CN104195082A - Planomicrobium as well as inoculant, preparation method and application thereof - Google Patents
Planomicrobium as well as inoculant, preparation method and application thereof Download PDFInfo
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- CN104195082A CN104195082A CN201410436033.0A CN201410436033A CN104195082A CN 104195082 A CN104195082 A CN 104195082A CN 201410436033 A CN201410436033 A CN 201410436033A CN 104195082 A CN104195082 A CN 104195082A
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Abstract
The invention relates to Planomicrobium as well as an inoculant, a preparation method and an application thereof. The Planomicrobium G14-7 (Planomicrobium sp.G14-7) is preserved in China General Microbiological Culture Collection Center with a preservation number CGMCC No.9602. An active ingredient of the microbial agent is Planomicrobium sp.G14-7. The Planomicrobium sp.G14-7 and the inoculant thereof can be used for remarkably improving salt resistance of crops; and the inoculant produced by virtue of the technology can be used in alkaline-resistant soil, so that the salt resistance of the crops and growth of the crops in the alkaline-resistant soil are improved.
Description
Technical field
The present invention relates to one move about germ belong to (Planomicrobium) bacterium, also relate to the microbiobacterial agent that utilizes this Planomicrobium bacterium to produce, and described Planomicrobium bacterium and the application of microbial inoculum in raising salt tolerance of crops.
Background technology
Saline-alkali soil refers to the soil salt content soil of too high (exceeding 0.3%), and the salinization meeting of soil increases agriculture production cost, even causes soil to fall into disuse.According to statistics, approximately 5.27 hundred million mu of the saltings resource areas of China, wherein 0.88 hundred million mu of Saline, is mainly distributed in North China, northwest and the Northeast.Salting of soil makes China cause every year a large amount of grain drop in production, and the grain drop in production that agricultural land soil salinization causes has not only caused serious financial loss, also national grain security has been formed to serious threat.
For this problem, people mainly adopt various physics, chemistry or biological method first to improve the physico-chemical property of salt affected soil at present, reduce soil salt content etc., realize the utilization in salinization soil.Administering alkaline land improving measure has physics, chemistry and biology and agricultural tillage measure etc., comprises water conservancy ameliorative measure (irrigation, draining, warp, plant rice, antiseepage etc.); Agricultural improvement measure (level land, improve farming, execute soil moved in to improve the original, fertilising, sowing, crop rotation, a kind interplanting etc.); Biological modification measure (plantation salt-tolerant plant and herbage, green manure, afforestation etc.); Chemical modifying measure (using upgrade materials, as gypsum, phosphogypsum, calcium sulfite etc.), and the fertilizer that utilizes the microorganism strains screening from soil to form improves soil property etc.
According to the study, in natural plant materials or rhizosphere there are a large amount of bacteriums, fungi and actinomycetes, these bacterial strains not only can not cause the disease of host plant, on the contrary can by with the interaction of plant, improve the ability that the anti-adverse environmental factor of plant self comprises that tolerance is saline and alkaline.Developing these Microbial resources has important practical significance to industrial and agricultural production.
Summary of the invention
The salinization of soil still lacks effective means administers, and technical problem solved by the invention is to improve the salt resistance ability of common crop by microorganism, realizes the utilization of salinized soil.
The present invention, mainly from having in the plant materials of stronger saline and alkaline tolerance, screens the strong bacterium that also can improve salt tolerance of crops of self salt resistance ability, to improving the salt resistance ability of common crop.The endophytic bacterium of this research can form mutualism relation plant rhizosphere and plant, make bacteria using amount low, easy and simple to handle, cost is low.
One of inventive point of the present invention is to provide a kind of Planomicrobium genus bacterium that has better effects to improving salt tolerance of crops.
Two of inventive point of the present invention is to provide a kind of microbiobacterial agent that utilizes above-mentioned Planomicrobium bacterium to produce.
Three of inventive point of the present invention is to provide above-mentioned Planomicrobium and belongs to bacterium and microbial inoculum at the application method that improves salt tolerance of crops.
Specifically, the present invention is achieved through the following technical solutions:
First, the invention provides the one germ that moves about and belong to bacterial strain G14-7 (Planomicrobium sp.G14-7), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.9602.
In addition, the present invention also provides the germ that moves about to belong to bacterial strain G14-7 in the application improving in salt tolerance of crops.
Wherein, the described germ that moves about belongs to bacterial strain G14-7 in the application improving in salt tolerance of crops, it is characterized in that the soil that it is 0～2.0% that described salt affected soil refers to containing soluble salt concentration.
The present invention also provides a kind of microbiobacterial agent, it is characterized in that, it utilizes the above-mentioned germ that moves about to belong to bacterial strain G14-7 production and obtains, and its activeconstituents is that the germ that moves about claimed in claim 1 belongs to bacterial strain G14-7.
Wherein, in described microbiobacterial agent of the present invention, its contain described moving property Bacillaceae bacterial strain G14-7 be 1,000,000,000/more than ml, the germ that moves about described in this microbial inoculum utilization belongs to bacterial strain G14-7 and fermention medium production obtains, and described fermention medium contains following component in mass: glucose 0.7～1.0%, (NH
4)
2sO
40.8～1.2%, K
2hPO
40.1～0.3%, MgSO
40.03～0.07%, NaCl0.005～0.015%, CaCO
30.2～0.4%, yeast powder 0.01～0.03%, all the other are water, pH value 7.2～7.5.
In addition, the present invention also provides described microbiobacterial agent in the application improving in salt tolerance of crops.
Wherein, the application of described microbiobacterial agent of the present invention in the salt resistance ability that improves crop, is characterized in that the soil that it is 0～2.0% that described salt affected soil refers to containing soluble salt concentration.
In addition, the invention provides the preparation method of described microbiobacterial agent, it is characterized in that, it obtains by the preparation method who comprises the steps:
1) inclined-plane kind is cultivated: will after moving property Bacillaceae bacterial strain G14-7 activation claimed in claim 1, be inoculated on test tube slant;
2) shaking flask kind cultivate: step 1) the test tube strains obtaining be inoculated in broth culture, constant-temperature shaking culture is to logarithmic phase;
3) seeding tank inoculation culture: by step 2) the shaking flask bacterial classification that obtains is inoculated in and in seeding tank, is cultured to logarithmic phase according to 10% volume ratio;
4) producing tank cultivates: by step 3) the seeding tank bacterial classification that obtains is inoculated in and produces in tank by 10% volume ratio, and temperature is controlled at 30-35 DEG C and cultivates, and obtains microbial inoculum.
Specifically, Planomicrobium sp.G14-7 provided by the invention, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 26th, 2014, and deposit number is CGMCC No.9602.
The morphological specificity of Planomicrobium sp.G14-7 of the present invention: thalline is spherical.
The physiological and biochemical property of Planomicrobium sp.G14-7 of the present invention: gram positive; The catalase positive, does not produce gemma, urase reaction negative; In the upper growth of LB solid medium (composition is as follows: yeast powder 5g/L, peptone 10g/L, NaCl10g/L, agar powder 20g/L, pH7.0～7.2) after 3 days, form diameter 0.3mm, redness, the circular bacterium colony that opaque, smooth surface is moistening.
The invention also discloses the microbiobacterial agent that utilizes above-mentioned Planomicrobium sp.G14-7 to produce, its activeconstituents is Planomicrobium sp.G14-7 thalline.
The preparation method of described microbiobacterial agent is as described below:
The technique of producing microbial inoculum is: inclined-plane kind-shaking flask kind-seeding tank-production tank-product, the packaging formulation of described product is liquid bacterial agent or solid absorption microbial inoculum.
One preferred embodiment in, the preparation of described microbial inoculum specifically can adopt following steps:
1, bacterial strain G14-7 (original seed) is cultivated on high salt LB substratum (composition is as follows: yeast powder 5g/L, peptone 10g/L, NaCl160g/L, agar powder 20g/L, pH7.0～7.2) flat board, for subsequent use.
2, the single colony inoculation on the high salt LB of picking culture medium flat plate is in 100ml broth culture (extractum carnis 3.0g/L, peptone 10.0g/L, NaCl5g/L, all the other are water, pH value 7.0～7.2) in, constant-temperature shaking culture, to logarithmic phase, is prepared inoculation seeding tank.
3, preparation fermention medium, its component and weight percent are: glucose 0.8%, (NH
4)
2sO
41.0%, K
2hPO
40.2%, MgSO
40.05%, NaCl0.01%, CaCO
30.3%, yeast powder 0.02%, all the other are water, pH value 7.2～7.5,400L fermention medium is added to 500L seeding tank, and 121 DEG C of high pressure moist heat sterilizations, are cooled to after 30 DEG C, shaking flask bacterial classification is inoculated into seeding tank by the inoculum size of 10% (volumn concentration), be cultured to logarithmic phase, stirring velocity is 220 revs/min, and sterile air intake is 1:0.8 (volume ratio of air and nutrient solution).
4, produce tank (producing 5 tons of tankages) used medium composition identical with fermention medium (4.5 tons of charging capacitys), the production tank after feeding intake is at 1.1kg/cm
2pressure under, 121 DEG C of high pressure moist heat sterilizations, are cooled to after sterilizing below 30 DEG C, logical sterile air keeps sterile state for subsequent use; The seed liquor that arrives logarithmic phase is produced to tank by the inoculum size access of 10% (volumn concentration), postvaccinal production tank temperature is controlled at 30～35 DEG C, in the culturing process of production tank, the air flow of sterile air is 1:(0.6～1.2) (volume ratio of air and nutrient solution), stirring velocity is 180～240 revs/min (can be preferably 240 revs/min), and whole technical process incubation time is 48～60 hours; After fermentation ends thalline quantity reach 1,000,000,000/more than ml.
5, the rear nutrient solution that fermented goes out tank and is directly distributed into liquid dosage form or adopts adsorption by peat packing bag to be distributed into solid fungicide formulation by plastic barrel or packing bottle.
Planomicrobium sp.G14-7 provided by the invention and microbial inoculum itself thereof can be to grow in the substratum such as 16% LB substratum in saliferous (NaCl) amount, containing in the aqueous solution of 0.5%, 1.0% and 2.0% salt (NaCl), can significantly improve corn seed germination rate; Seed is put in microbial inoculum and is soaked after 4 hours, in the soil that plantation is 1% to saltiness, can significantly improve the percentage of germination of seeding corn and other crops, and obviously improve the biomass of crop; Using bacterial strain containing in the soil of 2% soluble salt, can improve the surviving rate of seeding corn and other crops.The microbiobacterial agent that the present invention can improve salt tolerance of crops has advantages of that production and application cost is low, easy to use, can significantly improve the salt resistance ability of seeding corn and other crops.Bacterial strain provided by the invention and microbial inoculum can be used as microbial fertilizer and use in salinization farmland, improve percentage of germination, biomass or the grain yield of crop in salt affected soil.Use this microbial inoculum can expand the planting range of seeding corn and other crops, improve the cultivated area of crop.The present invention has successfully solved the poor and problem such as grain drop in production of causing of in the salinized soil common salt tolerance of crops such as corn, reduce the workload in production and use procedure, production and use cost are reduced, for safeguarding national food safety, the utilising efficiency that improves salinization soil is significant.
Preservation information
Planomicrobium sp.G14-7 provided by the invention, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101; Deposit number is CGMCC No.9602, and preservation date is on August 26th, 2014.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is from routine biochemistry reagent shop and buys, specifically as shown in table 1.Corn seed used is ordinary maize seed.Below experiment all arranges 3 repetitions, results averaged.The saltiness of indication in following examples, if no special instructions, is quality percentage composition, and the salt of indication is NaCl.
The each reagent of table 1 be purchased source-information table
Acquisition and the qualification of embodiment 1:Planomicrobium sp.G14-7
One, the acquisition of Planomicrobium sp.G14-7
Get in June, 2013 from Inner Mongol Ulansuhai Nur periphery Suaeda heteroptera plant one strain, Suaeda heteroptera root system is cut as after several sections, do surface sterilization with clorox, 70% ethanol, after testing after surface sterile, pulverize with aseptic mortar, collect plant tissue residue, with being coated with containing on the LB culture medium flat plate of 2.0% salt concn after sterilized water dilution, cultivate after 7 days, after picking list bacterium colony line purifying, preserve.
LB medium component containing 2.0% salt concn: yeast powder 5.0g, peptone 10.0g and NaCl20.0g, agar powder 20g, water is settled to 1L, and pH is 7.0 left and right.
Two, the qualification of Planomicrobium sp.G14-7
(1) morphological specificity: thalline is spherical.
(2) physiological and biochemical property: gram positive; In LB culture medium culturing after 3 days, the circular bacterium colony that formation diameter is 0.3mm, redness, opaque, smooth surface is moistening.
(3) 16S rDNA Sequence Identification
Adopt following primer: 8F (5 '-AGAGTTTGATCCTGGCTCAG-3 '), 1492 (5 '-TACCTTGTTA CGACTT-3 '), carry out pcr amplification, from bacterial strain G14-7, amplification, to 16SrDNA gene fragment, is cloned into obtained 16S rDNA after pGEM T-easy carrier and send Bo Aiyuanshang bio tech ltd, Beijing to check order.The partial sequence of 16S rDNA is shown in the base sequence shown in the SEQ ID No:1 of sequence table, finds with the sequence alignment in GenBank, and the 16S rDNA sequence of G14-7 is the highest with the homology of Planomicrobium genus bacterial strain sequence, reaches 99.8%.
Three, the preservation of Planomicrobium sp.G14-7
By above qualification result, confirm that above-mentioned bacterial strains is to come from the new bacterium that Planomicrobium belongs to, by its called after Planomicrobium sp.G14-7, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.9602.
Embodiment 2:Planomicrobium sp.G14-7 improves the percentage of germination of corn seed in the aqueous solution of different salt concn
The mono-bacterium colony of picking Planomicrobium sp.G14-7 is in 3ml LB liquid nutrient medium, and 30 DEG C, 165 revs/min shaking culture 24 hours, obtain fresh bacterium liquid.(its component and weight percent are: glucose 0.8%, (NH to be inoculated into 200mL fermention medium by the inoculum size of 2% (volume ratio)
4)
2sO
41%, K
2hPO
40.2%, MgSO
40.05%, NaCl 0.01%, CaCO
30.3%, yeast powder 0.02%, all the other are water, pH value 7.2～7.5) in enlarged culturing, 120r/min, 30 DEG C cultivate 48 hours.
Corn seed of uniform size is divided into two groups, one group is placed in fresh bacterium liquid inoculation and soaks 4 hours, another group is soaked after 4 hours with above-mentioned fermention medium, naturally after drying, the corn seed after soaking is placed in respectively containing on the filter paper of 0.5%, 1.0%, 2.0% salt solution, then seed is put in respectively in the incubator of 28 DEG C to lucifuge and leaves standstill and cultivate, the germination of observing its corn seed 7 days time.
Percentage of germination=(germination corn seed quantity/experiment seed amount) × 100% of corn.
Result (table 2) shows, the corn seed after bacterial strain soaks can germinate fast in the aqueous solution of 0.5～2.0% salt (NaCl) concentration, and its percentage of germination is apparently higher than the corn seed of not inoculating Planomicrobium sp.G14-7.
The impact of Planomicrobium sp.G14-7 seed soaking percentage of germination in the salts solution of different concns on corn for table 2
Embodiment 3:Planomicrobium sp.G14-7 improves biomass, the surviving rate of corn in salty soil
The mono-bacterium colony of picking Planomicrobium sp.G14-7 is in 3ml LB liquid nutrient medium, and 30 DEG C, 165 revs/min shaking culture 24 hours, obtain fresh bacterium liquid.(its component and weight percent are: glucose 0.8%, (NH to be inoculated into 200mL fermention medium by the inoculum size of 2% (volume ratio)
4)
2sO
41%, K
2hPO
40.2%, MgSO
40.05%, NaCl0.01%, CaCO
30.3%, yeast powder 0.02%, all the other are water, pH value 7.2～7.5) in enlarged culturing, 120r/min, 30 DEG C cultivate 48 hours.
Choose corn seed of the same size, be put in the fresh bacterium liquid of G14-7 and soak 4 hours, contrast seed soaks 4 hours in LB substratum.Then seed is seeded into respectively in soluble salt concentration is 0.5%, 1.0%, 2.0% soil.Basin alms bowl is put under sunlight and is cultivated, and regular watering maintains soil moisture content 20%.10 days time, observe the growing state of corn.
Result (table 3) shows, the speed of growth of the corn seed after bacterial strain soaks in the salty soil of 2.0% salt concn is obviously faster than the corn seed of not inoculating Planomicrobium; In the soil of the salt concn 2.0%, in the soil of inoculating strain, corn can not grown, and after seed soaking, corn can be grown.
Table 3Planomicrobium sp.G14-7 is improving the biomass of corn in salty soil
Embodiment 4
Utilize above-mentioned Planomicrobium sp.G14-7 to produce microbiobacterial agent
1, bacterial strain G14-7 (original seed) is cultivated on high salt LB substratum (composition is as follows: yeast powder 5g/L, peptone 10g/L, NaCl160g/L, agar powder 20g/L, pH7.0～7.2) flat board, for subsequent use.
2, the single colony inoculation on the high salt LB of picking culture medium flat plate is in 100ml broth culture (extractum carnis 3.0g/L, peptone 10.0g/L, NaCl5g/L, all the other are water, pH value 7.0～7.2) in, constant-temperature shaking culture, to logarithmic phase, is prepared inoculation seeding tank.
3, preparation fermention medium, its component and weight percent are: glucose 0.8%, (NH
4)
2sO
41%, K
2hPO
40.2%, MgSO
40.05%, NaCl0.01%, CaCO
30.3%, yeast powder 0.02%, all the other are water, pH value 7.2～7.5,400L fermention medium is added to 500L seeding tank, and 121 DEG C of high pressure moist heat sterilizations, are cooled to after 30 DEG C, shaking flask bacterial classification is inoculated into seeding tank by the inoculum size of 10% (volumn concentration), be cultured to logarithmic phase, stirring velocity is 220 revs/min, and sterile air intake is 1:0.8 (volume ratio of air and nutrient solution).
4, produce tank (producing 5 tons of tankages) used medium composition identical with fermention medium (4.5 tons of charging capacitys), the production tank after feeding intake is at 1.1kg/cm
2pressure under, 121 DEG C of high pressure moist heat sterilizations, are cooled to after sterilizing below 30 DEG C, logical sterile air keeps sterile state for subsequent use; The seed liquor that arrives logarithmic phase is produced to tank by the inoculum size access of 10% (volumn concentration), postvaccinal production tank temperature is controlled at 30～35 DEG C, in the culturing process of production tank, the air flow of sterile air is 1:1.0 (volume ratio of air and nutrient solution), stirring velocity is 180～240 revs/min (can be preferably 240 revs/min), and whole technical process incubation time is 60 hours; After fermentation ends thalline quantity reach 1,000,000,000/more than ml.
5, the rear nutrient solution that fermented goes out tank and is directly distributed into liquid dosage form or adopts adsorption by peat packing bag to be distributed into solid fungicide formulation by plastic barrel or packing bottle.
From upper embodiment, Planomicrobium sp.G14-7 provided by the invention and microbial inoculum thereof can improve the surviving rate of percentage of germination, the speed of growth or the corn of the farm crop such as corn under saliferous condition.The present invention has successfully solved and can not grow in the higher soil of saltiness common crops such as corns or problem that growing way is poor, significantly reduce the workload in production and use procedure, production and use cost are low, expand soil type and the area of the common food crop such as suitable planting corn, to guaranteeing staple food supply, safeguard that national food is safely etc. significant.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the claims in the present invention scope change and modify, and all should belong to the covering scope of the claims in the present invention.
Claims (8)
1. the germ that moves about belongs to bacterial strain G14-7(
planomicrobiumsp. G14-7), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.9602.
2. the germ that moves about claimed in claim 1 belongs to bacterial strain G14-7 in the application improving in salt tolerance of crops.
3. the germ that moves about as claimed in claim 2 belongs to bacterial strain G14-7 in the application improving in salt tolerance of crops, it is characterized in that the soil that it is 0 ~ 2.0% that described salt affected soil refers to containing soluble salt concentration.
4. a microbiobacterial agent, is characterized in that, it utilizes the germ that moves about described in claim 1 to belong to bacterial strain G14-7 and produces and obtain, and its activeconstituents is that the germ that moves about claimed in claim 1 belongs to bacterial strain G14-7.
5. microbiobacterial agent as claimed in claim 4, its contain moving property Bacillaceae bacterial strain G14-7 claimed in claim 1 be 1,000,000,000/more than ml, this microbial inoculum utilizes moving property Bacillaceae bacterial strain G14-7 and the fermention medium production described in claim 1 to obtain, and described fermention medium contains following component in mass: glucose 0.7 ~ 1.0%, (NH
4)
2sO
40.8 ~ 1.2%, K
2hPO
40.1 ~ 0.3%, MgSO
40.03 ~ 0.07%, NaCl 0.005 ~ 0.015%, CaCO
30.2 ~ 0.4%, yeast powder 0.01 ~ 0.03%, all the other are water, pH value 7.2 ~ 7.5.
6. the microbiobacterial agent described in claim 4 or 5 is in the application improving in salt tolerance of crops.
7. the application of microbiobacterial agent claimed in claim 6 in the salt resistance ability that improves crop, is characterized in that, the soil that it is 0 ~ 2.0% that described salt affected soil refers to containing soluble salt concentration.
8. the preparation method of the microbiobacterial agent described in claim 4 or 5, is characterized in that, it obtains by the preparation method who comprises the steps:
1) inclined-plane kind is cultivated: will after moving property Bacillaceae bacterial strain G14-7 activation claimed in claim 1, be inoculated on test tube slant;
2) shaking flask kind cultivate: step 1) the test tube strains obtaining be inoculated in broth culture, isothermal vibration is cultured to logarithmic phase;
3) seeding tank inoculation culture: by step 2) the shaking flask bacterial classification that obtains is inoculated in and in seeding tank, is cultured to logarithmic phase according to 10 volume %;
4) producing tank cultivates: the seeding tank bacterial classification that step 3) is obtained is inoculated in and produces in tank by 10 volume %, and temperature is controlled at 30-35 DEG C and cultivates, and obtains microbial inoculum.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111925945A (en) * | 2020-08-04 | 2020-11-13 | 贵州大学 | Tobacco pipe strain G14 and application |
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2014
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| 刘柯澜等: "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", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111925945A (en) * | 2020-08-04 | 2020-11-13 | 贵州大学 | Tobacco pipe strain G14 and application |
| CN111925945B (en) * | 2020-08-04 | 2022-11-22 | 贵州大学 | Smoke tube bacterium G14 and application thereof |
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Address after: Peking University Science Park 014010 the Inner Mongolia Autonomous Region Jiuyuan District of Baotou city planning new equipment Park District 2 Building 2 layer Applicant after: Packet header innovation research institute of Peking University Address before: 014010 room B, block, management committee, equipment manufacturing park, Qingshan District, the Inner Mongolia Autonomous Region, China Applicant before: BAOTOU RESEARCH INSTITUTE, COLLEGE OF ENGINEERING, PKU |
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