CN104611252B - A kind of tobacco rhizosphere Promoting bacteria TC6 and its application - Google Patents
A kind of tobacco rhizosphere Promoting bacteria TC6 and its application Download PDFInfo
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Abstract
The present invention relates to tobacco rhizosphere Promoting bacteria TC6 and its application, tobacco rhizosphere Promoting bacteria is tobacco rhizosphere Promoting bacteria TC6, Classification And Nomenclature be bacillus subtilis (Bacillus subtilis), on October 31st, 2014 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.9894, can effectively solve for slightly solubility to be converted into soluble potassium salt containing potassium silicate, solution organophosphor, improves the utilization rate of fertilizer, effective for the cultivation of tobacco, promote the growth of tobacco and improve yield, be the big innovation on microorganism and tobacco planting.
Description
Technical field
The present invention relates to microorganism, particularly a kind of tobacco rhizosphere Promoting bacteria TC6 and its application.
Background technology
Moisture soil is the soil that river deposit is influenceed and formed by ground water movement and farming activity, because there is Evening Tide phenomenon and
Gain the name.In China, in being distributed in the Yellow River more, the alluvial plain in downstream and its on the south Jiangsu, the plains region in Anhui and the Changjiang river stream
In domain, the river in downstream, lake plain and Delta Area.Moisture soil distributed area physical features is flat, and soil layer is deep, and hydrothermal resources relatively enriches,
Make kind of property wide, be the main upland soil of China, abound with grain and cotton.But the maximum Huang-Huai-Hai plain of moisture soil distribution area, drought and waterlogging calamity
Evil happen occasionally, still have perniciousness harm, in addition soil nutrient it is low or lack, major part category in, low productive soil, crop yield it is low and
It is unstable.The reasonable of moisture soil must be strengthened to utilize and improvement.
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 produce plant hormone, such as auxin, gibberellin, the basic element of cell division and ethene.Additionally, 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, the focus as current research, but so far there are no tobacco is exclusively used in
The open report of plant growth-promoting rhizobacteria.
The content of the invention
For above-mentioned situation, to overcome the defect of prior art, the purpose of the present invention to be just to provide a kind of tobacco rhizosphere and promote
Raw bacterium TC6 and its application, that is to say, that it is an object of the present invention to provide a kind of tobacco rhizosphere Promoting bacteria, another purpose
The application of the plant growth-promoting rhizobacteria is to provide, can effectively solve for slightly solubility to be converted into soluble potassium salt containing potassium silicate, solved organic
Phosphorus, improves the utilization rate of fertilizer, promotes the growth of tobacco and improves yield.
The technical scheme that the present invention is solved is that tobacco rhizosphere Promoting bacteria is tobacco rhizosphere Promoting bacteria TC6, and Classification And Nomenclature is withered
Careless bacillus (Bacillus subtilis), on October 31st, 2014 is preserved in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center, preserving number is CGMCC No.9894, depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
No. 3.
Plant growth-promoting rhizobacteria TC6（CGMCC No.9894）Bacterium colony is small, circular or irregular shape, opaque, rough surface, no
Smooth, edge roughness produces gemma.
The physio-biochemical characteristics of plant growth-promoting rhizobacteria TC6 are:Gram-positive, amphimicrobian, catalase reaction
The positive, M.R negatives, VP experiments are positive, and Starch Hydrolysis are positive, gelatin liquefaction positive, and nitrate reduction is positive, citrate
Using feminine gender.
Main nitrogen including but not limited to peptone, dusty yeast, glutamic acid, nitre that plant growth-promoting rhizobacteria TC6 is used when cultivating
Sour potassium, ammonium nitrate, ammonium sulfate, urea；The primary carbon source for using including but not limited to glucose, sucrose, fructose, xylose, sweet dew
Alcohol, lactose, maltose；The inorganic component for using including but not limited to potassium chloride, sodium chloride, sodium dihydrogen phosphate, dipotassium hydrogen phosphate,
Tricalcium phosphate, calcium chloride dihydrate, bitter salt, seven water and ferrous sulfate.Bacillus subtilis (Bacillus subtilis) ferment and can be carried out in the environment of pH5 ~ 9 at 28 ~ 32 DEG C.
Described preserving number is applications of the tobacco rhizosphere Promoting bacteria TC6 of CGMCC No.9894 in tobacco growing is promoted.
Described preserving number is applications of the tobacco rhizosphere Promoting bacteria TC6 of CGMCC No.9894 in tobacco cultivation.
The plant growth-promoting rhizobacteria TC6 energy high yield heteroauxins simultaneously can contain potassium silicate using slightly solubility（Potassium feldspar）Slightly solubility
Inorganic phosphate（Tricalcium phosphate）The organophosphor being difficult by is grown as potassium resource, phosphorus source.
Plant growth-promoting rhizobacteria TC6 of the present invention secretes heteroauxin（IAA）Ability it is strong, up to 28.63mgL-1.Indoles
Acetic acid is one kind of plant hormone, can promote the development of root.The strain of heteroauxin is produced, root system of plant or leaf is often attached to
Surface, IAA and a small amount of GA is produced while producing secretion using plant metabolism3The physiology mistake of plant is influenceed 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
It is prioritization scheme of the invention, the fermentation of the plant growth-promoting rhizobacteria is carried out under pH6 ~ 7, IAA amount highests is produced under the environment.
Used as further optimization of the invention, the carbon source that the plant growth-promoting rhizobacteria TC6 is used is mannitol, the nitrogen source of use
It is the combination of dusty yeast or peptone or both.Using culture medium obtained in above-mentioned carbon source and nitrogen source, the rhizosphere growth-promoting cultivated
Bacterium produces the amount highest of IAA.
Plant growth-promoting rhizobacteria TC6 of the present invention is with slightly solubility Inorganic phosphate（Tricalcium phosphate）For phosphorus source is grown, and
It is translated into soluble microcosmic salt.Under the conditions of laboratory shake flask, the plant growth-promoting rhizobacteria TC6 reaches to the inversion quantity of tricalcium phosphate
To 221.65 mgL-1 .Illustrate that TC6 bacterium have dissolution to tricalcium phosphate, Inorganic phosphate is contained as phosphorus source is carried out with slightly solubility
Growth, and it is translated into soluble microcosmic salt.
Plant growth-promoting rhizobacteria TC6 of the present invention is converted with the organophosphor that is difficult by as phosphorus source is grown
It is available phosphorus.Under the conditions of laboratory shake flask, the plant growth-promoting rhizobacteria TC6 reaches to the inversion quantity of solvable lecithin
0.63mg•L-1.Illustrate that TC6 bacterium have transformation to solvable lecithin, with the organophosphor that is difficult by as phosphorus source is given birth to
It is long, and it is translated into available phosphorus.
Plant growth-promoting rhizobacteria TC6 of the present invention contains potassium silicate with slightly solubility（Potassium feldspar）For potassium resource is grown, and
It is translated into soluble potassium salt.Under the conditions of laboratory shake flask, inversion quantities of the plant growth-promoting rhizobacteria YC-4 to feldspar in powder
Reach 19.88mgL-1.Illustrate that TC6 bacterium have dissolution to feldspar in powder, potassium silicate is contained as potassium resource is carried out with slightly solubility
Growth, and it is translated into soluble potassium salt.
, effectively containing potassium silicate can be converted into slightly solubility by the plant growth-promoting rhizobacteria TC6 high yield heteroauxins that the present invention is provided
Soluble potassium salt, soluble microcosmic salt is converted into by slightly solubility Inorganic phosphate and the difficult organophosphor for utilizing, and improves the utilization rate of fertilizer,
Promote plant root system development and the absorption to fertilizer, increase available potassium in soils, available phosphorus content；The present invention has good for tobacco
Good growth-promoting effect, the heteroauxin of high yield promotes growing for tobacco, available potassium in soils, the raising of available phosphorus content
So that tobacco is higher to the utilization rate of potash fertilizer, phosphate fertilizer, effective for the cultivation of tobacco, promotes the growth of tobacco and improve yield,
It is the big innovation on microorganism and tobacco planting.
Brief description of the drawings
Fig. 1 is the bacterium colony figure of bacterial strain TC6 of the present invention；
Fig. 2 is the influence situation map that different liquid amounts produce IAA to bacterial strain TC6；
Fig. 3 is the influence situation map that different initial pH produce IAA to TC6 bacterial strains；
Fig. 4 is the influence situation map that different carbon source produces IAA to bacterial strain TC6；
Fig. 5 is the influence situation map that different nitrogen sources produce IAA to TC6 bacterial strains；
Fig. 6 is bacterial strain TC6 to utilization power situation map of the slightly solubility containing potassium silicate；
Fig. 7 is utilization power figures of the bacterial strain TC6 to organophosphor；
Fig. 8 is utilization power figures of the bacterial strain TC6 to slightly solubility Inorganic phosphate；
Fig. 9 is that plantation tobacco is inoculated with influence situation map of the TC6 bacterial strains to soil IAA contents after 30 days.
Specific embodiment
Specific embodiment of the invention is elaborated below in conjunction with concrete condition.
Biomaterial preservation：Plant growth-promoting rhizobacteria TC6, Classification And Nomenclature is bacillus subtilis（Bacillus subtilis）,
On October 31st, 2014 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is court of Beijing
No. 3 Institute of Microorganism, Academia Sinica of institute of positive area's North Star West Road 1, preserving number is CGMCC No.9894.
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) |
| Moisture soil | 6.45 | 0.80 | 9.77 | 18.56 | 111.17 | 7.27 |
The physio-biochemical characteristics of the TC6 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 is utilized | - |
| 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 ibid；
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 moisture soil taken with fertilising scientific observation experiment station from the Wheat and maize rotation nutrition of Zhengzhou City North China is weighed into l0g to put
In the triangular flask of 250 ml of 100 ml aqua sterilisas is filled, 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, after being diluted using dilution method
LB culture mediums are applied to, flat board is inverted, in 30 DEG C, after cultivating 24h in insulating box, picking different type typical case's single bacterium colony, warp
After purification, 4 DEG C to be stored in LB inclined-planes stand-by for flat board.
The plant-growth promoting rhizobacteria that can secrete heteroauxin is filtered out by qualitative determination and quantitative determination again below.
Qualitative determination：Microbionation after isolating and purifying contains L-Trp in use（100 mg/L）LB liquid training
Support base, 30 DEG C, 180 rmin-1Shaking table culture 1d.50 μ L bacteria suspensions are taken to drip on whiteware plate, while plus 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 used as positive control.In being observed after the min of room temperature avoid light place 30, the color person of reddening represents can secrete Yin to whiteware plate
Indolylbutyric acid.
Quantitative determination：The bacterium of the producing IAA obtained to primary dcreening operation quantitative determines, and condition of culture is ibid.Use first and divide
Light photometry determines the OD600 values of bacteria suspension, then by bacteria suspension with 10000 rmin-110 min are centrifuged and take supernatant addition
Isometric Salkowski color solutions, lucifuge stands 30min, determines 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.
The product IAA bacterium for obtaining solve the screening test of organophosphor situation, strains tested is inoculated in and is filled 30ml and is had
The triangular flask of machine phosphorus fluid nutrient medium, 30 DEG C, 180 rmin-1After culture 4d, nutrient solution is loaded at 4 DEG C of centrifuge tube
10000r/min is centrifuged 15min, takes supernatant molybdenum blue colorimetric method and determines available phosphorus content.
The product IAA bacterium for obtaining are carried out the screening test of phosphorus decomposing situation, strains tested is inoculated in and fills 50mL Phos
Bacterial liquid culture medium（PKO）250 mL triangular flasks, 30 DEG C, 180 rmin-1After culture 4d, nutrient solution is loaded into centrifuge tube
10000r/min centrifugations 15min at 4 DEG C, takes supernatant molybdenum blue colorimetric method and determines available phosphorus content.
The product IAA bacterium for obtaining are carried out the screening test of potassium decomposing situation, strains tested is inoculated in fill 50mL potassium decomposings thin
250 mL triangular flasks of bacteria liquid culture medium, 30 DEG C, 200 rmin-1After culture 72h, the nutrient solution 20mL of culture 72h is taken,
6000r/min is centrifuged 20min, takes supernatant flame spectrophotometer and determines wherein K+ contents.
Being determined more than can filter out high yield heteroauxin, insoluble inorganic phosphorus of solving problem, the difficult phosphorus-containing matter for utilizing,
The strong bacterial strain of ability of dissolving potassium, is named as TC6.As shown in figure 1, the bacterium colony is small, and circular or irregular shape, opaque, rough surface,
Rough, edge roughness produces gemma..As shown in fig. 6, bacterial strain TC6 contains potassium silicate as potassium resource is grown with slightly solubility, and
It is translated into soluble potassium salt.Under the conditions of laboratory shake flask, the plant growth-promoting rhizobacteria TC6 reaches to the inversion quantity of feldspar in powder
To 19.88 mgL-1.Illustrate that TC6 bacterium have dissolution to feldspar in powder, potassium silicate is contained as potassium resource is carried out with slightly solubility
Growth, and it is translated into soluble potassium salt.As shown in fig. 7, bacterial strain TC6 bacterium are with the organophosphor that is difficult by as phosphorus source is given birth to
It is long, and it is translated into available phosphorus.Under the conditions of laboratory shake flask, the plant growth-promoting rhizobacteria TC6 is to solvable lecithin
Inversion quantity reach 0.63mgL-1.Illustrate that TC6 bacterium have transformation to solvable lecithin, it is organic with what is be difficult by
Phosphorus is grown for phosphorus source, and is translated into available phosphorus.As shown in figure 8, bacterial strain TC6 is containing Inorganic phosphate with slightly solubility
Phosphorus source is grown, and is translated into soluble microcosmic salt.Under the conditions of laboratory shake flask, the plant growth-promoting rhizobacteria TC6 is to phosphorus
The inversion quantity of sour DFP reaches 221.65 mgL-1.Illustrate that TC6 bacterium have dissolution to tricalcium phosphate, nothing is contained with slightly solubility
Machine microcosmic salt is grown for phosphorus source, and is translated into soluble microcosmic salt.
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 is carried out with other 16S rDNA sequences, the sequence MEGA version 3 of close sequence and TC6 are selected
The 16SrDNA systematic evolution trees of software building TC6.According to the physiological and biochemical property of the bacterial strain, bacillus subtilis is accredited as
（Bacillus subtilis）.The bacterial strain is general in China Committee for Culture Collection of Microorganisms on October 31st, 2014
Logical microorganism center preservation, preserving number CGMCC No.9894.
The bacterial strain is in Gram-positive, and amphimicrobian, bacterium colony is small, circular or irregular shape, and opaque, surface is thick
Rough, rough, edge roughness produces gemma.Catalase reaction is positive, M.R negatives, and VP experiments are positive, Starch Hydrolysis
The positive, nitrate reduction is positive, and citrate is using negative.Producing IAA content ability is strong, reaches 28.63mgL-1With indissoluble
Property grown for phosphorus source containing Inorganic phosphate, and be translated into soluble microcosmic salt.
Aerobic is tested
Sterilized LB culture mediums are poured into 3 sterilized test tubes, about at 2/3, on aseptic operating platform, is used
The bacterial strain TC6 of transfer needle picking inclined-plane culture, in percutaneous puncture-inoculation to above-mentioned culture medium（Ttom of pipe must be punctured to）.30 DEG C of trainings
Support, observed result at 3 days to 7 days respectively.Raw elder preferably oxygen bacterium, is anaerobism such as along line life elder is punctured on agar column surface
Bacterium or facultative anaerobic bacteria.
Result of the test is showed, and along agar column superficial growth, puncture in line also has colony growth to bacterial strain TC6 bacterium colonies, is facultative
Anaerobism.
The measure of catalase
Drop 1 drips 3%H on clean slide2O2, the ring of bacterial strain TC6 LB slant cultures 1 of 18 ~ 24 h cultures is taken,
H2O2Middle smearing, is the positive if having bubble to produce, and is otherwise feminine gender.
Result of the test display bacterial strain TC6 is 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 observe inoculating strain TC6 in above-mentioned nutrient solution, and 30 DEG C are cultivated l ~ 2 day.In training
Add a few drop methyl red reagents, such as nutrient solution to be presented red in nutrient solution, be positive methyl red, yellow is feminine gender（Methyl red changes colour
Red ~ 6.0 yellow of scope 4.4）.
Result of the test display bacterial strain TC6 is M.R negative.
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 and the same methyl red test of culture.Do
When VP is tested, nutrient solution is taken（About 2mL）40 %NaOH with equivalent are mixed, plus 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 display bacterial strain TC6 is VP positive.
Starch Hydrolysis are tested
A. culture medium and reagent add 0.2% soluble starch in meat soup peptone agar, dispense triangular flask, 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）Distillation water dissolves KI, it is existing
The crystalline flake of iodine is added, after iodine is completely dissolved, 300mL is diluted with water to.
B. Spawn incubation and result observation takes TC6 strains point and is connected on flat board, and 30 DEG C are cultivated 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 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 display bacterial strain TC6 is positive Starch Hydrolysis.
Gelatin hydrolysis are tested
A. culture medium and reagent peptone 5g, gelatin 120g, distilled water 1000mL.Regulation pH7.2 ~ 7.4, packing examination
Pipe, culture medium is highly about 4 ~ 5cm, 121 DEG C of sterilizing 20min.
B. Spawn incubation and result observation puncture method inoculating strain TC6 are in test tube center.Cultivated in 30 DEG C of incubators
One month, whether observation gelatin liquefied.
Result of the test display bacterial strain TC6 is 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：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,
Use 20mL distilled water dilutings.
Be inoculated in bacterial strain TC6 in nitrate fluid nutrient medium by b. 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., indicate that nitrite is present, be positive nitrate reduction, be otherwise the moon
Property.
Result of the test bacterial strain display TC6 is positive 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%
Aqueous solution 10mL, agar 20g, distilled water 1000mL, pH6.8-7.0,121 DEG C of sterilizing 20min of Australia's thymol blue.
B. Spawn incubation and result observation takes young age TC6 strains and is inoculated on inclined-plane, and 30 DEG C are cultivated 3-7 days, culture medium
In alkalescence（It is blue）Person is positive reaction, and constant person is then feminine gender.
The result of the test display bacterial strain TC6 that citrate is utilized is feminine gender.
In order to further verify plant growth-promoting rhizobacteria TC6 produce heteroauxin ability and optimum condition, below for different pH,
Liquid amount, different carbon source, different nitrogen sources explore the influence to heteroauxin yield.
25ml will be pressed containing L-Trp (100mg/L) LB fluid nutrient mediums, 50ml, 75ml, 100ml, 150ml is loaded on
In the triangular flask of 250mL, by 1%（v/v）After TC6 of the inoculum concentration inoculation in exponential phase, 30 DEG C, 180rmin are placed in-1
The h of shaking table culture 24, the amount for producing IAA is determined by the method for quantitative determination.Result is as shown in Fig. 2 because bacterial strain TC6 is facultative to detest
Oxygen metabolism, throughput influence bacterial strain produces the efficiency of IAA, and during 50mL liquid amounts, bacterial strain produces IAA amounts at most, afterwards with liquid amount
Increase, 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 50mL to be loaded in the triangular flask of 250mL, by 1%（v/v）After TC6 of the inoculum concentration inoculation in exponential phase, 30 DEG C are placed in,
180r·min-1Shaking table culture 24h, the amount for producing IAA is determined by the method for quantitative determination, as a result as shown in figure 3, showing that pH is 10
When do not produce IAA, in strong acid and strong base environment, thalline cannot carry out growth metabolism, and strain produces IAA more than alkalescence in micro- acid environment
Environment, the optimal pH of strain high yield IAA is 6 ~ 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 50ml and are loaded in the triangular flask of 250ml, by 1%（v/v）Inoculation
After TC6 of the amount inoculation in exponential phase, 30 DEG C, 180 rmin are placed in-1The h of shaking table culture 24, by the side of quantitative determination
Method determines the amount for producing IAA.Result as shown in figure 4, the bacterial strain supply mannitol when, produce IAA ability it is most strong, next to that newborn
Sugar, the utilization rate of fructose is minimum.
0.1% is separately added into containing L-Trp (100mg/L) minimal medium (not including ammonium sulfate)（w/v）
Nitrogen source, nitrogen source takes 50ml and is loaded on including ammonium nitrate, ammonium sulfate, potassium nitrate, peptone, dusty yeast, alanine, urea etc.
1% is pressed in the triangular flask of 250ml（v/v）After TC6 of the inoculum concentration inoculation in exponential phase, 30 DEG C, 180rmin are placed in-1Shake
Bed 24 h of culture, the amount for producing IAA is determined by the method for quantitative determination.Result as shown in figure 5, explanation take dusty yeast for nitrogen source when,
The amount of IAA is produced at most, next to that peptone.
Bacterial strain TC6 of the present invention has obvious growth promoting function to tobacco, is illustrated below by pot experiment.
The fresh soil of moisture soil 0 ~ 20cm soil layers under collection natural conditions, crosses 5mm sieves, and soil 700g is filled per basin, plants tobacco,
Regulation water content used root scanner to the 60% of maxmun field capacity, 30 day post-sampling（LA1600+ scanner,
Canada）After scanning obtains root system image, root system analysis software is used（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
The full potassium content of total nitrogen and total phosphor phosphorus.
Tobacco seed：Tobacco seed carries out 20% hydrogen peroxide surface sterilization 20min, and repeatedly, vernalization 2d is selected aseptic water washing
Take the consistent seed of germination standby.
Connect bacterium treatment：TC6 of the invention is inoculated in LB fluid nutrient mediums, 30 DEG C, 180rmin-1Shaking table culture, culture
Bacterium is long to exponential phase, then by bacteria suspension 10000rmin-1Centrifugation 10min, then it is resuspended with sterilized water, equally it is centrifuged three
Secondary, inoculum concentration is 108CFU·g-1 （I.e. every gram dry ground inoculation 108CFU·g-1 TC6 strains）.
Control treatment：Used as control, soil does not spray TC6 bacterium solutions, plus equivalent sterilized water.
Result sees below Lie Gebiao：
Influences of the inoculating strain TC6 of table 3 to tobacco root
| Treatment | Root is long（cm） | Tip of a root number（It is individual） | ||
| CK | 378.35±98.89 | 50.18±13.34 | 0.50±0.10 | 636±90.59 |
| TC6 | 661.6±59.89 |
Note：* indicates significant difference in same row（p<0.05）, * * represent pole significant difference（p<0.01）；Similarly hereinafter.
Influences of the inoculating strain TC6 of table 4 to tobacco plant
| Treatment | Fresh weight（g) | Plant height（cm） | SPAD | Full nitrogen（g/kg） | Full phosphorus（g/kg） | Full potassium（g/kg） |
| CK | 5.47±1.80 | 17.12±1.83 | 3.35±0.16 | 0.93±0.09 | 1.64±0.19 | 24.08±1.66 |
| TC6 |
Influences of the bacterial strain TC6 of table 5 to soil quick-effective phosphor available potassium
| Treatment | Rapid available phosphorus（mg/kg） | Available potassium（mg/kg） |
| CK | 5.04±0.08 | 149.00±1.73 |
| TC6 |
As can be seen from Table 4, the overground part fresh weight of the tobacco plant that TC6 soil-growns go out is vaccinated with, and plant height is compared with CK all
There is obvious growth trend；There is the effect of dissolving phosphor and dissolving potassium due to TC6, make rapid available phosphorus and quick-acting potassium content increase in soil
（Table 5）, so as to promote plant to P, the absorption of the element such as K, from table 3 it can be seen that inoculation TC6 treatment with do not connect bacterium process into
Row contrast, tobacco root total length, root surface area, average root diameter and tip of a root number are all dramatically increased, and promote tobacco root
Development；From fig. 9, it can be seen that after connecing bacterium treatment, soil IAA contents are dramatically increased, and 3 times or so are higher by than control group.With reference to
Result above can be seen that plant growth-promoting rhizobacteria TC6 of the invention to growth, the development of foundation with positive effect, IAA yield
Height, can effectively facilitate crop growth.Continuous in 3 mu of Tobacco Farms administrations through 3 years, yield of tobacco improves 15-20%, its
The good of effect was 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, 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 tobacco rhizosphere Promoting bacteria TC6, Classification And Nomenclature be bacillus subtilis (Bacillus subtilis), 2014
October 31 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC
No.9894。
2. the preserving number described in claim 1 is the tobacco rhizosphere Promoting bacteria TC6 of CGMCC No.9894 in tobacco growing is promoted
Application.
3. the preserving number described in claim 1 is tobacco rhizosphere Promoting bacteria TC6 the answering in tobacco cultivation of CGMCC No.9894
With.
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| CN101544523A (en) * | 2009-04-30 | 2009-09-30 | 谢群英 | Fertilizer with biological ferment and preparation method and use method thereof |
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| CN101531971A (en) * | 2008-03-14 | 2009-09-16 | 天津现代职业技术学院 | Bacillus subtilis and application thereof in prevention and treatment of continuous cropping obstacle of greenhouse vegetable |
| CN101544523A (en) * | 2009-04-30 | 2009-09-30 | 谢群英 | Fertilizer with biological ferment and preparation method and use method thereof |
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