CN108893435A - A kind of microbial liquid microbial inoculum of the general bacterium containing camel thorn and its preparation method and application - Google Patents
A kind of microbial liquid microbial inoculum of the general bacterium containing camel thorn and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of microbial liquid microbial inoculum of general bacterium containing camel thorn, the component including following mass percent：Promoting bacteria bacterium solution 1%-10%, polyglutamic acid 0.1%-10%, biological organic matter 50%-70%, surplus are water or aqueous solution；The Promoting bacteria bacterium solution includes the general bacterium of camel thorn (Pantoea alhagi) bacterium solution.The invention also discloses the preparation methods and application of above-mentioned complex microorganism liquid bacterial agent.Complex microorganism liquid bacterial agent of the invention, has the function of high-activity biological, wherein polyglutamic acid, biological organic matter and microbial bacterial agent have synergistic function, the micropopulation environment of crop root can be improved, increase beneficial microorganism quantity in soil, the quality of crop can be improved, improves yield, and reinforce crop to the anti-adversity ability of drought resisting, salt resistance, cold-resistant, disease-resistant etc..
Description
Technical field
The present invention relates to microbial bacterial agents, and in particular to a kind of microbial liquid microbial inoculum of the general bacterium containing camel thorn and its preparation
Methods and applications.
Background technique
China is world agriculture big country, and with the rapid development of China's agricultural, the usage amount of chemical fertilizer is also increasing, every year
Dosage reach 1,800,000 tons.Although this solves the problems, such as increases in grain production and supply, causes environment very serious and ask
Topic.The Use out of range of chemical fertilizer has aggravated the hardened of soil and pollution, leads to the decline and degeneration of soil quality, this has soil
Machine matter content is seriously lower than the average level in the whole world.Once soil organic matter content is low, will be greatly reduced to heavy metal fixation
Ability increases the harmfulness of heavy metal-polluted soil, compromises the sustainable development of China's agricultural.
Microbial bacterial agent be it is a kind of can optimize soil physico-chemical property, improve absorption and utilization efficiency of the crop to nutrient, into
And promoting the novel agricultural biological agent of plant growth, it has economic and environment-friendly, the improvement effectiveness such as soil and fertilizer synergistic.This
Outside, moreover it is possible to enhance the drought-resistant of crop, low-temperature resistance, the anti-adversity abilities such as high salt tolerance.In recent years, using microbial bacterial agent as bio-fertilizer
The research of material is more and more, and especially microbial liquid microbial inoculum has low production cost, easy to use, growth-promoting and degeneration-resistant effect bright
The advantages that aobvious.
But because state of the art limits, all there is effective bacterium number content in the microbial liquid microbial inoculum sold currently on the market
Low, the defects of effective bacterium easy in inactivation, especially commercially available microbial inoculum is Yi Dan sealed off, and effective bacterial content drops cliff of displacement formula, significantly
The effectiveness for reducing microbial inoculum increases the use cost of peasant.
Summary of the invention
Goal of the invention：In order to solve the problems, such as that the existing effective bacterial content of microbial liquid microbial inoculum is low and easy in inactivation, the present invention
A kind of microbial liquid microbial inoculum of general bacterium containing camel thorn is provided, invention further provides the preparations of microbial liquid microbial inoculum
Method, the present invention further provide the application of microbial liquid microbial inoculum.
Technical solution：A kind of microbial liquid microbial inoculum of general bacterium containing camel thorn of the present invention, including following quality percentage
The component of ratio：Promoting bacteria bacterium solution 1%-10%, polyglutamic acid 0.1%-10%, biological organic matter 50%-70%, surplus be water or
Aqueous solution；The Promoting bacteria bacterium solution includes the general bacterium of camel thorn (Pantoea alhagi) bacterium solution.
Preferably, Promoting bacteria bacterium solution 5%-10%, polyglutamic acid 0.5%-8%, biological organic matter 50%-65%, surplus
For water or aqueous solution.
It is highly preferred that Promoting bacteria bacterium solution 8%-10%, polyglutamic acid 1%-5%, biological organic matter 55%-65%, surplus
For water or aqueous solution.
Preferably, the general bacterium bacterium solution of the camel thorn is the general fermented liquid of camel thorn, and viable count is 1 × 10 in fermentation liquid9-9
×1010CFU/mL；Its classification naming of the general bacterium of camel thorn is the general bacterium of camel thorn (Pantoea alhagi), and bacterial strain number is XK-
11, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the preservation time is March 29 in 2018
Day, deposit number is CGMCC NO.15525, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences
Institute of microbiology's collection, postcode 100101.The bacterial strain is inventor in October, 2017 from West Zhejiang Province Grand Canyon breeding
Bacterial strain.
The general bacterium XK-11 of camel thorn has the following properties：
1, colonial morphology feature
Microscope observes that cell is unicellular, rod-shaped, the long 1.4-2.0 of thallus after 30 DEG C of culture 16-24h of LB culture medium
μm, it is 0.7-1.0 μm wide.Belong to Gram-negative bacteria, there is flagellum can move, amphimicrobian.30 DEG C in above-mentioned culture medium
Cultivating 12h thallus can be in ecru with raised growth, bacterium colony, and round, surface is smooth, glues shape, intermediate projections.Its temperature grown
Range is 7-48 DEG C, and optimum temperature is 35-39 DEG C, and the pH range of growth is 5.0-9.5, optimal pH 6.5-7.5.With ammonia benzyl
Chloramphenicol resistance (100 μ g/mL), can in the culture mediums such as LB, R2A, NA, TSA, MC normal growth.
2,16SrDNA sequence is analyzed
16SrDNA sequence length 1488bp, nucleotide sequence are shown in SEQ ID NO.1.By 16SrDNA sequence and GeneBank
Related kind in database is compared, and constructs the phylogenetic tree based on 16SrDNA complete sequence.The result shows that：Strain X K-
11 with the general bacterium of camel thorn to reach 100% homologous.So assert that bacterial strain of the invention is the general bacterium of camel thorn (Pantoea alhagi),
Bacterial strain number is Pantoea alhagi XK-11.
Wherein, the Promoting bacteria bacterium solution further includes bacillus subtilis (Bacillus subtilis), gel-shaped gemma bar
Bacterium (Bacillus mucilaginosus) and bacillus amyloliquefaciens (Bacillus amyloliquefaciens) fermentation liquid
In any one or the combination of several of them, bacillus subtilis fermentation liquor, bacillusmusilaginosiengineering fermentation liquid or solution starch gemma
Viable count is 1 × 10 in bacillus fermentation liquid9-9×1010CFU/mL, viable count >=5 of the Promoting bacteria in microbial liquid preparation ×
109CFU/mL.The additive amount of Promoting bacteria bacterium solution is according to the general bacterium of camel thorn, bacillus subtilis, gel-shaped bacillus, solution starch
Any one in bacillus or combined summation calculate, living bacteria count >=5 × 10 in microbial liquid microbial inoculum9CFU/mL。
Bacillus subtilis, gel-shaped bacillus, bacillus amyloliquefaciens make thallus by refrigeration obtained by high density fermentation
Into gemma form, gemma state is the dormant state of bacillus, can be survived in the state of not sprouting several years and to tens
Year.
Preferably, the bacillus subtilis (Bacillus subtilis) is in China typical culture collection center
Deposit number is CCTCC NO:M 2016264, bacillusmusilaginosiengineering (Bacillus mucilaginosus) is in Chinese Typical Representative
The deposit number of culture collection is CCTCC NO:M 2016265, bacillus amyloliquefaciens (Bacillus
It amyloliquefaciens is) CCTCC NO in the deposit number of China typical culture collection center:M 2016266 is recorded
In patent 2016106658324.
Preferably, the Promoting bacteria bacterium solution is the general fermented liquid of camel thorn, bacillus subtilis fermentation liquor, gel-shaped gemma
The combination of bacillus fermentation liquid and bacillus amyloliquefaciens fermentation liquid, volume ratio 0.1-10:0.1-10:0.1-10:0.1-10.
Wherein, the polyglutamic acid weight average molecular weight is 5-3000kDa, preferred molecular weight 5-50kDa, the polyglutamic
Acid is polyglutamic acid sterling, polyglutamate sterling, polyglutamic acid fermentation liquid, polyglutamic acid fermentation liquid dried object and polyglutamic acid
Any one or the combination of several of them in wettable powder.The additive amount of polyglutamic acid is with polyglutamic acid sterling or polyglutamic acid
Gather contained in salt sterling or polyglutamic acid fermentation liquid or polyglutamic acid fermentation liquid dried object or polyglutamic acid wettable powder
Glutamic acid calculates.The Related product of above-mentioned polyglutamic acid is prepared by existing fermentation technique.When polyglutamic acid is polyglutamic acid
When fermentation liquid, the quality of polyglutamic acid is calculated in microbial liquid microbial inoculum with polyglutamic acid content in fermentation liquid, and aqueous solution is
Polyglutamic acid fermentation liquid (not including polyglutamic acid therein).
Polyglutamic acid is a kind of special anionic polymer polymer, and side chain contains a large amount of carboxylic groups, these groups
Its special bioactivity is assigned, as a kind of substance of adhesiveness, polyglutamic acid can provide folder for bacillus in Promoting bacteria
Film protection promotes bacillus to form a kind of external wrapping biological membranous system, thus separates the substance of Promoting bacteria intraor extracellular
Exchange, reaches a kind of balance stable state.
Wherein, the biological organic matter is the group of any one or a few in biochemical fulvic acid, molasses and yeast extract
It closes.The additive amount of biological organic matter is according to any one in yeast extract, molasses, biochemical fulvic acid or combined summation meter
It calculates.Above-mentioned biological organic matter can be bought or according to prior art preparation from market.
Wherein, yeast extract main component is polypeptide, amino acid, microelement etc..
Biological organic matter can inhibit the bud of bacillus by forming hyperosmosis environment under use ratio of the present invention
Spore is sprouted, and the bacillus in microbial liquid microbial inoculum is maintained to be in gemma state.And farming is applied to after microbial inoculum is diluted
When object, biological organic matter concentration is reduced, and changes its " identity " immediately, becomes the nutritional ingredient of gemma sprouting and microbial growth.
In addition, biological organic matter can also improve soil texture, the problems such as reducing soil compaction rate, solve soil acidification.
The present invention further provides the preparation methods of the microbial liquid microbial inoculum, and steps are as follows：By Promoting bacteria bacterium solution, gather
Glutamic acid and biological organic matter are mixed according to mass percent to get microbial liquid microbial inoculum.
Wherein, the preparation step of the general fermented liquid of camel thorn is as follows：The general bacterium of camel thorn, which is inoculated in LB culture medium, activates 12-
For 24 hours, activation temperature is 25-35 DEG C, the general bacterium of the camel thorn of activation is inoculated in liquid fermentation medium, 30-35 DEG C of culture 36-
50h obtains fermentation liquid；The fermentative medium formula is as follows：Carbon source 10-100g/L, nitrogen source 1-30g/L, inorganic salts 0.01-
50g/L, pH5.0-9.0.
Wherein, the preparation step of bacillus subtilis, bacillusmusilaginosiengineering or bacillus amyloliquefaciens fermentation liquid is as follows：
The Promoting bacteria that inclined-plane saves is inoculated on activation medium, 18-24h is cultivated；Bacterium on picking activation medium
Kind, it is inoculated in fermentation medium, 30-35 DEG C of culture 12-24h, fermentation liquid or fermentation concentrated liquor is placed in low in Cool Room 4 DEG C
Temperature processing 35-55h；The activation medium is LB culture medium, is formulated as follows：Peptone 10g/L, yeast powder 5g/L, sodium chloride
10g/L, pH6.0-8.0, the solvent are water.The formula of the fermentation medium is as follows：Carbon source 10-100g/L, nitrogen source 1-
40g/L, inorganic salts 0.01-50g/L, solvent are water, pH5.0-9.0.
The general fermented liquid of camel thorn, bacillus subtilis fermentation liquor, bacillusmusilaginosiengineering fermentation liquid or solution starch gemma
Bacillus fermentation liquid is kept separately or mixes in proportion preservation.
Preferably, the carbon source be glucose, sucrose, maltose, lactose, xylose, fructose, lactic acid, citric acid, glycerol,
Any one or the combination of several of them in starch and molasses；The nitrogen source is yeast powder, beef extract, peptone, yeast extract, corn
Slurry, beancake powder, cottonseed meal, urea, (NH4)2SO4、NH4Cl、(NH4)2HPO4And NH4NO3In any one or a few group
It closes；The inorganic salts are any one or a few in sulfate, phosphate, dihydric phosphate, phosphoric acid hydrogen disalt and hydrochloride
Combination.
The present invention further provide the microbial liquid microbial inoculum improve crop yield, promoted crops quality,
Enhance the application in any one or a few in crops resistance.
Wherein, the microbial liquid microbial inoculum can be used cooperatively with conventional chemical fertilizers, also be can be used as bacterial manure and directly applied；
Microbial liquid microbial inoculum is diluted 10-1000 times, by water planting, is sprayed or root irrigation crops.
The crops are cereal crops, greengrocery crop or melon or fruit type crops.
Beneficial effect：(1) polyglutamic acid provides pod membrane protection in microbial liquid microbial inoculum for Promoting bacteria, and makes gemma bar
Bacterium forms biofilm system, reaches a kind of stable equilibrium state, is at gemma state；Polyglutamic acid is produced by microbial fermentation
It is raw, it is formed by D-Glu and Pidolidone monomer by glutamy amine key homopolymerization, there is nontoxic, biodegradable and environment friend
The advantages that good, has the absorption and other effects for improving plant to nutrient.(2) raw when microbial liquid microbial inoculum is in packed state
Object organic matter inhibits the growth of Promoting bacteria by providing high organic environment and hyperosmosis environment, so that Promoting bacteria is in
Dormant state；When microbial liquid microbial inoculum is diluted application, organic matter can provide nutrition again for the growth of Promoting bacteria, promote to promote
The fast breeding of raw bacterium；When biological organic matter and polyglutamic acid are used in combination, in addition to nutrition synergistic function, mainly pass through
Synergistic effect maintains the vigor of Promoting bacteria in liquid bacterial agent, substantially prolongs the shelf life of microbial liquid microbial inoculum, makes microorganism
The number of viable of Promoting bacteria in liquid bacterial agent maintains >=5 × 10 in 24 months9CFU/mL.(3) heretofore described four
Kind microorganism, crop root biological community structure can be improved by being applied in crops, increase quantity of useful microbe in soil,
Soil texture can be improved, is kept soil from packing together, can be improved crop yield, enhancing crop drought resistance, salt resistance, disease-resistant, cold-resistant etc.
Anti-adversity ability.
Detailed description of the invention
Fig. 1 is the number of viable survival curve of different microbial inoculums.
Specific embodiment
A kind of microbial liquid microbial inoculum of general bacterium containing camel thorn of the present invention, the component including following mass percent：
Promoting bacteria bacterium solution 1%-10%, polyglutamic acid 0.1%-10%, biological organic matter 50%-70%, surplus are water or aqueous solution；Institute
Stating Promoting bacteria bacterium solution includes the general bacterium of camel thorn (Pantoea alhagi) bacterium solution.
Preferably, Promoting bacteria bacterium solution 5%-10%, polyglutamic acid 0.5%-8%, biological organic matter 50%-65%, surplus
For water or aqueous solution.
It is highly preferred that Promoting bacteria bacterium solution 8%-10%, polyglutamic acid 1%-5%, biological organic matter 55%-65%, surplus
For water or aqueous solution.
Preferably, the general bacterium bacterium solution of the camel thorn is the general fermented liquid of camel thorn, and viable count is 1 × 10 in fermentation liquid9-9
×1010CFU/mL；The general bacterium of camel thorn (Pantoea alhagi) is common in China Committee for Culture Collection of Microorganisms
The deposit number at microorganism center is CGMCC NO.15525.
Wherein, the Promoting bacteria bacterium solution further includes bacillus subtilis (Bacillus subtilis), gel-shaped gemma bar
Bacterium (Bacillus mucilaginosus) and bacillus amyloliquefaciens (Bacillus amyloliquefaciens) fermentation liquid
In any one or the combination of several of them, bacillus subtilis fermentation liquor, bacillusmusilaginosiengineering fermentation liquid or solution starch gemma
Viable count is 1 × 10 in bacillus fermentation liquid9-9×1010CFU/mL, viable count >=5 of the Promoting bacteria in microbial liquid preparation ×
109CFU/mL.The additive amount of Promoting bacteria bacterium solution is according to the general bacterium of camel thorn, bacillus subtilis, gel-shaped bacillus, solution starch
Any one in bacillus or combined summation calculate, living bacteria count >=5 × 10 in microbial liquid microbial inoculum9CFU/mL。
Bacillus subtilis, gel-shaped bacillus, bacillus amyloliquefaciens make thallus by refrigeration obtained by high density fermentation
Into gemma form, gemma state is the dormant state of bacillus, can be survived in the state of not sprouting several years and to tens
Year.
Preferably, the bacillus subtilis (Bacillus subtilis) is in China typical culture collection center
Deposit number is CCTCC NO:M 2016264, bacillusmusilaginosiengineering (Bacillus mucilaginosus) is in Chinese Typical Representative
The deposit number of culture collection is CCTCC NO:M 2016265, bacillus amyloliquefaciens (Bacillus
It amyloliquefaciens is) CCTCC NO in the deposit number of China typical culture collection center:M 2016266 is recorded
In patent 2016106658324.
Preferably, the Promoting bacteria bacterium solution is the general fermented liquid of camel thorn, bacillus subtilis fermentation liquor, gel-shaped gemma
The combination of bacillus fermentation liquid and bacillus amyloliquefaciens fermentation liquid, volume ratio 0.1-10:0.1-10:0.1-10:0.1-10.
Wherein, the polyglutamic acid weight average molecular weight is 5-3000kDa, preferred molecular weight 5-50kDa, the polyglutamic
Acid is polyglutamic acid sterling, polyglutamate sterling, polyglutamic acid fermentation liquid, polyglutamic acid fermentation liquid dried object and polyglutamic acid
Any one or the combination of several of them in wettable powder.The additive amount of polyglutamic acid is with polyglutamic acid sterling or polyglutamic acid
Gather contained in salt sterling or polyglutamic acid fermentation liquid or polyglutamic acid fermentation liquid dried object or polyglutamic acid wettable powder
Glutamic acid calculates.The Related product of above-mentioned polyglutamic acid is prepared by existing fermentation technique.When polyglutamic acid is polyglutamic acid
When fermentation liquid, the quality of polyglutamic acid is calculated in microbial liquid microbial inoculum with polyglutamic acid content in fermentation liquid, and aqueous solution is
Polyglutamic acid fermentation liquid (not including polyglutamic acid therein).
Polyglutamic acid is a kind of special anionic polymer polymer, and side chain contains a large amount of carboxylic groups, these groups
Its special bioactivity is assigned, as a kind of substance of adhesiveness, polyglutamic acid can provide folder for bacillus in Promoting bacteria
Film protection promotes bacillus to form a kind of external wrapping biological membranous system, thus separates the substance of Promoting bacteria intraor extracellular
Exchange, reaches a kind of balance stable state.
Wherein, the biological organic matter is the group of any one or a few in biochemical fulvic acid, molasses and yeast extract
It closes.The additive amount of biological organic matter is according to any one in yeast extract, molasses, biochemical fulvic acid or combined summation meter
It calculates.Above-mentioned biological organic matter can be bought or according to prior art preparation from market.
Wherein, yeast extract main component is polypeptide, amino acid, microelement etc..
Biological organic matter can inhibit the bud of bacillus by forming hyperosmosis environment under use ratio of the present invention
Spore is sprouted, and the bacillus in microbial liquid microbial inoculum is maintained to be in gemma state.And farming is applied to after microbial inoculum is diluted
When object, biological organic matter concentration is reduced, and changes its " identity " immediately, becomes the nutritional ingredient of gemma sprouting and microbial growth.
In addition, biological organic matter can also improve soil texture, the problems such as reducing soil compaction rate, solve soil acidification.
The fermentation of 1 Promoting bacteria of embodiment
Microbial bacterial agent of the present invention is prepared as follows：
The general bacterium of camel thorn (Pantoea alhagi) CGMCC NO.15525 that 4 DEG C of inclined-planes save is inoculated in eggplant shape culture
On bottle culture medium (i.e. activation medium), 30 DEG C of culture 16h.Strain on picking activation medium, is inoculated in fermentation medium
In, 30 DEG C, 40h is cultivated, fermentation liquid or fermentation concentrated liquor are placed in low-temperature treatment 48h in Cool Room 4 DEG C, is coated with and surveys through plate
Fixed, the number of viable in the general fermented liquid of camel thorn can reach 1 × 1010CFU/mL or more.Culture in the eggplant shape culture bottle
Base is LB culture medium；Fermentation medium group is divided into：Sucrose 50g/L, beef extract 3g/L, NaCl 8g/L, peptone 8g/L, pH are
6.0-8.0。
The bacillus subtilis CCTCC NO that 4 DEG C of inclined-planes are saved:M 2016264, gel-shaped bacillus CCTCCNO:M
2016265, bacillus amyloliquefaciens CCTCC NO:M 2016266 is activated, and it is (i.e. living to be inoculated in eggplant shape culture bottle culture medium respectively
Change culture medium) on, 30 DEG C of culture 18h.The strain grown is transferred in fermentation medium respectively, after 30 DEG C of cultures for 24 hours, will be sent out
Zymotic fluid is placed in low-temperature treatment 48h in Cool Room 4 DEG C, is coated with and measures through plate, and the number of viable in every kind of microbial fermentation solution is reachable
To 1 × 1010CFU/mL or more.Culture medium in the eggplant shape culture bottle is LB culture medium, and fermentation medium group is divided into：Glucose
50g/L, peptone 10g/L, (NH4)2SO40.05g/L, NaCl 1g/L, MnSO4·4H2O 0.5g/L, KH2PO41.0g/L
PH is 6.0-8.0.
By four kinds of microbial bacterial agents according to volume ratio 1:1:1:1 ratio mixes compounding.
The ventilatory capacity of filtrated air is 0.6vvm during the fermented and cultured of above-described production tank, and mixing speed is
220rpm。
The preparation of 2 complex micro organism fungicide of embodiment
It is prepared as follows the composite microbial of the invention combined containing polyglutamic acid, biological organic matter, growth promoting bacteria agent
Object liquid bacterial agent：
1) polyglutamic acid prepares (bibliography Xu Z, Feng X, Dan Z, et al.Enhanced using fermentation method
poly(γ-glutamic acid)fermentation by Bacillus subtilis,NX-2immobilized in an
aerobic plant fibrous-bed bioreactor[J].Bioresour Technol,2014,155:8-14), it ferments
Polyglutamic acid content is 15% after liquid is concentrated；
2) molasses are purchased by sugar refinery；
It 3) is 1 according to mass ratio by the compound Promoting bacteria bacterium solution of embodiment 1 and polyglutamic acid Fermented Condensed liquid, molasses:
2.5:6.5 ratio is uniformly mixed, and complex micro organism fungicide of the present invention is prepared, through detecting, polyglutamic acid content
Mass percent is 4.0%, and content of organic matter mass percent is 63%, and living bacteria count is 7.6 × 109cfu/mL。
3 complex microorganism liquid bacterial agent of embodiment can open preservation up to 24 months
Microbial inoculum used in the present embodiment is as follows：
1) mix bacterium agent being prepared in embodiment 1 is diluted to living bacteria count is 7.6 × 109Cfu/mL, as
Microbial inoculum 1；
2) mix bacterium agent in embodiment 1 is mixed in proportion with polyglutamic acid Fermented Condensed liquid, makes polyglutamic acid content
It is 4.0%, living bacteria count is 7.6 × 109Cfu/mL, as microbial inoculum 2；
3) mix bacterium agent in embodiment 1 is mixed in proportion with molasses, makes the content of organic matter 63%, it is effectively living
Bacterium number is 7.6 × 109Cfu/mL, as microbial inoculum 3；
4) mix bacterium agent that embodiment 1 is prepared is mixed in proportion with polyglutamic acid, molasses, makes polyglutamic acid
Content is 4.0%, the content of organic matter 15%, and living bacteria count is 7.6 × 109CFU/mL, as microbial inoculum 4；
5) the complex microorganism liquid bacterial agent containing polyglutamic acid, biological organic matter and Promoting bacteria for preparing embodiment 2, i.e.,
Complex microorganism liquid bacterial agent of the present invention, as microbial inoculum 5.
Above-mentioned 5 kinds of microbial inoculums are placed in 25 DEG C, is stored in the environment of relative humidity 40%, 1mL microbial inoculum was respectively taken every 2 months,
Dilution 108Times, it in applying solid LB culture medium, is placed under 32 DEG C of environment, cultivates 18-24h, obtained using colony counting method effective
Number of viable, continuous sampling 24 months.If three groups of parallel laboratory tests, living bacteria count is changed over time such as Fig. 1 institute in different microbial inoculums
Show.
As shown in Figure 1, its living bacteria count amount of microbial inoculum 1, microbial inoculum 2, microbial inoculum 3, microbial inoculum 4 only maintains 8 months, 12 months, 14
A month and 14 months, fail to reach 24 months standards.Microbial inoculum 5, i.e. the living bacteria count amount of complex microorganism liquid bacterial agent
Reached >=5 × 10 before 24 months9The level of cfu/mL.It can be seen that the complex microorganism liquid bacterial agent of this patent protection
Validity period be 24 months.
4 complex microorganism liquid bacterial agent of embodiment can reduce the application of chemical fertilizer
The complex microorganism liquid bacterial agent preparation containing polyglutamic acid, biological organic matter and Promoting bacteria in the present embodiment is the same as real
Apply example 2.Using potting pakchoi as experimental subjects, test sets 3 processing, 3 repetitions of each processing, wherein processing 1 is test pair
According to common fertilizer, processing 2 is common fertilizer+complex microorganism liquid bacterial agent, processing 3 be subtract apply the common fertilizer of 40% mass+
Complex microorganism liquid bacterial agent.The fertilizer applied is every kilogram of soil 150mg, and the dosage of complex microorganism liquid bacterial agent is every thousand
Gram soil 0.2mL, the results are shown in Table 1.
Influence of the 1 complex microorganism liquid bacterial agent of table to potting Growth of Cabbage characteristic and yield
It mixes and applies containing polyglutamic acid, biological organic matter, the complex microorganism liquid bacterial agent of Promoting bacteria and fertilizer in the present invention, energy
The development for enough promoting pakchoi root makes root thickening, increases, all to pakchoi plant plant height, underground part fresh weight, overground part fresh weight
There is apparent facilitation, 54.8% can be increased production.It, still can be to Chinese cabbage output increased when subtracting the fertilizer for applying 40% mass
20.8%, this illustrate polyglutamic acid in the present invention, biological organic matter, growth promoting bacteria agent complex microorganism liquid bacterial agent can promote
Plant growth, and effectively reduce the usage amount of chemical fertilizer.
5 complex microorganism liquid bacterial agent of embodiment significantly improves the quality of crops
The complex microorganism liquid bacterial agent preparation containing polyglutamic acid, biological organic matter and Promoting bacteria in the present embodiment is the same as real
Apply example 2.Using muskmelon as experimental subjects, test sets 3 processing, 3 repetitions of each processing altogether.Processing 1 is application Common compound fertilizer
(450kg/ha2), processing 2 is application Common compound fertilizer (450kg/ha2)+complex microorganism liquid bacterial agent (6L/ha2), processing 3
For the Common compound fertilizer+complex microorganism liquid bacterial agent (315kg/ha for reducing 40% mass of application2), muskmelon management is daily pipe
Reason, the results are shown in Table 2.
Influence of the 2 complex microorganism liquid bacterial agent of table to muskmelon Vc and nitrate content
Compared with processing 1, Vc content 48.8% is can be improved in complex microorganism liquid bacterial agent, reduces nitrate content
24.3%.When reducing the compound fertilizer of 40% mass of application, Vc content 29.3% is can be improved in complex microorganism liquid bacterial agent, drop
Protonitrate content 46.6% improves the quality of muskmelon.Test result shows the complex microorganism liquid bacterial agent in the present invention not
But the usage amount of chemical fertilizer can be reduced, and the quality of muskmelon can also be significantly improved.
6 complex microorganism liquid bacterial agent of embodiment significantly improves the anti-adversity ability of plant
Preparing for the complex microorganism liquid bacterial agent containing polyglutamic acid, biological organic matter and Promoting bacteria in the present embodiment is same
Embodiment 2.Using Xiaoqinling Nature Reserve as experimental subjects, potting is carried out, experiment sets 3 processing, 3 repetitions of each processing altogether.Water control method
Using Hsiao (1973) mesad gradation of moisture partitioning, processing 1 is that control 75%-80% (accounts for maximum water holding capacity
Percentage), processing 2 coerces 40%-45% for moderate, and processing 3 is severe water stress 30%-35%.Potting Xiaoqinling Nature Reserve is carried out just
It often manages, controls moisture before test observation, when reaching 3 gradations of moisture, in processing 2,3 addition complex microorganism liquid bacterias of processing
Agent, applied amount are every kilogram of soil 0.2mL.Period measures volumetric water content of soil daily, and the moisture that the supplement same day loses makes everywhere
Reason level reaches the water content of setting, and experimental result is shown in Table 3.
Influence of the 3 complex microorganism liquid bacterial agent of table to Xiaoqinling Nature Reserve chlorophyll and fresh weight
As the result is shown：Under the environmental condition of mild drought, by applying complex microorganism liquid bacterial agent, with control group phase
Than Xiaoqinling Nature Reserve can increase production 23.2%, under the environmental condition of Severe drought, by applying complex microorganism liquid bacterial agent, small blueness
Dish yield remains to raising 11.0% compared with the control group.The experimental results showed that complex microorganism liquid bacterial agent can improve plant
Drought-resistant ability, to promote the growth of plant.
Sequence table
<110>Nanjing Shinekingbiotech, Ltd.
<120>A kind of microbial liquid microbial inoculum of the general bacterium containing camel thorn and its preparation method and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1488
<212> DNA
<213> Pantoea alhagi
<400> 1
tctggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgaac ggtaacaggg 60
agcagcttgc tgctctgctg acgagtggcg gacgggtgag taatgtctgg ggatctgccc 120
gatggagggg gataaccact ggaaacggtg gctaataccg cataacgtcg caagaccaaa 180
gtgggggacc ttcgggcctc acaccatcgg atgaacccag atgggattag ctagtaggtg 240
aggtaacggc tcacctaggc gacgatccct agctggtctg agaggatgac cagccacact 300
ggaactgaga cacggtccag actcctacgg gaggcagcag tggggaatat tgcacaatgg 360
gcgcaagcct gatgcagcca tgccgcgtgt atgaagaagg ccttcgggtt gtaaagtact 420
ttcagcgggg aggaagggag caaggttaat aacctggttc attgacgtta cccgcagaag 480
aagcaccggc taactccgtg ccagcagccg cggtaatacg gagggtgcaa gcgttaatcg 540
gaattactgg gcgtaaagcg cacgcaggcg gtctgtcaag tcggatgtga aatccccggg 600
ctcaacccgg gaactgcatt cgaaactggc aggctagagt cttgtagagg ggggtagaat 660
tccaggtgta gcggtgaaat gcgtagagat ctggaggaat accggtggcg aaggcggccc 720
cctggacaaa gactgacgct caggtgcgaa agcgtgggga gcaaacagga ttagataccc 780
tggtagtcca cgccgtaaac gatgtcgact tggaggctgt gagcttgact cgtggcttcc 840
ggagctaacg cgttaagtcg accgcctggg gagtacggcc gcaaggttaa aactcaaatg 900
aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgatgc aacgcgaaga 960
accttacctg gccttgacat ccacggaatt ctgcagagat gcggaagtgc cttcgggaac 1020
cgtgagacag gtgctgcatg gctgtcgtca gctcgtgttg tgaaatgttg ggttaagtcc 1080
cgcaacgagc gcaaccctta tcctttgttg ccagcgattc ggtcgggaac tcaagggaga 1140
ctgccggtga taaaccggag gaaggtgggg atgacgtcaa gtcatcatgg cccttacggc 1200
cagggctaca cacgtgctac aatggcgcat acaaagagaa gcgacctcgc gagagcaagc 1260
ggacctcata aagtgcgtcg tagtccggat cggagtctgc aactcgactc cgtgaagtcg 1320
gaatcgctag taatcgtgga tcagaatgcc acggtgaata cgttcccggg ccttgtacac 1380
accgcccgtc acaccatggg agtgggttgc aaaagaagta ggtagcttaa ccttcgggag 1440
ggcgcttacc actttgtgat tcatgactgg ggtgaagtcg taacaagg 1488
Claims (10)
1. a kind of microbial liquid microbial inoculum of general bacterium containing camel thorn, which is characterized in that the component including following mass percent：Promote
Raw bacterium bacterium solution 1%-10%, polyglutamic acid 0.1%-10%, biological organic matter 50%-70%, surplus are water or aqueous solution；It is described
Promoting bacteria bacterium solution includes the general bacterium of camel thorn (Pantoea alhagi) bacterium solution.
2. microbial liquid microbial inoculum according to claim 1, which is characterized in that the general bacterium bacterium solution of camel thorn is camel thorn
General fermented liquid, viable count is 1 × 10 in fermentation liquid9-9×1010CFU/mL；The general bacterium of camel thorn (Pantoea alhagi)
It is CGMCC NO.15525 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.
3. microbial liquid microbial inoculum according to claim 2, which is characterized in that the Promoting bacteria bacterium solution further includes withered grass bud
Spore bacillus (Bacillus subtilis), bacillusmusilaginosiengineering (Bacillus mucilaginosus) conciliate starch gemma
The combination of any one fermentation liquid or several fermentation liquids, bacillus subtilis in bacillus (Bacillus amyloliquefaciens)
Viable count is 1 × 10 in fermented liquid, bacillusmusilaginosiengineering fermentation liquid or bacillus amyloliquefaciens fermentation liquid10-9×
1010CFU/mL, viable count >=5 × 10 of the Promoting bacteria in microbial liquid preparation9CFU/mL。
4. microbial liquid microbial inoculum according to claim 3, which is characterized in that the bacillus subtilis (Bacillus
It subtilis is) CCTCC NO in the deposit number of China typical culture collection center:M 2016264, gel-shaped gemma bar
Bacterium (Bacillus mucilaginosus) is CCTCC NO in the deposit number of China typical culture collection center:
M2016265, bacillus amyloliquefaciens (Bacillus amyloliquefaciens) are in China typical culture collection center
Deposit number is CCTCC NO:M 2016266.
5. microbial liquid microbial inoculum according to claim 4, which is characterized in that the Promoting bacteria bacterium solution is the general bacterium of camel thorn
The combination of fermentation liquid, bacillus subtilis fermentation liquor, bacillusmusilaginosiengineering fermentation liquid and bacillus amyloliquefaciens fermentation liquid,
Volume ratio is 0.1-10:0.1-10:0.1-10:0.1-10.
6. microbial liquid microbial inoculum according to claim 1, which is characterized in that the polyglutamic acid weight average molecular weight is 5-
3000kDa, the polyglutamic acid are polyglutamic acid sterling, polyglutamate sterling, polyglutamic acid fermentation liquid, polyglutamic acid fermentation
Any one or the combination of several of them in liquid dried object and polyglutamic acid wettable powder.
7. microbial liquid microbial inoculum according to claim 1, which is characterized in that the biological organic matter is biochemical yellow rotten
Any one or the combination of several of them in acid, molasses and yeast extract.
8. the preparation method of microbial liquid microbial inoculum described in claim 1-7 any one, which is characterized in that steps are as follows：
Promoting bacteria bacterium solution, polyglutamic acid and biological organic matter are mixed according to mass percent to get microbial liquid microbial inoculum.
9. microbial liquid microbial inoculum described in claim 1-7 any one improve crop yield, promoted crops quality,
Enhance the application in any one or a few in crops resistance.
10. application according to claim 9, which is characterized in that the microbial liquid microbial inoculum passes through water planting, sprays or fill
Root application is used for crops.
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| CN110317754A (en) * | 2019-06-28 | 2019-10-11 | 轩凯生物科技(滁州)有限公司 | A kind of microbial liquid microbial inoculum and preparation method thereof containing polyglutamic acid |
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