positive, does not produce gemma, urase reaction negative；In LB solid mediums, (composition is as follows：Dusty yeast 5g/L, peptone 10g/L,
On NaCl10g/L, agar powder 20g/L, pH7.0~7.2) after growth 3 days, diameter about 0.3mm, red, opaque, surface are formed
The circular colonies of smooth moistening.
The invention also discloses the microbial bacterial agent produced using above-mentioned Planomicrobium sp.G14-7, its activity
Composition is Planomicrobium sp.G14-7 thalline.
The preparation method of the microbial bacterial agent is as described below：
Production microbial inoculum technique be：Inclined-plane kind-shaking flask kind-seeding tank-production tank-product, the packaging of the product
Formulation is liquid bacterial agent or solid absorption microbial inoculum.
In a preferred embodiment, the preparation of the microbial inoculum can specifically use following steps：
1st, by bacterial strain G14-7 (original seed), in high salt LB culture mediums, (composition is as follows：Dusty yeast 5g/L, peptone 10g/L,
Cultivated on the flat board of NaCl160g/L, agar powder 20g/L, pH7.0~7.2), it is standby.
2nd, the single bacterium colony on picking high salt LB culture medium flat plates is inoculated in 100ml broth bouillons (beef extract 3.0g/L, egg
White peptone 10.0g/L, NaCl5g/L, remaining is water, and in pH value 7.0~7.2), constant-temperature shaking culture prepares to connect to exponential phase
Plant seeding tank.
3rd, fermentation medium is prepared, its component and percentage by weight are：Glucose 0.8%, (NH4)2SO41.0%th,
K2HPO40.2%th, MgSO40.05%th, NaCl0.01%, CaCO30.3%th, dusty yeast 0.02%, remaining is water, pH value 7.2~
7.5,400L fermentation mediums are added into 500L seeding tanks, 121 DEG C of high pressure moist heat sterilizations are cooled to after 30 DEG C, by shaking flask strain
It is inoculated with by the inoculum concentration of 10% (volumn concentration) into seeding tank, culture to exponential phase, mixing speed is 220 revs/min,
Filtrated air intake is 1：0.8 (volume ratio of air and nutrient solution).
4th, production tank (5 tons of tankage size of production) used medium composition is identical with fermentation medium (4.5 tons of inventory), throws
Production tank after material is in 1.1kg/cm2Pressure under, 121 DEG C of high pressure moist heat sterilizations are cooled to less than 30 DEG C after sterilizing, lead to it is sterile
Air keeps germ-free condition standby；The seed liquor for reaching logarithmic phase is accessed by the inoculum concentration of 10% (volumn concentration) and produced
Tank, the production tank temperature control after inoculation is at 30~35 DEG C, and the throughput for producing filtrated air in the incubation of tank is 1：
(0.6~1.2) (volume ratio of air and nutrient solution), mixing speed is 180~240 revs/min (being preferably 240 revs/min), whole
Individual technological process incubation time is 48~60 hours；Thalline quantity reaches 1,000,000,000/more than ml after fermentation ends.
5th, nutrient solution goes out tank and is directly distributed into liquid dosage form with plastic barrel or Packaging Bottle or uses mud after the completion of fermenting
Charcoal absorption is distributed into solid fungicide formulation with packaging bag.
The Planomicrobium sp.G14-7 and its microbial inoculum that the present invention is provided can be in itself 16% in saliferous (NaCl) amount
The culture medium such as LB culture mediums in grow, in the aqueous solution containing 0.5%, 1.0% and 2.0% salt (NaCl), can significantly carry
High corn seed germination rate；Seed is put into after being soaked 4 hours in microbial inoculum, planted in the soil for being 1% to salt content, Ke Yixian
The germination percentage for improving seeding corn and other crops is write, and significantly improves the biomass of crop；Bacterium is used in the soil containing 2% soluble salt
Strain, can improve the survival rate of seeding corn and other crops.The microbial bacterial agent that the present invention can improve salt tolerance of crops makes with production
With low, the easy to use advantage of cost, the salt resistance ability of seeding corn and other crops can be significantly improved.Bacterial strain and bacterium that the present invention is provided
Agent can be used as microbial manure in salinization of soil farmland, improve germination percentage of the crop in salt affected soil, biomass or
Grain yield.The planting range of seeding corn and other crops can be expanded using the microbial inoculum, the cultivated area of crop is improved.Present invention success
The problems such as ground solves grain drop in production caused by the common crops salt resistance ability such as corn is poor in salinized soil, reduces
Workload during production and use, reduces production and use cost, for safeguarding national food security, improves salinization of soil
The utilization ratio in soil is significant.
Preservation information
The Planomicrobium sp.G14-7 that the present invention is provided, are preserved in Chinese microorganism strain preservation conservator
Can common micro-organisms center (CGMCC), address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism is ground
Study carefully institute, postcode 100101；Deposit number is CGMCC No.9602, and preservation date is August in 2014 26.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used in following embodiments, is certainly unless otherwise specified
Routine biochemistry reagent shop is bought, specific as shown in table 1.Corn seed used is ordinary maize seed.Experiment is respectively provided with 3 below
Secondary repetition, results averaged.Signified salt content in following examples, is weight/mass percentage composition unless otherwise specified, institute
The salt of finger is NaCl.
The commercial source information table of each reagent of table 1
Embodiment 1：Planomicrobium sp.G14-7 acquisition and identification
First, Planomicrobium sp.G14-7 acquisition
Take in June, 2013 from one plant of Inner Mongol Ulansuhai Nur periphery Suaeda heteroptera plant, Suaeda heteroptera root system be cut to after several sections,
Surface sterilization is done with sodium hypochlorite, 70% ethanol, after testing after surface sterile, is pulverized with sterile mortar, plant tissue is collected residual
Slag, is coated with after being diluted with sterilized water on the LB culture medium flat plates containing 2.0% salinity, after cultivating 7 days, the line of picking single bacterium colony
Preserve after purification.
LB medium components containing 2.0% salinity：Dusty yeast 5.0g, peptone 10.0g and NaCl20.0g, agar powder
20g, 1L is settled to water, and pH is 7.0 or so.
2nd, Planomicrobium sp.G14-7 identification
(1) morphological feature：Thalline is spherical.
(2) physiological and biochemical property：Gram positive；After LB medium cultures 3 days, formed a diameter of
0.3mm, red, the circular colonies of the smooth moistening in opaque, surface.
(3) 16S rDNA Sequence Identifications
Using following primer：8F (5 '-AGAGTTTGATCCTGGCTCAG-3 '), 1492 (5 '-TACCTTGTTA
CGACTT-3 '), enter performing PCR amplification, expanded from bacterial strain G14-7 to 16SrDNA genetic fragments, by obtained 16S rDNA gram
Beijing Bo Aiyuanshang bio tech ltd is sent to be sequenced after the grand T-easy carriers to pGEM.16S rDNA partial sequence is shown in
The SEQ ID No of sequence table：Base sequence shown in 1, finds, G14-7 16S rDNA sequences with the sequence alignment in GenBank
Row belong to the homology highest of strain sequence with Planomicrobium, reach 99.8%.
3rd, Planomicrobium sp.G14-7 preservation
By above qualification result, it is the new bacterium for coming from Planomicrobium category to confirm above-mentioned bacterial strains, is named
For Planomicrobium sp.G14-7, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved in, is protected
Tibetan number is CGMCC No.9602.
Embodiment 2：Planomicrobium sp.G14-7 improve hair of the corn seed in the aqueous solution of different salinity
Bud rate
Picking Planomicrobium sp.G14-7 single bacteriums are fallen within 3ml LB fluid nutrient mediums, and 30 DEG C, 165 revs/min are shaken
Culture 24 hours is swung, fresh bacterium solution is obtained.By the inoculum concentration of 2% (volume ratio) be inoculated into 200mL fermentation mediums (its component and
Percentage by weight is：Glucose 0.8%, (NH4)2SO41%th, K2HPO40.2%th, MgSO40.05%th, NaCl 0.01%,
CaCO30.3%th, dusty yeast 0.02%, remaining is water, pH value 7.2~7.5) middle expansion culture, 120r/min, 30 DEG C of cultures 48
Hour.
Corn seed of uniform size is divided into two groups, one group is placed in inoculation immersion 4 hours, another group of use in fresh bacterium solution
After above-mentioned fermentation medium soaks 4 hours, the corn seed after immersion is respectively placed in containing 0.5% after drying naturally, 1.0%,
On the filter paper of 2.0% salt solution, then seed is put in lucifuge quiescent culture in 28 DEG C of incubator respectively, it is observed at 7 days beautiful
The germination of rice seed.
The germination percentage of corn=(germination corn seed quantity/Tests Seeds quantity) × 100%.
As a result (table 2) shows, the corn seed after bacterial strain immersion is in the aqueous solution of 0.5~2.0% salt (NaCl) concentration
Can fast germination, its germination percentage is apparently higher than the corn seed for not being inoculated with Planomicrobium sp.G14-7.
Germination percentage of the table 2 with Planomicrobium sp.G14-7 seed soaking to corn in the salting liquid of various concentrations
Influence
Embodiment 3：Planomicrobium sp.G14-7 improve biomass of the corn in salty soil, survival rate
Picking Planomicrobium sp.G14-7 single bacteriums are fallen within 3ml LB fluid nutrient mediums, and 30 DEG C, 165 revs/min are shaken
Culture 24 hours is swung, fresh bacterium solution is obtained.By the inoculum concentration of 2% (volume ratio) be inoculated into 200mL fermentation mediums (its component and
Percentage by weight is：Glucose 0.8%, (NH4)2SO41%th, K2HPO40.2%th, MgSO40.05%th, NaCl0.01%,
CaCO30.3%th, dusty yeast 0.02%, remaining is water, pH value 7.2~7.5) middle expansion culture, 120r/min, 30 DEG C of cultures 48
Hour.
Corn seed of the same size is chosen, is put into G14-7 fresh bacterium solution and soaks 4 hours, control seed is trained in LB
Support in base and soak 4 hours.Then seed is seeded into respectively in the soil that soluble-salt concentration is 0.5%, 1.0%, 2.0%
In.Basin alms bowl is put under sunlight and cultivated, regular watering maintains soil moisture content 20%.The growth feelings of corn are observed at 10 days
Condition.
As a result (table 3) shows, the speed of growth of the corn seed after bacterial strain immersion in the salty soil of 2.0% salinity
It is significantly faster than that the corn seed for not being inoculated with Planomicrobium；In the soil of 2.0% salinity, the not soil of inoculating strain
Corn can not grow in earth, and corn can grow after soaking seed.
Table 3Planomicrobium sp.G14-7 are improving biomass of the corn in salty soil
Embodiment 4
Microbial bacterial agent is produced using above-mentioned Planomicrobium sp.G14-7
1st, by bacterial strain G14-7 (original seed), in high salt LB culture mediums, (composition is as follows：Dusty yeast 5g/L, peptone 10g/L,
Cultivated on the flat board of NaCl160g/L, agar powder 20g/L, pH7.0~7.2), it is standby.
2nd, the single bacterium colony on picking high salt LB culture medium flat plates is inoculated in 100ml broth bouillons (beef extract 3.0g/L, egg
White peptone 10.0g/L, NaCl5g/L, remaining is water, and in pH value 7.0~7.2), constant-temperature shaking culture prepares to connect to exponential phase
Plant seeding tank.
3rd, fermentation medium is prepared, its component and percentage by weight are：Glucose 0.8%, (NH4)2SO41%th,
K2HPO40.2%th, MgSO40.05%th, NaCl0.01%, CaCO30.3%th, dusty yeast 0.02%, remaining is water, pH value 7.2~
7.5,400L fermentation mediums are added into 500L seeding tanks, 121 DEG C of high pressure moist heat sterilizations are cooled to after 30 DEG C, by shaking flask strain
It is inoculated with by the inoculum concentration of 10% (volumn concentration) into seeding tank, culture to exponential phase, mixing speed is 220 revs/min,
Filtrated air intake is 1：0.8 (volume ratio of air and nutrient solution).
4th, production tank (5 tons of tankage size of production) used medium composition is identical with fermentation medium (4.5 tons of inventory), throws
Production tank after material is in 1.1kg/cm2Pressure under, 121 DEG C of high pressure moist heat sterilizations are cooled to less than 30 DEG C after sterilizing, lead to it is sterile
Air keeps germ-free condition standby；The seed liquor for reaching logarithmic phase is accessed by the inoculum concentration of 10% (volumn concentration) and produced
Tank, the production tank temperature control after inoculation is at 30~35 DEG C, and the throughput for producing filtrated air in the incubation of tank is 1：1.0
(volume ratio of air and nutrient solution), mixing speed is 180~240 revs/min (being preferably 240 revs/min), whole technological process
Incubation time is 60 hours；Thalline quantity reaches 1,000,000,000/more than ml after fermentation ends.
5th, nutrient solution goes out tank and is directly distributed into liquid dosage form with plastic barrel or Packaging Bottle or uses mud after the completion of fermenting
Charcoal absorption is distributed into solid fungicide formulation with packaging bag.
The Planomicrobium sp.G14-7 and its microbial inoculum provided from upper embodiment, the present invention can improve jade
The survival rate of germination percentage of the crops such as rice under the conditions of saliferous, the speed of growth or corn.Present invention successfully solves in jade
The problem of common crops such as rice can not grow in the higher soil of salt content or growing way is poor, significantly reduces production and used
Workload in journey, production and use cost are low, expand the soil types and area of the common cereal crops such as suitable planting corn,
To ensuring staple food supply, safeguarding that national food security etc. is significant.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the invention as claimed with
Modification, should all belong to the covering scope of the claims in the present invention.
Claims (7)
1. a kind of travelling germ that can be grown in salt content is 16% culture medium belongs to Planomicrobium sp. bacterial strains
Applications of the G14-7 in salt tolerance of crops is improved, wherein travelling germ, which belongs to bacterial strain G14-7, is preserved in Chinese microorganism strain guarantor
Administration committee's common micro-organisms center is hidden, deposit number is CGMCC No.9602.
2. travelling germ belongs to Planomicrobium sp. bacterial strains G14-7 in raising crop tolerance to salt energy as claimed in claim 1
Application in power, it is characterised in that the salt affected soil of plant growth refers to the soil that concentration containing soluble-salt is 0~2.0 mass %
Earth.
3. application of a kind of microbial bacterial agent in salt tolerance of crops is improved, wherein the microbial bacterial agent utilizes claim
Travelling germ described in 1 belongs to Planomicrobium sp. bacterial strains G14-7 productions and obtained, the active component of the microbial bacterial agent
Belong to Planomicrobium sp. bacterial strains G14-7 for the travelling germ described in claim 1.
4. application of the microbial bacterial agent as claimed in claim 3 in salt tolerance of crops is improved, wherein, the microbial bacteria
The travelling germ that agent contains described in claim 1 belongs to Planomicrobium sp. bacterial strains G14-7 for 1,000,000,000/more than mL, should
Microbial bacterial agent belongs to Planomicrobium sp. bacterial strains G14-7 and fermented and cultured using the travelling germ described in claim 1
Base production is obtained, and the fermentation medium contains following component in mass：Glucose 0.7~1.0%, (NH4)2SO40.8~
1.2%th, K2HPO40.1~0.3%, MgSO40.03~0.07%, NaCl 0.005~0.015%, CaCO30.2~
0.4%th, dusty yeast 0.01~0.03%, remaining is water, pH value 7.2~7.5.
5. application of the microbial bacterial agent in the salt resistance ability for improving crop as described in claim 3 or 4, it is characterised in that
The salt affected soil of plant growth refers to the soil that concentration containing soluble-salt is 0~2.0 mass %.
6. application of the microbial bacterial agent in the salt resistance ability for improving crop as described in claim 3 or 4, it is characterised in that
Described microbial bacterial agent is obtained by the preparation method comprised the steps：
1) inclined-plane kind culture：Travelling germ described in claim 1 is belonged to after Planomicrobium sp. bacterial strains G14-7 activation
It is inoculated on test tube slant；
2) shaking flask kind culture：Step 1) obtained test tube strains be inoculated in broth bouillon, isothermal vibration culture to logarithm
Growth period；
3) seeding tank inoculated and cultured：By step 2) obtained shaking flask strain is inoculated in seeding tank according to 10 volume % and cultivates to right
Number growth period；
4) tank culture is produced：By step 3) obtained seeding tank strain is inoculated in production tank by 10 volume %, and temperature control exists
30-35 DEG C is cultivated, and obtains microbial inoculum.
7. application of the microbial bacterial agent as claimed in claim 6 in the salt resistance ability for improving crop, it is characterised in that step
1) the travelling germ described in claim 1 is belonged into Planomicrobium sp. bacterial strains G14-7 on high salt LB culture medium flat plates
Culture, the high salt LB culture mediums include dusty yeast 5g/L, peptone 10g/L, NaCl 160g/L and agar powder 20g/L,
pH7.0-7.2。
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Non-Patent Citations (3)
| Title |
|---|
| 内蒙古荒漠生物结皮可培养细菌及两个盐碱湖中不产氧光合细菌群落结构分析;刘柯澜等;《中国优秀硕士学位论文全文数据库 基础科学辑》;20111215;A006-115 * |
| 松嫩平原盐碱地中耐(嗜)盐菌的生物多样性;潘媛媛等;《微生物学报》;20121004;第52卷(第10期);第1187-1194页，主要涉及表1 * |
| 种植盐地碱蓬改良滨海盐渍土对土壤微生物区系的影响;林学政等;《生态学报》;20060331;第26卷(第3期);第801-807页 * |
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