CN104630087B - A kind of Rhizosphere of Crops Promoting bacteria YM4 and its application - Google Patents
A kind of Rhizosphere of Crops Promoting bacteria YM4 and its application Download PDFInfo
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Abstract
The present invention relates to Rhizosphere of Crops Promoting bacteria YM4 and its application, Rhizosphere of Crops Promoting bacteria is Rhizosphere of Crops Promoting bacteria YM4, Classification And Nomenclature for bacillus pumilus (Bacillus pumilus), on October 31st, 2014 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.9898, it can effectively solve slightly solubility being converted into soluble potassium salt containing potassium silicate, organophosphor is solved, the utilization rate of fertilizer is improved, effective for the plantation of corn, promote the growth of corn and improve yield, be the big innovation on microorganism and corn planting.
Description
Technical field
The present invention relates to agriculture microorganism, particularly a kind of Rhizosphere of Crops Promoting bacteria YM4 and its application.
Background technology
Sandstone area permeability is strong, and ventilation is good, and aerobic microorganism activity is dominant, can promote organic matter decomposition,
The mineralization quickening of organic matter.And loosing soil, easy farming.Soil capillarity is strong, and moisture operation is fast, there is " Evening Tide " phenomenon.It is suitable
The cultivated phase is also grown, Yi Limiao；But nutrient content is low, fertilizer conservation poor performance, the easily de- fertile early ageing of crop later stage.At present, the soil is most of
Make cultivated land utilization, its utilization rate yields two crops a year up to 92.3%, general year per mu yield grain 600kg or so.Targetedly improvement is arranged from now on
Apply：Because soil is planted, rational use of chemical fertilizer, carries out rational application of fertilizers；Multipath applying organic manure；Farm field and forest network is carried out, is being ensured
On the premise of grain-production grows steadily, the industrial crops such as development Chinese yam, watermelon.
Plant growth-promoting rhizobacteria (Plant Growth Promoting Bacteria, abbreviation PGPB) is defined as in certain bar
Be conducive under part the free living of plant growth soil, rhizosphere, root table, phyllosphere bacterium.These bacteriums being capable of fixed nitrogen, molten
Phosphorus, molten iron, and plant hormone is produced, such as auxin, gibberellin, the basic element of cell division and ethene.In addition, they can also improve plant
Resistance, including arid, high salt, heavy metallic poison and pesticide.Therefore the isolated plant growth-promoting rhizobacteria from moisture soil, and crop
Syntaxial system is formed, using biological prosthetic improvement moisture soil, becomes the hot spot of current research, but so far there are no Rhizosphere of Crops growth-promoting
The open report of bacterium.
The content of the invention
For the above situation, to overcome the defect of the prior art, the purpose of the present invention is just to provide a kind of Rhizosphere of Crops and promotees
Raw bacterium YM4 and its application, that is to say, that it is an object of the present invention to provide a kind of plant growth-promoting rhizobacteria, another purpose is to carry
For the application of the plant growth-promoting rhizobacteria, it can effectively solve slightly solubility being converted into soluble potassium salt containing potassium silicate, solve organophosphor, carry
The utilization rate of high fertilizer, the problem of promoting the growth of corn and improve yield.
The technical solution that the present invention solves is that Rhizosphere of Crops Promoting bacteria is Rhizosphere of Crops Promoting bacteria YM4, and Classification And Nomenclature is short
Bacillus pumilus (Bacillus pumilus), on October 31st, 2014 is preserved in China Committee for Culture Collection of Microorganisms
Common micro-organisms center, preserving number are CGMCC No.9898, depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number.
Rhizosphere of Crops Promoting bacteria YM4（CGMCC No.9898）Bacterium colony is small, milky, opaque, rough surface, and edge is thick
Rough, irregular rod-shaped arrangement, produces gemma.
The physio-biochemical characteristics of Rhizosphere of Crops Promoting bacteria YM4 are：Gram-positive, amphimicrobian, chemoheterotrophy, catalase
The positive, M.R negatives, VP experiments are positive, and Starch Hydrolysis is positive, gelatin liquefaction positive, and nitrate reduction is positive, citrate
Utilize feminine gender.
The main nitrogen that Rhizosphere of Crops Promoting bacteria YM4 is used when cultivating includes but not limited to peptone, dusty yeast, the third ammonia
Acid, potassium nitrate, ammonium nitrate, ammonium sulfate, urea；The primary carbon source used include but not limited to glucose, sucrose, fructose, xylose,
Mannitol, lactose, maltose；The inorganic component used includes but not limited to potassium chloride, sodium chloride, sodium dihydrogen phosphate, phosphoric acid hydrogen
Dipotassium, tricalcium phosphate, calcium chloride dihydrate, bitter salt, seven water and ferrous sulfate.YM4 fermentations can be at 28 ~ 32 DEG C, pH5 ~ 9
In the environment of carry out.
The preserving number is applications of the Rhizosphere of Crops Promoting bacteria YM4 of CGMCC No.9898 in corn growth is promoted；
The preserving number is the Rhizosphere of Crops Promoting bacteria YM4 of CGMCC No.9898 answering in plant culture or plantation
With；
The preferred corn of the plant.
The plant growth-promoting rhizobacteria YM4 energy high yields heteroauxin can simultaneously be given birth to using slightly solubility containing potassium silicate for potassium resource
It is long, while there is insoluble inorganic microcosmic salt of solving problem, organophosphor ability.
Plant growth-promoting rhizobacteria YM4 of the present invention secretes heteroauxin（IAA）Ability it is strong, up to 37.18mgL-1.Indoles
Acetic acid is one kind of plant hormone, can promote the development of root.The strain of heteroauxin is produced, is often attached to root system of plant or leaf
Surface, IAA and a small amount of GA is produced while producing secretion using plant metabolism3The physiology mistake of plant is influenced Deng plant hormone
Journey and metamorphosis.Show as directly facilitating the elongation of root, so as to increase the chance with the contact of nutriment in soil；Can
Improve the content of plant Endogenous IAA；The expression of inducing plant defense gene, raising plant is disease-resistant, the resistance such as drought resisting.Make
For the prioritization scheme of the present invention, the fermentation of the plant growth-promoting rhizobacteria produces IAA amount highests under the lower progress in pH8 ~ 9, the environment.
As the further optimization of the present invention, the carbon source that the plant growth-promoting rhizobacteria YM4 is used for sucrose, the nitrogen source that uses for
The combination of dusty yeast or glutamic acid or both.Using culture medium made from above-mentioned carbon source and nitrogen source, the plant growth-promoting rhizobacteria cultivated
Produce the amount highest of IAA.
Plant growth-promoting rhizobacteria YM4 of the present invention contains potassium silicate as potassium resource using slightly solubility, with slightly solubility Inorganic phosphate, has
Machine phosphorus is grown for phosphorus source, and is translated into soluble potassium salt, microcosmic salt.Under the conditions of laboratory shake flask, the rhizosphere growth-promoting
Raw bacterium YM4 reaches 17.40mgL to the inversion quantity of feldspar in powder-1, 128.90 mgL are reached to the inversion quantity of tricalcium phosphate-1, 0.65 mgL is reached to organophosphor inversion quantity-1.Illustrate that YM4 bacterium have dissolving to feldspar in powder, tricalcium phosphate, organophosphor
Effect, can be grown, and be translated into soluble-salt as potassium resource, phosphorus source.
Beneficial effect：Plant growth-promoting rhizobacteria YM4 high yield heteroauxins provided by the invention, can be effectively by slightly solubility silicic acid containing potassium
Salt is converted into soluble potassium salt, solve problem insoluble inorganic phosphorus, organophosphor, improves the utilization rate of fertilizer, promote plant root system development and
Absorption to fertilizer, increase available potassium in soils, phosphorus content；The present invention has good growth-promoting effect for corn, high yield
Heteroauxin promotes the growth and development of corn, and the raising of available potassium in soils, phosphorus content is also so that utilization of the corn to potash fertilizer, phosphate fertilizer
Rate higher, effective for the plantation of corn, improves yield, is the big innovation on microorganism and corn planting.
Brief description of the drawings
Fig. 1 is the bacterium colony figure of bacterial strain YM4 of the present invention；
Fig. 2 is influence figure of the different liquid amounts of the invention to bacterial strain YM4 productions IAA；
Fig. 3 is influence figures of the different initial pH of the invention to YM4 bacterial strains production IAA；
Fig. 4 is influence figure of the different carbon source of the present invention to bacterial strain YM4 productions IAA；
Fig. 5 is influence figure of the different nitrogen sources of the present invention to YM4 bacterial strains production IAA；
Fig. 6 is utilization power figures of the bacterial strain YM4 of the present invention to slightly solubility feldspar in powder；
Fig. 7 is utilization power figures of the bacterial strain YM4 of the present invention to organophosphor；
Fig. 8 is utilization power figures of the bacterial strain YM4 of the present invention to slightly solubility Phos；
Fig. 9 is that maize planting of the present invention is inoculated with influence figure of the YM4 bacterial strains to soil IAA contents after 30 days.
Embodiment
Elaborate below in conjunction with concrete condition to the embodiment of the present invention.
Biomaterial preservation information
Plant growth-promoting rhizobacteria YM4, Classification And Nomenclature are bacillus pumilus (Bacillus pumilus), on October 31st, 2014
China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved in, address is BeiChen West Road, Chaoyang District, BeiJing City 1
Number No. 3 Institute of Microorganism, Academia Sinica of institute, preserving number is CGMCC No.9898.
Table 1 is for examination soil labile organic matter
| Soil | Organic carbon（g/kg） | Full phosphorus（g/kg） | Rapid available phosphorus（mg/kg） | Full potassium（g/kg） | Available potassium（mg/kg） | pH(H2O) |
| Sandstone area | 1.91 | 0.29 | 3.44 | 19.56 | 20.42 | 7.39 |
The physio-biochemical characteristics of 2 YM4 bacterial strains of table
| Project | As a result | Project | As a result |
| Gram's staining | + | Starch Hydrolysis | + |
| Aerobic is tested | Amphimicrobian | Gelatin liquefaction | + |
| Catalase test | + | Nitrate reduction | + |
| Methyl red（M.R）Reaction | - | Citrate utilizes | - |
| V-P is tested | + |
Note：+ :Positive reaction； -:Negative reaction
In specific implementation, following culture medium is prepared first：
LB culture mediums：Peptone 10g, yeast extract 5g, sodium chloride 10g, agar 20g, distilled water 1000ml, pH
7.0-7.2,121 DEG C of sterilizings, 20min.
LB fluid nutrient mediums：Agar is not added with, other conditions are same as above.
Meng Jinna culture mediums：Glucose 10.0g, (NH4)2SO4 0.5g, MgSO4·7H2O 0.3g, NaCl 0.3g, KCl
0.3g, FeSO4 0.03g, MnSO4·H2O 0.03g, distilled water 1000ml, 115 DEG C of sterilizing 30min.
Organophosphor fluid nutrient medium：1000ml Meng Jinna culture mediums add 0.4g yeast extracts, then add the solvable lecithins of 0.2g.
Inorganic phosphorus bacteria fluid nutrient medium（PKO culture mediums）：Tricalcium phosphate 5g, glucose 10g, ammonium sulfate 0.5g, chlorination
Sodium 0.3g, bitter salt 0.3g, potassium chloride 0.3g manganese sulfate 0.03g, green vitriol 0.03g, distilled water
1000ml, pH7.0 ~ 7.2,121 DEG C of sterilizings, 20min.
Potassium bacterium fluid nutrient medium：Sucrose 10.0g, yeast extract 0.5g,（NH4）2SO41.0g, Na2HPO4
2.0g, MgSO4·7H2O 0.5g, CaCO31.0g, feldspar in powder 1.0g, distilled water 1000mL, 121 DEG C go out
Bacterium, 20min.
Nitrogen fixing capacity uses Ashby nitrogen-free agars：Mannitol 10g, KH2PO40.2g, MgSO40.2g, NaCl
0.2g, K2SO40.3g, CaCO35g, distilled water 1000mL, Agar 15g, 121 DEG C of sterilizings, 20min.
Minimal medium：Ammonium sulfate 2.0g；Sodium dihydrogen phosphate 0.5g；Dipotassium hydrogen phosphate 0.5g；Epsom salt 0.2
g；7.0,121 DEG C of sterilizings of calcium chloride dihydrate 0.1g, distilled water 1000mL, pH, 20min.
The sand taken from the North China Wheat and maize rotation nutrition of the Ministry of Agriculture of Zhengzhou City with fertilising scientific observation experiment station is claimed
L0g is taken to be placed in the triangular flask for 250 ml for filling 100 ml aqua sterilisas, in shaking table, 30 DEG C, 150rmin-1Vibration
20min, stands 10min, obtains soil bacterium suspension.Contain several plant growth-promoting rhizobacteria in the soil bacterium suspension, use is dilute
LB culture mediums are applied to after interpretation of the law dilution, tablet is inverted, in 30 DEG C, after cultivating 24h in insulating box, picking different type typical case is single
A bacterium colony, 4 DEG C to be stored in LB inclined-planes stand-by through tablet after purification.
The plant-growth promoting rhizobacteria of heteroauxin can be secreted by being filtered out again by qualitative determination and quantitative determination below.
Qualitative determination：Microbionation after isolating and purifying is contained into L-Trp in use（100 mg/L）LB liquid training
Foster base, 30 DEG C, 180 rmin-1Shaking table culture 1d.50 μ L bacteria suspensions drop is taken on whiteware plate, while adds 50 μ L
Salkowski color solutions（50mL 35%HClO4+1mL 0.5M FeCl3）.The colorimetric of 50 μ L, 50 mg/L heteroauxins will be added
Liquid is as positive control.Whiteware plate is observed after 30 min of room temperature avoid light place, and the color person of reddening represents that Yin can be secreted
Indolylbutyric acid.
Quantitative determination：The bacterium of the producing IAA obtained to primary dcreening operation quantitative determines, and condition of culture is same as above.Use and divide first
Light photometry measures the OD600 values of bacteria suspension, then by bacteria suspension with 10000 rmin-1Centrifuging 10 min takes supernatant to add
Isometric Salkowski color solutions, lucifuge stand 30min, measure its OD530Value.Calculate bacteria concentration OD600Be worth for 1 when, unit bodies
The content of heteroauxin in product zymotic fluid.The drafting of standard curve is prepared using analytically pure heteroauxin gradient dilution.
Obtained production IAA bacterium are carried out the screening test of phosphorus decomposing situation, strains tested is inoculated in and fills 50mL organophosphors
The 250mL triangular flasks of fluid nutrient medium, 30 DEG C, 200 rmin-1After cultivating 72h, the nutrient solution 10mL of culture 72h is taken,
6000r/min centrifuges 20min, takes the content of supernatant ultraviolet specrophotometer measure wherein phosphorus.
Obtained production IAA bacterium are carried out the screening test of phosphorus decomposing situation, strains tested is inoculated in and fills 50mL Phos
Fluid nutrient medium（PKO culture mediums）250mL triangular flasks, 30 DEG C, 200 rmin-1After cultivating 72h, the culture of culture 72h is taken
Liquid 10mL, 6000r/min centrifugation 20min, takes the content of supernatant ultraviolet specrophotometer measure wherein phosphorus.
Obtained production IAA bacterium are carried out the screening test of phosphorus decomposing situation, strains tested is inoculated in and fills 50mL organophosphors
250 mL triangular flasks of fluid nutrient medium, 30 DEG C, 200 rmin-1After cultivating 72h, the nutrient solution 20mL of culture 72h is taken,
6000r/min centrifuges 20min, takes supernatant ultraviolet specrophotometer to measure wherein K+Content.
Measure can filter out high yield heteroauxin more than, insoluble inorganic phosphorus of solving problem, phosphorus-containing matter, ability of dissolving potassium
Strong bacterial strain, is named as YM4.As shown in Figure 1, the bacterium colony that the bacterial strain is formed is small, milky is opaque, rough surface, and edge is thick
Rough, irregular rod-shaped arrangement, produces gemma.As shown in fig. 6, bacterial strain YM4 is grown using slightly solubility feldspar in powder as potassium resource, and will
It is converted into soluble potassium salt.Under the conditions of laboratory shake flask, the plant growth-promoting rhizobacteria YM4 reaches the inversion quantity of feldspar in powder
17.40mg·L-1.Illustrate that YM4 bacterium have dissolution to feldspar in powder, grown using slightly solubility containing potassium silicate as potassium resource,
And it is translated into soluble potassium salt.As shown in fig. 7, bacterial strain YM4 using it is difficult using organophosphor grown as phosphorus source, and by its
It is converted into available phosphorus.Under the conditions of laboratory shake flask, the plant growth-promoting rhizobacteria YM4 reaches the inversion quantity of feldspar in powder
0.65mg·L-1.Illustrate that YM4 bacterium have transformation to solvable lecithin, using it is difficult using organophosphor given birth to as phosphorus source
It is long, and it is translated into available phosphorus.As shown in figure 8, bacterial strain YM4 is grown using slightly solubility Phos as phosphorus source, and will
It is converted into soluble microcosmic salt.Under the conditions of laboratory shake flask, the plant growth-promoting rhizobacteria YM4 reaches the inversion quantity of tricalcium phosphate
128.90mg·L-1.Illustrate that YM4 bacterium have dissolution to tricalcium phosphate, grown using slightly solubility Inorganic phosphate as phosphorus source,
And it is translated into titanium pigment.
The quantitative determination of nitrogen-fixing bacteria nitrogen fixing capacity：The azotobacteria obtained to primary dcreening operation quantitative determines, will be purified
Nitrogen-fixing bacteria are respectively connected in nitrogen-free fluid nutrient medium, when culture 24 is small after with centrifugal process collect bacterium cell, then in the bacterium being collected into
45 ml sterile waters and 5 ml nitrogen-free fluid nutrient mediums are added in cell, form 50 ml fixed nitrogen bacterium suspensions (about 107 every milliliter
Bacterium cell) it is used as bacterium solution to be seeded.
Nitrogenase activity is measured using acetylene reduction method, and concrete operations are as follows：Bacterial strain after purification is seeded in equipped with 2
In the penicillin bottle of ml nitrogen-free fluid nutrient mediums, when culture 18~24 is small at 30 DEG C；Tampon is changed to anti-chewing-gum plug sealing,
The penicillin bottle for first having bacterium from culture with the syringe of good airproof performance extracts 0.5 ml air, reinjects 0.5 ml C2H2, use
Adhesive plaster seals pinprick；Continue after cultivating 24h, take 100 μ L gas samples to measure C on gas chromatograph2H4Peak value, Standard Gases C2H4It is dense
Spend for 130 mg/L.
The HP6890 type gas chromatographs produced using Hewlett-Packard Corporation of the U.S., its operating condition are arranged to：Hydrogen flameionization
Detector, 250 DEG C of temperature, H230 ml of flow/min, pressure 20kPa；Chromatographic column is capillary, 15.0 m of column length, internal diameter
320 μm, 30 DEG C of furnace temperature；Carrier gas N2, 30 ml of flow/min, pressure 20kPa；250 ml of air mass flow/min；Preceding injection port
250 DEG C of temperature, 20 kPa of pressure.Acetyiene reduction activity（ARA）, computational methods are：
Actual C2H4Peak area × standard Gas content × penicillin bottle volume
ARA= ——————————————————————
Standard Gases peak area × sample size × incubation time × sample size
（Unit：nmol C2H4/h·ml）
The bacterial strain that above method screening is isolated, through Shanghai, handsome bioengineering Co., Ltd is sequenced, according to 16SrDNA
Sequencing result, in http://www.ncbi.nlm.nih.gov online queries are analyzed, using Blast softwares in GenBank
Tetraploid rice, sequence similar in selection and the sequence of YM4 MEGA version 3 are carried out with other 16S rDNA sequences
The 16SrDNA systematic evolution trees of software building YM4.According to the physiological and biochemical property of the bacterial strain, bacillus pumilus is accredited as
(Bacillus pumilus).The bacterial strain is common in China Committee for Culture Collection of Microorganisms on October 31st, 2014
The preservation of microorganism center, preserving number CGMCC No.9898.
The strain is small in Gram-positive, sporiferous irregular rod-shaped bacterium colony, and milky is opaque, rough surface, side
Edge is coarse, and irregular rod-shaped arrangement, produces gemma.Amphimicrobian, chemoheterotrophy.Optimum growth temperature is 30 DEG C.Contact enzyme positive,
Nitrate reduction is positive.Producing IAA ability is strong, reaches 37.18 μ gmL-1, grown using slightly solubility feldspar in powder as potassium resource,
And soluble potassium salt is translated into, there is nitrogen fixing capacity.
Aerobic is tested
Sterilized LB culture mediums are poured into 3 sterilized test tubes, about at 2/3, on aseptic operating platform, are used
The bacterial strain YM4 of transfer needle picking inclined-plane culture（CGMCC No.9898）, percutaneous puncture-inoculation is into above-mentioned culture medium（It must be punctured to
Tube bottom）.30 DEG C of cultures, observed result at 3 days to 7 days respectively.Preferably oxygen bacterium, such as edge puncture raw elder on agar column surface
Line life elder is anaerobic bacteria or facultative anaerobic bacteria.
Result of the test shows, and bacterial strain YM4 bacterium colonies are grown along agar column surface, also has colony growth in puncture line, is facultative
Anaerobism.
The measure of catalase
The drop of drop 1 3%H on clean slide2O2, take 18 ~ 24 h cultivate 1 ring of bacterial strain YM4 LB slant cultures,
H2O2Middle smearing, is the positive if having bubble generation, is otherwise feminine gender.
Result of the test shows bacterial strain YM4 for contact enzyme positive.
Methyl red test（M.R is tested）
A. culture medium and reagent：Peptone 5g, glucose 5g, sodium chloride 5g, distilled water 1000mL, adjusting pH7.0 ~
7.2, test tube is dispensed, often pipe fills 4 ~ 5 mL, 121 DEG C of 20 min of sterilizing.Reagent：Methyl red 0.1g, 95 % alcohol, 300 mL, steam
200 mL of distilled water.
B. Spawn incubation and result observation inoculating strain YM4（CGMCC No.9898）In above-mentioned nutrient solution, 30 DEG C of trainings
Support l ~ 2 day.A few drop methyl red reagents are added in nutrient solution, are that methyl red is positive such as nutrient solution presentation red, yellow is cloudy
Property（Methyl red color change interval 4.4 is red ~ 6.0 yellow）.
Result of the test shows that bacterial strain YM4 is negative for M.R.
Second phthalein carbinol methine is tested（VP is tested）
A. the same methyl red test of culture medium.B. Spawn incubation and result observation inoculation are with cultivating same methyl red test.Do
When VP is tested, nutrient solution is taken（About 2mL）Mixed with 40 %NaOH of equivalent, add a small amount of creatine, fully vibrate 2 ~ 5 min
Afterwards, as red, as the VP positives occurs in nutrient solution.
Result of the test shows that bacterial strain YM4 is positive for VP.
Starch Hydrolysis is tested
A. culture medium and reagent add 0.2% soluble starch in meat soup peptone agar, dispense triangular flask, and 121
DEG C sterilizing 20min it is spare.Road Ge Shi iodine solutions：Crystalline flake of iodine 1g, potassium iodide 2g, first with a small amount of（3~5mL）Distilled water dissolves potassium iodide, existing
The crystalline flake of iodine is added, after iodine is completely dissolved, is diluted with water to 300mL.
B. Spawn incubation and result observation take YM4 strains point to be connected on tablet, and 30 DEG C are cultivated 2 ~ 4 days, after forming bacterium colony,
Road Ge Shi iodine solutions are added dropwise on tablet, to be paved with periphery of bacterial colonies as degree, tablet is in blueness, and periphery of bacterial colonies is if any water white transparency circle
Occur, illustrate that starch has been hydrolyzed.The size of the size general remark hydrolysis starch ability of transparent circle.
Result of the test shows that bacterial strain YM4 is positive for Starch Hydrolysis.
Nitrate reduction test
A. culture medium and reagent nitrate fluid nutrient medium：Peptone 10g, KNO31g, distilled water 1000mL, pH7.0 ~
7.4.Ge Lisishi（Gries）Reagent：A liquid：P-aminobenzene sulfonic acid 0.5g, spirit of vinegar（10% or so）150mL；B liquid：Cai's amine
0.1g, distilled water 20mL, spirit of vinegar（10% or so）150mL.Diphenylamines reagent:Diphenylamines 0.5g is dissolved in the l00mL concentrated sulfuric acids,
Diluted with 20mL distilled water.
B. bacterial strain YM4 is inoculated in nitrate fluid nutrient medium by Spawn incubation and result observation, 30 DEG C of cultures 1,3,5
My god.A little nutrient solution is poured into white porcelain dish aperture, then drop 1 drips reagent A and B liquid respectively wherein, when nutrient solution is changed into
When pink, rose, orange or brown etc., nitrite presence is indicated, be that nitrate reduction is positive, be otherwise the moon
Property.
Result of the test bacterial strain shows that YM4 is positive for nitrate reduction.
The utilization of citrate
A. culture medium and reagents citric acid sodium 2g, NaCl 5g, MgSO4·7H2O 0.2g, (NH4)2·HPO4 1g, 1%
Bromothymol blue aqueous solution 10mL, agar 20g, 1000mL, pH6.8-7.0,121 DEG C of sterilizing 20min of distilled water.
B. Spawn incubation and result observation take young age YM4 strains to be inoculated on inclined-plane, and 30 DEG C are cultivated 3-7 days, culture medium
In alkalescence（Blueness）Person is positive reaction, and constant person is then feminine gender.
The result of the test that citrate utilizes shows bacterial strain YM4 for feminine gender.
In order to further verify plant growth-promoting rhizobacteria YM4 production heteroauxin ability and optimum condition, below for different pH,
Liquid amount, different carbon source, different nitrogen sources explore the influence to heteroauxin yield.
25ml, 50ml, 75ml will be pressed containing L-Trp (100mg/L) LB fluid nutrient mediums, 100ml, 150ml are loaded on
In the triangular flask of 250mL, by 1%（v/v）After YM4 of the inoculum concentration inoculation in exponential phase, 30 DEG C are placed in, 180rmin-1
24 h of shaking table culture, by the amount of the method measure production IAA of quantitative determination.The results are shown in Figure 2, since bacterial strain YM4 is facultative to detest
Oxygen metabolism, throughput influences the efficiency of bacterial strain production IAA, during 25mL liquid amounts, bacterial strain production IAA amounts at most, afterwards with liquid amount
Increase, yield are fewer.
LB culture mediums containing L-Trp (100mg/L) are respectively adjusted to different pH（4、5、6、7、8、9、10）,
Take in triangular flasks of the 50mL loaded on 250mL, by 1%（v/v）After YM4 of the inoculum concentration inoculation in exponential phase, 30 DEG C are placed in,
180r·min-1Shaking table culture 24h, by the amount of the method measure production IAA of quantitative determination, the results are shown in Figure 3, and it is 4 Hes to show pH
IAA is not produced when 10, in strong acid and strong base environment, thalline can not carry out growth metabolism, and strain produces IAA more than acid in micro- alkali environment
Property environment, the optimal pH of strain high yield IAA is 7 ~ 9.
1% is separately added into containing L-Trp (100mg/L) minimal medium（w/v）Carbon source, carbon source has grape
Sugar, xylose, sucrose, fructose, mannitol, lactose, maltose, take in triangular flasks of the 50ml loaded on 250ml, by 1%（v/v）Inoculation
After YM4 of the amount inoculation in exponential phase, 30 DEG C are placed in, 180 rmin-124 h of shaking table culture, by the side of quantitative determination
The amount of method measure production IAA.The results are shown in Figure 4, and for the bacterial strain when supplying sucrose, it is most strong to produce the ability of IAA, secondly xylose.
0.1% is separately added into containing L-Trp (100mg/L) minimal medium (not including ammonium sulfate)（w/v）
Nitrogen source, nitrogen source includes ammonium nitrate, ammonium sulfate, potassium nitrate, peptone, dusty yeast, alanine, urea etc., takes 50ml to be loaded on
1% is pressed in the triangular flask of 250ml（v/v）After YM4 of the inoculum concentration inoculation in exponential phase, 30 DEG C are placed in, 180rmin-1Shake
Bed 24 h of culture, by the amount of the method measure production IAA of quantitative determination.The results are shown in Figure 5, when illustrating that it is nitrogen source to take dusty yeast,
It is most to produce the amount of IAA, secondly glutamic acid.
Bacterial strain YM4 of the present invention has obvious growth promoting function to corn, is illustrated below by pot experiment.
The fresh soil of sand 0 ~ 20cm soil layers under natural conditions is gathered, crosses 5mm sieves, the dress soil 700g per basin, maize planting,
Water content is adjusted to the 60% of maxmun field capacity, 30 day post-sampling, uses root scanner（LA1600+ scanner,
Canada）After scanning obtains root system image, with root system analysis software（Winrhizo2003b, Canada）Associated root is carried out to mean
Mark analysis, soil IAA contents are measured with HPLC methods, and measure soil water-storage, rapid available phosphorus, quick-acting potassium content, plant fresh weight, strain
The high and full potassium of total nitrogen and total phosphor phosphorus.
Corn seed：Corn seed carries out 20% hydrogen peroxide surface sterilization 20min, and aseptic water washing is multiple, vernalization 2d, choosing
Take the consistent seed that germinates spare.
Connect bacterium processing：The YM4 of the present invention is inoculated in LB fluid nutrient mediums, 30 DEG C, 180rmin-1Shaking table culture, culture
Bacterium is grown to exponential phase, then by bacteria suspension 10000rmin-13min is centrifuged, then is resuspended with sterile water, equally centrifuges three
It is secondary, inoculum concentration 108CFU·g-1（That is every gram of dry ground inoculation 108CFU·g-1 YM4 strains）.
Control treatment：As control, soil does not spray YM4 bacterium solutions, adds equivalent sterile water.
The result is shown in following each table：
Influences of the 3 inoculating strain YM4 of table to maize root system
| Processing | Root long（cm） | Root surface area（cm2） | Root volume（cm3） | Tip of a root number（It is a） |
| CK | 773.33±138.08 | 115.87±15.67 | 1.40±0.26 | 4357.00±967.08 |
| YM4 | 2105.31±318.64** | 280.09±41.56** | 2.97±0.50** | 8911.67±1189.81** |
Note：* indicates significant difference in same row（p<0.05）, * * expression pole significant differences（p<0.01）；Similarly hereinafter.
Influences of the 4 inoculating strain YM4 of table to plant
| Processing | Fresh weight（g) | Plant height（cm） | SPAD | Full nitrogen（g/kg） | Full phosphorus（g/kg） | Full potassium（g/kg） |
| CK | 1.43±0.18 | 22.37±1.56 | 21.18±1.51 | 1.81±0.05 | 1.99±0.56 | 14.29±1.84 |
| YM4 | 3.29±0.50** | 33.70±3.20** | 32.10±0.87** | 2.19±0.10** | 3.31±0.31** | 19.75±0.30** |
Influences of the 5 inoculating strain YM4 of table to soil mineral state nitrogen, rapid available phosphorus and available potassium
| Processing | Ammonium nitrogen（mg/kg） | Nitrate nitrogen（mg/kg） | Mineral nitrogen（mg/kg） | Rapid available phosphorus（mg/kg） | Available potassium（mg/kg） |
| CK | 22.35±1.45 | 5.42±5.19 | 26.72±0.32 | 2.24±0.31 | 35.00±1.73 |
| YM4 | 31.63±0.76** | 6.17±0.14* | 37.97±0.50** | 3.75±0.35** | 40.00±1.73* |
As can be seen from Table 4, the overground part fresh weight for the plant that YM4 soil-growns go out is vaccinated with, and plant height is compared with CK all
There is obvious growth trend；Since YM4 has the function that fixing nitrogen, dissolving phosphor and dissolving potassium, make soil Mineral N, rapid available phosphorus and quick-acting
Potassium content increase（It is shown in Table 5）, so that absorption of the plant to elements such as N, P, K is promoted, from table 3 it can be seen that inoculation YM4 processing
Contrasted with not connecing bacterium processing, maize root system total length, root surface area, average root diameter and tip of a root number are all dramatically increased, promoted
Into the development of maize root system；From fig. 9, it can be seen that after connecing bacterium processing, soil IAA contents dramatically increase, higher than control group
Go out 2.5 times or so.With reference to result above as can be seen that growths of the plant growth-promoting rhizobacteria YM4 to foundation of the present invention, development have it is bright
Effective fruit, IAA yield is high, can effectively facilitate crop growth, improves yield.
With reference to result above as can be seen that growth, developments of the plant growth-promoting rhizobacteria YM4 of the present invention to foundation have obvious effect
Fruit, IAA yield is high, can promote crop growth effective for corn planting, yield be improved, through 3 years continuously in 3 mu of jade
Rice field is applied, and yield improves more than 10%, and the good of its effect is not expected.
The above is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (3)
1. a kind of Rhizosphere of Crops Promoting bacteria YM4, Classification And Nomenclature is bacillus pumilus (Bacillus pumilus), 2014 10
The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 31st, and preserving number is CGMCC No.9898.
2. applications of the Rhizosphere of Crops Promoting bacteria YM4 in corn growth is promoted described in claim 1.
3. applications of the Rhizosphere of Crops Promoting bacteria YM4 in corn planting described in claim 1.
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