CN104560788B - A kind of peanut plant growth-promoting rhizobacteria HS9 and its application - Google Patents
A kind of peanut plant growth-promoting rhizobacteria HS9 and its application Download PDFInfo
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Abstract
The present invention relates to peanut plant growth-promoting rhizobacteria HS9 and its application, peanut plant growth-promoting rhizobacteria is peanut plant growth-promoting rhizobacteria HS9, and Classification And Nomenclature is Bacillus flexus（Bacillus flexus）On October 31st, 2014 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.9889, 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 peanut, promote the growth of peanut and improve yield, be the big innovation on microorganism and peanut cultivation.
Description
Technical field
The present invention relates to microorganism, particularly a kind of peanut plant growth-promoting rhizobacteria HS9 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 crop later stage easily takes off fertile early ageing.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, rational application of fertilizers is carried out；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 advantageous 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 agricultural chemicals.Therefore the isolated plant growth-promoting rhizobacteria from moisture soil, and crop
Syntaxial system is formed, using biological prosthetic improvement moisture soil, turns into the focus of current research, but so far there are no be exclusively used in peanut
The open report of plant growth-promoting rhizobacteria.
The content of the invention
For the above situation, to overcome the defect of prior art, the purpose of the present invention is just to provide a kind of peanut rhizosphere growth-promoting
Raw bacterium HS9 and its application, that is to say, that it is an object of the present invention to provide a kind of peanut plant growth-promoting rhizobacteria, another purpose
It is to provide the application of the plant growth-promoting rhizobacteria, can effectively solves slightly solubility being converted into soluble potassium salt containing potassium silicate, solution is organic
Phosphorus, the utilization rate of fertilizer is improved, promote the growth of peanut and improve yield.
The technical scheme that the present invention solves is that peanut plant growth-promoting rhizobacteria is peanut plant growth-promoting rhizobacteria HS9, and Classification And Nomenclature is curved
Bent bacillus（Bacillus flexus）, it is preserved in China Committee for Culture Collection of Microorganisms on October 31st, 2014
Common micro-organisms center, preserving number are CGMCC No.9889.
Peanut plant growth-promoting rhizobacteria HS9（CGMCC No.9889）Bacterium colony concentric circles, it is opaque, rough surface, do not moisten,
Produce gemma.
Peanut plant growth-promoting rhizobacteria HS9 physio-biochemical characteristics 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 peanut plant growth-promoting rhizobacteria HS9 is used when cultivating includes but is not limited to peptone, dusty yeast, the third ammonia
Acid, potassium nitrate, ammonium nitrate, ammonium sulfate, urea；The primary carbon source used include but is not limited to glucose, sucrose, fructose, xylose,
Mannitol, lactose, maltose；The inorganic component used includes but is 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.Bacillus flexus HS9 fermentations can be
28 ~ 32 DEG C, carried out in the environment of pH5 ~ 9.
Described preserving number is applications of the CGMCC No.9889 peanut plant growth-promoting rhizobacteria HS9 in peanut growth is promoted；
Described preserving number is applications of the CGMCC No.9889 peanut plant growth-promoting rhizobacteria HS9 in peanut cultivation；
The peanut plant growth-promoting rhizobacteria HS9 can produce heteroauxin, and solve problem insoluble inorganic microcosmic salt, phosphorus-containing matter can simultaneously utilize
Slightly solubility is that potassium resource is grown containing potassium silicate.
Peanut plant growth-promoting rhizobacteria HS9 of the present invention secretes heteroauxin（IAA）Ability it is strong, up to 41.53 μ g
mL-1.Heteroauxin is one kind of plant hormone, can promote the development of root.The strain of heteroauxin is produced, is often attached to plant
Root system or leaf surface, IAA and a small amount of GA is produced while producing secretion using plant metabolism3Plant is influenceed Deng plant hormone
Physiology course and metamorphosis.Show as directly facilitating the elongation of root, so as to increase the contact with nutriment in soil
Chance；The content of plant Endogenous IAA can be improved；The expression of inducing plant defense gene, raising plant is disease-resistant, drought resisting etc.
Resistance.As the prioritization scheme of the present invention, the fermentation of the plant growth-promoting rhizobacteria produces IAA amounts under the lower progress in pH5 ~ 6, the environment
Highest.
As the present invention further optimization, the carbon source that the peanut plant growth-promoting rhizobacteria HS9 is used for glucose, use
Nitrogen source is the combination of urea or dusty yeast or both.Using culture medium made from above-mentioned carbon source and nitrogen source, the rhizosphere growth-promoting cultivated
Raw bacterium production IAA amount highest.
Peanut plant growth-promoting rhizobacteria HS9 of the present invention is grown using slightly solubility containing Inorganic phosphate as phosphorus source, and by its
It is converted into soluble microcosmic salt.Under the conditions of laboratory shake flask, the peanut plant growth-promoting rhizobacteria HS9 reaches to the inversion quantity of tricalcium phosphate
To 279.23 mgL-1.Illustrate that HS9 bacterium have dissolution to tricalcium phosphate, given birth to using slightly solubility Inorganic phosphate as phosphorus source
It is long, and it is translated into soluble microcosmic salt.
Peanut plant growth-promoting rhizobacteria HS9 of the present invention is grown using phosphorus-containing matter using hardly possible as phosphorus source, and by its
It is converted into using phosphorus.Under the conditions of laboratory shake flask, inversion quantities of the peanut plant growth-promoting rhizobacteria HS9 to solvable lecithin
Reach 0.48 mgL-1.Illustrate that HS9 bacterium have decomposition to solvable lecithin, phosphorus-containing matter is utilized as phosphorus source using hardly possible
Grown, and be translated into using phosphorus.
Peanut plant growth-promoting rhizobacteria HS9 of the present invention is grown using slightly solubility containing potassium silicate as potassium resource, and by its
It is converted into soluble potassium salt.Under the conditions of laboratory shake flask, the peanut plant growth-promoting rhizobacteria HS9 reaches to the inversion quantity of feldspar in powder
To 15.26 mgL-1.Illustrate that HS9 bacterium have dissolution to feldspar in powder, given birth to using slightly solubility containing potassium silicate as potassium resource
It is long, and it is translated into soluble potassium salt.
Slightly solubility effectively can be converted into soluble potassium by peanut plant growth-promoting rhizobacteria HS9 provided by the invention containing potassium silicate
Salt, the utilization rate of fertilizer is improved, promote plant root system development and the absorption to fertilizer, increase available potassium in soils content, soil has
The raising for imitating potassium content also make it that peanut is higher to the utilization rate of potash fertilizer.By slightly solubility Inorganic phosphate and it can be difficult by organic
Phosphorus is converted into available phosphorus, increases the content of soil available phosphorus, improves the utilization rate of fertilizer, promote plant grow and
Absorption to fertilizer;The present invention has good growth-promoting effect for peanut, and the heteroauxin of high yield promotes the growth of peanut
Development and raising yield, effective for the plantation of peanut, promote the growth of peanut and improve yield, be microorganism and peanut cultivation
On a big innovation.
Brief description of the drawings
Fig. 1 is bacterial strain HS9 of the present invention bacterium colony figure；
Fig. 2 is the influence situation map that different liquid amounts produce IAA to bacterial strain HS9；
Fig. 3 is the influence situation map that different initial pH produce IAA to HS9 bacterial strains；
Fig. 4 is the influence situation map that different carbon source produces IAA to bacterial strain HS9；
Fig. 5 is the influence situation map that different nitrogen sources produce IAA to HS9 bacterial strains；
Fig. 6 is bacterial strain HS9 to utilization power figure of the slightly solubility containing potassium silicate；
Fig. 7 is utilization power figures of the bacterial strain HS9 to organophosphor；
Fig. 8 is utilization power figures of the bacterial strain HS9 to slightly solubility Phos；
Fig. 9 is inoculated with influence situation map of the HS9 bacterial strains to soil IAA contents for plantation peanut after 30 days.
Embodiment
The embodiment of the present invention is elaborated below in conjunction with concrete condition.
Biomaterial preservation：Peanut plant growth-promoting rhizobacteria HS9, Classification And Nomenclature are Bacillus flexus（Bacillus flexus）, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 31st, 2014, address is
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, preserving number are CGMCC No.9889.
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 the HS9 bacterial strains of table 2
| 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, prepare following culture medium 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 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, 10min is stood, 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, flat board is inverted, in 30 DEG C, after cultivating 24h in insulating box, picking different type typical case is single
Individual bacterium colony, 4 DEG C to be stored in LB inclined-planes stand-by through flat board after purification.
The plant of heteroauxin can be secreted by being filtered out again by qualitative determination and quantitative determination below（Peanut）Growth-promoting bacterium.
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.Take 50 μ L bacteria suspensions to drip on whiteware plate, while add 50 μ L
Salkowski color solutions（50mL 35%HClO4+1mL 0.5M FeCl3）.The colorimetric of the mg/L heteroauxins of 50 μ L 50 will be added
Liquid is as positive control.Whiteware plate is observed after the min of room temperature avoid light place 30, 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 is quantitative determined, and condition of culture is same as above.Use and divide first
Light photometry determines the OD of bacteria suspension600Value, then by bacteria suspension with 10000 rmin-1Centrifuging 10 min takes supernatant to add
Isometric Salkowski color solutions, lucifuge stand 30min, determine 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, say that strains tested is inoculated in and fill 50mL organophosphors
The 250mL triangular flasks of fluid nutrient medium, 30 DEG C, 200 rmin-1After cultivating 72h, culture 72h nutrient solution 10mL 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, say that strains tested is inoculated in and fill 50mL Phos
Fluid nutrient medium（PKO culture mediums）250mL triangular flasks, 30 DEG C, 200 rmin-1After cultivating 72h, culture 72h culture is taken
Liquid 10mL, 6000r/min centrifugation 20min, take the content of supernatant ultraviolet specrophotometer measure wherein phosphorus.
Obtained production IAA bacterium are carried out the screening test of potassium decomposing situation, strains tested is inoculated in to fill 50mL potassium decomposings thin
250 mL triangular flasks of bacteria liquid culture medium, 30 DEG C, 200 rmin-1After cultivating 72h, culture 72h nutrient solution 20mL is taken,
6000r/min centrifuges 20min, takes supernatant flame spectrophotometer to determine wherein K+Content.
Measure can filter out high yield heteroauxin more than, the strong bacterial strain of ability of dissolving potassium, be named as HS9.Such as Fig. 1 institutes
Show, the bacterium colony concentric circles that the bacterial strain is formed are opaque, rough surface, do not moisten, and produce gemma.As shown in fig. 6, bacterial strain HS9 with
Slightly solubility is that potassium resource is grown containing potassium silicate, and is translated into soluble potassium salt.It is described under the conditions of laboratory shake flask
Peanut plant growth-promoting rhizobacteria HS9 reaches 15.26 mgL to the inversion quantity of feldspar in powder-1.It is molten to illustrate that HS9 bacterium have to feldspar in powder
Solution acts on, and is grown using slightly solubility containing potassium silicate as potassium resource, and be translated into soluble potassium salt.As shown in fig. 7, bacterial strain
HS9 using it is difficult using phosphorus-containing matter grown as phosphorus source, and be translated into available phosphorus.In laboratory shake flask condition
Under, the inversion quantity for the organophosphor that the peanut plant growth-promoting rhizobacteria HS9 utilizes to hardly possible reaches 0.48 mg L-1.Illustrate HS9 bacterium to containing
Phosphorus organic matter has transformation, using it is difficult using phosphorus-containing matter grown as phosphorus source, and be translated into available
Phosphorus.As shown in figure 8, bacterial strain HS9 is grown using slightly solubility Phos as phosphorus source, and it is translated into the phosphorus of solubility.In reality
Test under the conditions of the shaking flask of room, the peanut plant growth-promoting rhizobacteria HS9 reaches 279.23mg L-1 to the inversion quantity of slightly solubility Phos.Say
Bright HS9 bacterium have dissolution to slightly solubility Phos, are grown using slightly solubility Phos as phosphorus source, and be translated into can
Dissolubility phosphorus.
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 HS9 sequence MEGA version 3 are carried out with other 16S rDNA sequences
Software building HS9 16SrDNA systematic evolution trees.According to the physiological and biochemical property of the bacterial strain, Bacillus flexus is accredited as
（Bacillus flexus）.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.9889.
The strain is in Gram-positive, sporiferous irregular shaft-like.Bacterium colony concentric circles, opaque, rough surface, no
Moistening, produce gemma.Amphimicrobian, chemoheterotrophy.Optimum growth temperature is 30 DEG C.Enzyme positive is contacted, nitrate reduction is positive.Point
It is strong to secrete IAA abilities, reaches 41.53 μ gmL-1, grown using slightly solubility containing potassium silicate as potassium resource, and be translated into can
Dissolubility sylvite.
Aerobic is tested
Sterilized LB culture mediums are poured into 3 sterilized test tubes, about at 2/3, on aseptic operating platform, used
The bacterial strain HS9 of transfer needle picking inclined-plane culture（CGMCC No.9889）, percutaneous puncture-inoculation is into above-mentioned culture medium（It must be punctured to
Ttom of pipe）.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, bacterial strain HS9（CGMCC No.9889）Bacterium colony is along agar column
Superficial growth, also there is colony growth in puncture line, be amphimicrobian.
The measure of catalase
The drop of drop 1 3%H on clean slide2O2, take 18 ~ 24 h cultivate bacterial strain HS9（CGMCC No.9889）LB is oblique
The ring of face culture 1, in H2O2Middle smearing, it is the positive if having bubble generation, is otherwise feminine gender.Result of the test shows bacterial strain HS9
（CGMCC No.9889）To 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, regulation 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, the mL of 95 % alcohol 300, steam
The mL of distilled water 200.
B. Spawn incubation and result observation inoculating strain HS9（CGMCC No.9889）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 bacterial strain HS9（CGMCC No.9889）It 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, the as VP positives occurs in nutrient solution.
Result of the test shows bacterial strain HS9（CGMCC No.9889）It 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 standby.Road Ge Shi iodine solutions：Crystalline flake of iodine 1g, KI 2g, first with a small amount of（3~5mL）Distilled water dissolves KI, 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 HS9 strains（CGMCC No.9889）Point is connected on flat board, and 30 DEG C of cultures 2 ~
4 days, after forming bacterium colony, road Ge Shi iodine solutions are added dropwise on flat board, to be paved with periphery of bacterial colonies as degree, flat board is in blueness, and bacterium colony is all
Enclose and irised out now if any water white transparency, illustrate that starch has been hydrolyzed.The size of the size general remark hydrolysis starch ability of transparent circle.
Result of the test shows bacterial strain HS9（CGMCC No.9889）It is positive for Starch Hydrolysis.
Gelatin hydrolysis is tested
A. culture medium and reagent peptone 5g, gelatin 120g, distilled water 1000mL.Adjust pH7.2 ~ 7.4, packing examination
Pipe, culture medium are highly about 4 ~ 5cm, 121 DEG C of sterilizing 20min.
B. puncture method inoculating strain HS9 is used in Spawn incubation and result observation（CGMCC No.9889）In test tube center.
Cultivated one month in 30 DEG C of incubators, whether observation gelatin liquefies.
Result of the test shows bacterial strain HS9（CGMCC No.9889）For gelatin liquefaction positive.
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：A- naphthols
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,
With 20mL distilled water dilutings.
B. Spawn incubation and result are observed bacterial strain HS9（CGMCC No.9889）It is inoculated in nitrate fluid nutrient medium
In, 30 DEG C are cultivated 1,3,5 day.A little nutrient solution is poured into white porcelain dish aperture, then drop 1 drips reagent A and B respectively wherein
Liquid, when nutrient solution is changed into pink, rose, orange or brown etc., nitrite presence is indicated, is nitrate reduction
The positive, it is otherwise feminine gender.
Result of the test bacterial strain shows HS9（CGMCC No.9889）It 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（Cultivate 18-24h）HS9 strains（CGMCC No.9889）It is inoculated in
On inclined-plane, 30 DEG C are cultivated 3-7 days, and culture medium is 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 HS9（CGMCC No.9889）For feminine gender.
In order to further verify peanut plant growth-promoting rhizobacteria HS9（CGMCC No.9889）Produce the ability of heteroauxin and most suitable
Condition, the influence to heteroauxin yield is explored below for different pH, liquid amount, different carbon source, different nitrogen sources.
25ml, 50ml, 75ml will be pressed containing L-Trp (100mg/L) LB fluid nutrient mediums, 100ml, 150ml are loaded on
In 250mL triangular flask, by 1%（v/v）HS9 of the inoculum concentration inoculation in exponential phase（CGMCC No.9889）Afterwards, it is placed in
30 DEG C, 180rmin-1The h of shaking table culture 24, by the method measure production IAA of quantitative determination amount.As a result as shown in Fig. 2 due to
Bacterial strain HS9（CGMCC No.9889）It is amphimicrobian metabolism, throughput influences bacterial strain production IAA efficiency, during 100mL liquid amounts,
Bacterial strain production IAA amounts are most, and afterwards as liquid amount is reduced, yield is 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）HS9 of the inoculum concentration inoculation in exponential phase（CGMCC
No.9889）Afterwards, 30 DEG C are placed in, 180rmin-1Shaking table culture 24h, IAA amount is produced by the method measure of quantitative determination, as a result
As shown in figure 3, showing not produce IAA when pH is 10, in strong acid and strong base environment, thalline can not carry out growth metabolism, and strain is micro-
IAA is produced in alkali environment and is more than sour environment, strain high yield IAA optimal pH is 5 ~ 7.
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
HS9 of the amount inoculation in exponential phase（CGMCC No.9889）Afterwards, 30 DEG C are placed in, 180 rmin-1The h of shaking table culture 24,
By the method measure production IAA of quantitative determination amount.As a result as shown in figure 4, the bacterial strain is when supplying glucose, IAA ability is produced
Most strong, next to that fructose, the utilization rate of lactose is minimum, hardly produces IAA.
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 250ml triangular flask（v/v）HS9 of the inoculum concentration inoculation in exponential phase（CGMCC No.9889）Afterwards, 30 are placed in
DEG C, 180rmin-1The h of shaking table culture 24, by the method measure production IAA of quantitative determination amount.As a result as shown in figure 5, explanation takes
When urea is nitrogen source, it is most to produce IAA amount, next to that dusty yeast.
Bacterial strain HS9 of the present invention（CGMCC No.9889）There is obvious growth promoting function to peanut, below by pot experiment
Illustrate.
The fresh soil of sand 0 ~ 20cm soil layers under natural conditions is gathered, 5mm sieves is crossed, native 700g is filled per basin, plant peanut,
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 determined with HPLC methods, and determine soil quick-effective phosphor, quick-acting potassium content, plant fresh weight, plant height and complete
The full potassium of the full phosphorus of nitrogen.
Peanut seed：Peanut seed carries out 20% hydrogen peroxide surface sterilization 20min, and aseptic water washing is multiple, vernalization 2d, choosing
Take the consistent seed that germinates standby.
Connect bacterium processing：By the HS9 of the present invention（CGMCC No.9889）It is inoculated in LB fluid nutrient mediums, 30 DEG C, 180r
min-1Shaking table culture, culture bacterium are grown to exponential phase, then by bacteria suspension 10000rmin-110min is centrifuged, then with sterile
Water is resuspended, and repeats three times, inoculum concentration 108CFU·g-1（That is every gram of dry ground inoculation 108CFU·g-1 HS9 strains）.
Control treatment：As control, soil does not spray HS9 bacterium solutions, adds equivalent sterilized water.
As a result Lie Gebiao is seen below：
Influences of the inoculating strain HS9 of table 3 to Peanut Root System
| Processing | Root long（cm） | Root surface area（cm2） | Root volume（cm3） | Tip of a root number（It is individual） |
| CK | 931.72±198.45 | 147.66±15.68 | 1.84±0.49 | 3356.14±931.59 |
| HS9 | 1952.83±280.00** | 272.14±64.57** | 3.24±0.65** | 16319.00±2272.71** |
Note：* indicates significant difference in same row（p<0.05）, * * expression pole significant differences（p<0.01）；Similarly hereinafter.
Influences of the inoculating strain HS9 of table 4 to peanut plant
| Processing | Fresh weight（g) | Plant height（cm） | SPAD | Full nitrogen（g/kg） | Full phosphorus（g/kg） | Full potassium（g/kg） |
| CK | 3.44±0.77 | 18.52±1.68 | 39.03±1.63 | 1.86±0.17 | 1.71±0.56 | 6.68±0.58 |
| HS9 | 5.85±1.01** | 23.77±1.66** | 45.30±2.97** | 2.29±0.06* | 3.13±1.06** | 8.28±1.54** |
Influences of the inoculating strain HS9 of table 5 to soil quick-effective phosphor available potassium
| Processing | Rapid available phosphorus（mg/kg） | Available potassium（mg/kg） |
| CK | 3.16±0.21 | 34.00±3.46 |
| HS9 | 3.69±0.07* | 43.50±2.12* |
As can be seen from Table 4, it is vaccinated with HS9（CGMCC No.9889）The overground part for the peanut plant that soil-grown goes out is fresh
Weight, and plant height have obvious growth trend compared with CK；Due to HS9（CGMCC No.9889）There is dissolving phosphor and dissolving potassium, make
Rapid available phosphorus and quick-acting potassium content increase in soil（Table 5）, so as to promote absorption of the plant to elements such as P, K（Table 5）, from table 3
As can be seen that inoculation HS9 processing with do not connect bacterium processing contrasted, Peanut Root System total length, root surface area, average root diameter with
Tip of a root number all dramatically increases, and promotes the development of Peanut Root System；From fig. 9, it can be seen that after connecing bacterium processing, soil IAA contents
Dramatically increase, 3 times or so are higher by than control group.With reference to result above as can be seen that HS9 pairs of the peanut plant growth-promoting rhizobacteria of the present invention
Growth, the development of foundation have positive effect, and IAA yield is high, can effectively facilitate crop growth and improve yield.Through 3
Year continuously applies in 3 mu of Peanut Fields, and peanut yield improves 10-15%, and the good of its effect is not expected.
Described 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, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (3)
1. a kind of peanut plant growth-promoting rhizobacteria HS9, Classification And Nomenclature are Bacillus flexus（Bacillusflexus）, in October, 2014
China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved within 31st, preserving number is CGMCC No.9889, should
Bacillus flexus（Bacillusflexus）It is from the North China Wheat and maize rotation nutrition of the Ministry of Agriculture of Zhengzhou City and the fertilising view of science
Survey in the sand that experiment station takes and screen the bacterial strain separated.
2. the preserving number described in claim 1 is CGMCC No.9889 peanut plant growth-promoting rhizobacteria HS9 in peanut growth is promoted
Application.
3. the preserving number described in claim 1 is CGMCC No.9889 peanut plant growth-promoting rhizobacteria HS9 answering in peanut cultivation
With.
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| 芽孢杆菌SH6-1 的分离鉴定及其生物活性测定;李纪顺;《河南农业科学》;20091231;97-102 * |
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