CN104560789A - Peanut growth promoting rhizobacteria HS2 and application thereof - Google Patents

Peanut growth promoting rhizobacteria HS2 and application thereof Download PDF

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CN104560789A
CN104560789A CN201410761474.8A CN201410761474A CN104560789A CN 104560789 A CN104560789 A CN 104560789A CN 201410761474 A CN201410761474 A CN 201410761474A CN 104560789 A CN104560789 A CN 104560789A
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promoting rhizobacteria
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谭云飞
高爱华
曹军
罗中玉
王小玲
王宏武
李雪
周鹏
巫伟
王阳
万晓瑞
董海涛
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Zhengzhou Sewage Purification Co Ltd
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Abstract

The invention relates to peanut growth promoting rhizobacteria HS2 and an application thereof. The peanut growth promoting rhizobacteria is peanut growth promoting rhizobacteria HS2 which is classified and named bacillus subtilis and collected in the China General Microbiological Culture Collection Center on October 31, 2014, with a collection number CGMCC No.9888; the insoluble potassium-containing silicate can be effectively converted into soluble potassium salt, organophosphorus is dissolved, and the utilization rate of fertilizer is improved; and the peanut growth promoting rhizobacteria HS2 is effectively applied to peanut planting to promote the peanut growth and increase the yield, and the application is a great innovation in microorganism and peanut planting.

Description

A kind of peanut plant growth-promoting rhizobacteria HS2 and application thereof
Technical field
The present invention relates to microorganism field, particularly a kind of peanut plant growth-promoting rhizobacteria HS2 and application thereof.
Background technology
Sandstone area permeability is strong, and ventilation is good, and aerobic microorganism activity is preponderated, and can promote organic matter decomposition, organic mineralization quickening.And loosing soil, easy farming.Soil capillarity is strong, and moisture runs fast, has " Evening Tide " phenomenon.The suitable cultivated phase is also long, Yi Limiao; But nutrient content is low, fertilizer conservation poor performance, the crop later stage is de-fertile early ageing easily.At present, this soil major part makes cultivated land utilization, and its availability reaches 92.3%, yields two crops a year, general year per mu yield grain about 600kg.Ameliorative measure targetedly from now on: because of soil plantation, rational use of chemical fertilizer, carries out rational application of fertilizers; Multipath applying organic manure; Carry out farm field and forest network, ensureing under the prerequisite that grain-production grows steadily, economic crops such as development Chinese yam, watermelon etc.
Plant growth-promoting rhizobacteria (Plant Growth Promoting Bacteria, be called for short PGPB) is defined as the free living that the is conducive to plant growth under certain condition bacterium at soil, rhizosphere, root table, phyllosphere.These bacteriums can fixed nitrogen, molten phosphorus, molten iron, and produce plant hormone, as growth hormone, gibberellin, the basic element of cell division and ethene.In addition, they can also improve the resistance of plant, comprise arid, high salt, heavy metallic poison and agricultural chemicals.Therefore be separated from moisture soil and obtain plant growth-promoting rhizobacteria, and crop forms syntaxial system, utilizes and biological prostheticly improves moisture soil, become the focus of current research, but so far there are no being exclusively used in the open report of plant growth-promoting rhizobacteria of peanut.
Summary of the invention
For above-mentioned situation, for overcoming the defect of prior art, the object of the present invention is just to provide a kind of peanut plant growth-promoting rhizobacteria HS2 and application thereof, that is, an object of the present invention is to provide a kind of peanut plant growth-promoting rhizobacteria, another object is to provide the application of this plant growth-promoting rhizobacteria, effectively can solve and slightly solubility is converted into soluble potassium salt containing potassium silicate, separate organic phosphor, improve the availability of fertilizer, promote the growth of peanut and improve output.
The present invention solve technical scheme be that peanut plant growth-promoting rhizobacteria is peanut plant growth-promoting rhizobacteria HS2, 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, and preserving number is CGMCC No.9888.
Peanut plant growth-promoting rhizobacteria HS2(CGMCC No.9888) bacteria colony white, irregular arrangement, dry tack free, opaque, edge roughness, produces gemma.
The physio-biochemical characteristics of peanut plant growth-promoting rhizobacteria HS2 are: Gram-positive, amphimicrobian, chemoheterotrophy, and catalase is positive, M.R negative, and VP tests the positive, and Starch Hydrolysis is positive, gelatin liquefaction positive, and nitrate reduction is positive, and citrate utilizes positive.
The main nitrogen used when peanut plant growth-promoting rhizobacteria HS2 cultivates includes but not limited to peptone, dusty yeast, alanine, potassium nitrate, ammonium nitrate, ammonium sulfate, urea; The primary carbon source used includes but not limited to glucose, sucrose, fructose, wood sugar, mannitol, lactose, maltose; The inorganic component used includes 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 HS2 fermentation at 28 ~ 32 DEG C, can be carried out under the environment of pH5 ~ 9.
Described preserving number is that the peanut plant growth-promoting rhizobacteria HS2 of CGMCC No.9888 is promoting the application in peanut growth;
Described preserving number is the application of peanut plant growth-promoting rhizobacteria HS2 in peanut cultivation of CGMCC No.9888;
Described peanut plant growth-promoting rhizobacteria HS2 can produce heteroauxin, separates organic phosphor and slightly solubility can be utilized to contain potassium silicate for potassium source to grow.
The ability that peanut plant growth-promoting rhizobacteria HS2 of the present invention secretes heteroauxin (IAA) is strong, reaches 16.31 μ gmL -1.Heteroauxin is the one of plant hormone, can promote the growth of root.Produce the bacterial classification of heteroauxin, be often attached to root system of plant or leaf surface, while utilizing plant metabolism to produce secretion, produce IAA and a small amount of GA 3physiology course and the metamorphosis of plant is affected Deng plant hormone.Show as the elongation directly promoting root, thus increase the chance with the contact of soil Middle nutrition material; The content of plant corpus Endogenous IAA can be improved; The expression of inducing plant defense gene, improves plant corpus disease-resistant, the resistance such as drought resisting.As prioritization scheme of the present invention, the fermentation of described plant growth-promoting rhizobacteria time to be carried out in pH7 ~ 8, and it is the highest to produce IAA amount under this environment.
As further optimization of the present invention, the carbon source that described peanut plant growth-promoting rhizobacteria HS2 adopts is maltose, and the nitrogenous source of employing is dusty yeast or peptone or both combinations.Utilize the medium that above-mentioned Carbon and nitrogen sources is obtained, the amount that the plant growth-promoting rhizobacteria cultivated produces IAA is the highest.
Peanut plant growth-promoting rhizobacteria HS2 of the present invention utilizes phosphorus-containing matter to grow for phosphorus source with difficulty, and is translated into and can utilizes phosphorus.Under laboratory shake flask condition, the inversion quantity of described peanut plant growth-promoting rhizobacteria HS2 to solubility lecithin reaches 0.49 mgL -1.Illustrate that HS2 bacterium has decomposition to solubility lecithin, utilize phosphorus-containing matter to grow for phosphorus source with difficulty, and be translated into and can utilize phosphorus.
Peanut plant growth-promoting rhizobacteria HS2 of the present invention grows for potassium source containing potassium silicate with slightly solubility, and is translated into soluble potassium salt.Under laboratory shake flask condition, the inversion quantity of described peanut plant growth-promoting rhizobacteria HS2 to feldspar in powder reaches 19.05 mgL -1.Illustrate that HS2 bacterium has dissolution to feldspar in powder, grow for potassium source containing potassium silicate with slightly solubility, and be translated into soluble potassium salt.
Bacterial strain HS2 point is connected to nitrogen-free agar flat board, can grows, it may have certain authigenic nitrogen fixation capacity.
Slightly solubility effectively can be converted into soluble potassium salt containing potassium silicate by peanut plant growth-promoting rhizobacteria HS2 provided by the invention, improve the availability of fertilizer, promote plant root system development and the absorption to fertilizer, increase available potassium in soils content, the raising of available potassium in soils content also makes the availability of peanut to potash fertilizer higher.The organic phosphor being difficult to utilize can be converted into available phosphorus, increase the content of soil available phosphorus, improve the availability of fertilizer, promote growing and absorption to fertilizer of plant; The present invention is directed to peanut and have good growth-promoting effect, the heteroauxin of product promotes growing of peanut, is effective to the plantation of peanut, promotes the growth of peanut and improves output, is that one on microorganism and peanut cultivation is innovated greatly.
attached caption
Fig. 1 is the bacterium colony figure of bacterial strain HS2 of the present invention;
To be different liquid amount produce IAA to bacterial strain HS2 to Fig. 2 affects situation map;
To be different initial pH produce IAA to HS2 bacterial strain to Fig. 3 affects situation map;
To be different carbon source produce IAA to bacterial strain HS2 to Fig. 4 affects situation map;
To be different nitrogen sources produce IAA to HS2 bacterial strain to Fig. 5 affects situation map;
Fig. 6 is that bacterial strain HS2 is to the utilization power figure of slightly solubility containing potassium silicate;
Fig. 7 is the utilization power figure of bacterial strain HS2 to organic phosphor;
Fig. 8 is that plantation peanut 30 days is inoculated HS2 bacterial strain afterwards and affected situation map to soil IAA content.
Embodiment
Below in conjunction with concrete condition, the specific embodiment of the present invention is elaborated.
Biomaterial preservation: peanut plant growth-promoting rhizobacteria HS2, 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, and address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and preserving number is CGMCC No.9888.
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(H 2O)
Sandstone area 1.91 0.29 3.44 19.56 20.42 7.39
The physio-biochemical characteristics of table 2 HS2 bacterial strain
Project Result Project Result
Gram’s staining + Starch Hydrolysis +
Aerobic is tested Amphimicrobian Gelatin liquefaction +
Catalase test + Nitrate reduction +
Methyl red (M.R) reacts - Citrate utilizes +
V-P tests +
Note :+: positive reaction;-: negative reaction
In concrete enforcement, first prepare following medium:
LB medium: 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 liquid nutrient medium: do not add agar, other condition is the same;
Meng Jinna medium: glucose 10.0g, (NH 4) 2sO 40.5g, MgSO 47H 2o 0.3g, NaCl 0.3g, KCl 0.3g, FeSO 40.03g, MnSO 4h 2o 0.03g, distilled water 1000ml, 115 DEG C of sterilizing 30min;
Organic phosphor liquid nutrient medium: 1000ml Meng Jinna medium adds 0.4g yeast extract, then add 0.2g solubility lecithin;
Potassium bacterium liquid nutrient medium: sucrose 10.0g, yeast extract 0.5g, (NH 4) 2sO 41.0g, Na 2hPO 42.0g, MgSO 47H 2o 0.5g, CaCO 31.0g, feldspar in powder 1.0g, distilled water 1000mL, 121 DEG C of sterilizings, 20min;
Nitrogen fixing capacity adopts Ashby nitrogen-free agar: mannitol 10g, KH 2pO 40.2g, MgSO 40.2g, NaCl 0.2g, K 2sO 40.3g, CaCO 35g, 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; Calcium chloride dihydrate 0.1g, distilled water 1000mL, pH 7.0,121 DEG C of sterilizings, 20min.
The sandstone area taked from the North China Wheat and maize rotation nutrition of the Ministry of Agriculture of Zhengzhou City and fertilising scientific observation experiment station is taken the triangular flask that l0g is placed in 250 ml filling 100 ml aqua sterilisas, in shaking table, 30 DEG C, 150rmin -1vibration 20min, leaves standstill 10min, obtains soil bacteria suspension.Containing several plant growth-promoting rhizobacteria in this soil bacteria suspension, be applied to LB medium after adopting dilution method dilution, flat board is inverted, in 30 DEG C, after cultivating 24h in insulating box, the single bacterium colony of the dissimilar typical case of picking, after dull and stereotyped purifying, 4 DEG C to be kept at LB inclined-plane stand-by.
Plant (peanut) the growth-promoting bacterium that can secrete heteroauxin is filtered out again below by qualitative determination and quantitative assay.
qualitative determination: by the microbionation after separation and purification in adopting the LB liquid nutrient medium containing L-Trp (100 mg/L), 30 DEG C, 180 rmin -11d cultivated by shaking table.Getting 50 μ L bacteria suspensions drips on whiteware plate, adds 50 μ L Salkowski color solution (50mL 35%HClO simultaneously 4+ 1mL 0.5M FeCl 3).To the color solution of 50 μ L 50 mg/L heteroauxins be added as positive control.Whiteware plate is observed after room temperature lucifuge places 30 min, and the color person of reddening represents and can secrete heteroauxin.
quantitative assay: carry out quantitative assay to the bacterium of the producing IAA that primary dcreening operation obtains, condition of culture is the same.First the OD of spectrophotometry bacteria suspension is used 600value, then by bacteria suspension with 10000 rmin -1centrifugal 10 min get supernatant and add equal-volume Salkowski color solution, and lucifuge leaves standstill 30min, measures its OD 530value.Calculate bacteria concentration OD 600when value is 1, the content of heteroauxin in unit volume zymotic fluid.The drafting of calibration curve adopts analytically pure heteroauxin gradient dilution to prepare.
The product IAA bacterium obtained is carried out the Screening test of phosphorus decomposing situation, say that strains tested is inoculated in the 250mL triangular flask filling 50mL organic phosphor liquid nutrient medium, 30 DEG C, 200 rmin -1after cultivating 72h, get the culture fluid 10mL cultivating 72h, the centrifugal 20min of 6000r/min, gets the content that supernatant ultraviolet specrophotometer measures wherein phosphorus.
The product IAA bacterium obtained is carried out the Screening test of potassium decomposing situation, strains tested is inoculated in the 250 mL triangular flasks filling 50mL potassium bacterium liquid nutrient medium, 30 DEG C, 200 rmin -1after cultivating 72h, get the culture fluid 20mL cultivating 72h, the centrifugal 20min of 6000r/min, gets supernatant flame spectrophotometer and measures wherein K +content.
Producing heteroauxin by measuring to screen above, separating organic phosphor, the bacterial strain that ability of dissolving potassium is strong, called after HS2.As shown in Figure 1, the bacteria colony white that this bacterial strain is formed, irregular arrangement, dry tack free, opaque, edge roughness, produces gemma.As shown in Figure 6, bacterial strain HS2 grows for potassium source containing potassium silicate with slightly solubility, and is translated into soluble potassium salt.Under laboratory shake flask condition, the inversion quantity of described peanut plant growth-promoting rhizobacteria HS2 to feldspar in powder reaches 19.05 mgL -1.Illustrate that HS2 bacterium has dissolution to feldspar in powder, grow for potassium source containing potassium silicate with slightly solubility, and be translated into soluble potassium salt.As shown in Figure 7, the organic phosphor that bacterial strain HS2 utilizes with difficulty grows for phosphorus source, and is translated into available phosphorus.Under laboratory shake flask condition, the inversion quantity of described peanut plant growth-promoting rhizobacteria HS2 to the organic phosphor that difficulty utilizes reaches 0.49 mgL-1.Illustrate that HS2 bacterium has dissolution to organic phosphor, with the organic phosphor of difficulty utilization for phosphorus source grows, and be translated into available phosphorus.
The quantitative assay of azotobacter nitrogen fixing capacity: quantitative assay is carried out to the azotobacteria that primary dcreening operation obtains, the azotobacter of purifying are accessed without in nitrogen liquid nutrient medium respectively, cultivate and collect bacterial cell by centrifugal process after 24 hours, in the bacterial cell collected, add 45 ml sterile waters and 5 ml again without nitrogen liquid nutrient medium, form 50 ml azotobacter suspension (every milliliter of about 107 bacterial cells) as bacterium liquid to be seeded.
Nitrogenase activity adopts acetylene reduction method to measure, and concrete operations are as follows: by the bacterial strain after purifying, are seeded in and 2 ml are housed without in the penicillin bottle of nitrogen liquid nutrient medium, cultivates 18 ~ 24 hours at 30 DEG C; Tampon is changed to the sealing of anti-chewing-gum plug, first have the penicillin bottle of bacterium to extract 0.5 ml air from cultivation with the syringe of good airproof performance, reinject 0.5 ml C 2h 2, seal pinprick with adhesive plaster; After continuing to cultivate 24h, get 100 μ L gas samples and measure C on gas chromatograph 2h 4peak value, Standard Gases C 2h 4concentration is 130 mg/L.
Adopt the HP6890 type gas chromatograph that Hewlett-Packard Corporation of the U.S. produces, its condition of work is set to: flame ionization ditector, temperature 250 DEG C, H 2flow 30 ml/min, pressure 20kPa; Chromatographic column is capillary, column length 15.0 m, internal diameter 320 μm, furnace temperature 30 DEG C; Carrier gas N 2, flow 30 ml/min, pressure 20kPa; Air mass flow 250 ml/min; Front injector temperature 250 DEG C, pressure 20 kPa.Acetyiene reduction activity (ARA), computational methods are:
Actual C 2h 4peak area × Standard Gases content × penicillin bottle volume
ARA= ——————————————————————
Standard Gases peak area × sample size × incubation time × sample size
(unit: nmol C 2h 4/ hml)
Said method is screened isolated bacterial strain, the handsome biotechnology Co., Ltd order-checking through Shanghai, according to the sequencing result of 16SrDNA, analyze in http://www.ncbi.nlm.nih.gov online query, utilize Blast software to carry out tetraploid rice with other 16S rDNA sequence in GenBank, select the 16SrDNA systematic evolution tree of the sequence MEGA version 3 software building HS2 of close sequence and HS2.According to the physiological and biochemical property of this bacterial strain, be accredited as bacillus subtilis ( bacillus subtilis).By this bacterial strain on October 31st, 2014 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number CGMCC No.9888.
This bacterial classification is Gram-positive, sporiferous irregular shaft-like.Bacteria colony white, dry tack free, opaque, edge roughness, produces gemma.Amphimicrobian, chemoheterotrophy.Optimum growth temperature is 30 DEG C.Catalase is positive, and nitrate reduction is positive.Producing IAA ability is strong, reaches 16.31 μ gmL -1, grow for potassium source containing potassium silicate with slightly solubility, and be translated into soluble potassium salt, there is nitrogen fixing capacity.
aerobic is tested
Sterilized LB medium is poured in 3 sterilized test tubes, and greatly about 2/3 place, on aseptic operating platform, the bacterial strain HS2 cultivated with transfer needle picking inclined-plane, percutaneous puncture-inoculation (must be punctured at the bottom of pipe) in above-mentioned medium.30 DEG C of cultivations, respectively 3 days to 7 days observed results.Be aerobic bacteria agar column surface-borne person, as being anaerobic bacteria or facultative anaerobe along the raw elder of puncture line.
Result of the test shows, and bacterial strain HS2 bacterium colony is along agar column superficial growth, and also having colony growth in puncture line, is amphimicrobian.
catalatic mensuration
Clean slide drips 1 3%H 2o 2, get bacterial strain HS2 LB slant culture 1 ring that 18 ~ 24 h cultivate, at H 2o 2in smear, if there is bubble to produce, be positive, otherwise be feminine gender.
Result of the test display bacterial strain HS2 is that catalase is positive.
methyl red test (M.R test)
A. medium and reagent: peptone 5g, glucose 5g, sodium chloride 5g, distilled water 1000mL, regulate pH7.0 ~ 7.2, packing test tube, often pipe fills 4 ~ 5 mL, 121 DEG C of sterilizing 20 min.Reagent: methyl red 0.1g, 95 % alcohol 300 mL, distilled water 200 mL.
B. Spawn incubation and result observe inoculating strain HS2 in above-mentioned culture fluid, cultivate l ~ 2 day for 30 DEG C.In culture fluid, add several methyl red reagent, as culture fluid presents redness, for methyl red is positive, yellow is negative (methyl red color change interval 4.4 redness ~ 6.0 is yellow).
Result of the test display bacterial strain HS2 is that M.R is negative.
second phthalein carbinol methine test (VP test)
A. the same methyl red test of medium.B. Spawn incubation and result are observed inoculation and are cultivated same methyl red test.When doing VP test, get culture fluid (about 2mL) and mix with 40 %NaOH phases of equivalent, add a small amount of creatine, after 2 ~ 5 min that fully vibrate, as culture fluid occurs red, be the VP positive.
Result of the test display bacterial strain HS2 is that VP is positive.
starch Hydrolysis is tested
A. medium and reagent add the soluble starch of 0.2% in meat soup peptone agar, and packing triangular flask, 121 DEG C of sterilizing 20min are for subsequent use.Road Ge Shi iodine liquid: crystalline flake of iodine 1g, potassium iodide 2g, first uses a small amount of (3 ~ 5mL) distilled water to dissolve potassium iodide, now adds the crystalline flake of iodine, after iodine dissolves completely, be diluted with water to 300mL.
B. Spawn incubation and result are observed and are got HS2 bacterial classification point and be connected on flat board, cultivate 2 ~ 4 days for 30 DEG C, after forming bacterium colony, flat board drips road Ge Shi iodine liquid, to be paved with periphery of bacterial colonies for degree, dull and stereotyped in blue, and periphery of bacterial colonies is irised out existing if any water white transparency, illustrate that starch is hydrolyzed.The size of the size general remark hydrolyzed starch ability of transparent circle.
Result of the test display bacterial strain HS2 is that Starch Hydrolysis is positive.
gelatin hydrolysis is tested
A. medium and reagent peptone 5g, gelatin 120g, distilled water 1000mL.Regulate pH7.2 ~ 7.4, packing test tube, medium height is about 4 ~ 5cm, 121 DEG C of sterilizing 20min.
B. Spawn incubation and result observation with puncture method inoculating strain HS2 in test tube central authorities.Cultivate one month in 30 DEG C of incubators, observe gelatin and whether liquefy.
Result of the test display bacterial strain HS2 is gelatin liquefaction positive.
nitrate reduction test
A. medium and reagent nitrate liquid nutrient medium: peptone 10g, KNO 31g, distilled water 1000mL, pH7.0 ~ 7.4.Ge Lisishi (Gries) reagent: A liquid: sulfanilic acid 0.5g, spirit of vinegar (about 10%) 150mL; B liquid: a-naphthols 0.1g, distilled water 20mL, spirit of vinegar (about 10%) 150mL.Diphenylamines reagent: diphenylamines 0.5g is dissolved in the l00mL concentrated sulfuric acid, uses 20mL distilled water diluting.
B. Spawn incubation and result are observed and are inoculated in nitrate liquid nutrient medium by bacterial strain HS2, cultivate 1,3,5 day for 30 DEG C.In white porcelain dish aperture, pour a little culture fluid into, then drip 1 reagent A and B liquid wherein respectively, when culture fluid become pink, rose, orange or brown etc. time, indicate that nitrite exists, be that nitrate reduction is positive, otherwise be feminine gender.
Result of the test bacterial strain display HS2 is that nitrate reduction is positive.
the utilization of citrate
A. medium and reagents citric acid sodium 2g, NaCl 5g, MgSO 47H 2o 0.2g, (NH 4) 2hPO 41g, 1% Bromothymol blue aqueous solution 10mL, agar 20g, distilled water 1000mL, pH6.8-7.0,121 DEG C of sterilizing 20min.
B. Spawn incubation and result are observed and are got children's age (cultivating 18-24h) HS2 strain inoculation on inclined-plane, and 30 DEG C of cultivation 3-7 days, medium is positive reaction in alkalescence (blueness) person, and constant person is then negative.
The result of the test display bacterial strain HS2 that citrate utilizes is the positive.
In order to verify that peanut plant growth-promoting rhizobacteria HS2 produces ability and the optimum condition of heteroauxin further, below for different pH, liquid amount, different carbon source, the impact of different nitrogen sources exploration on heteroauxin output.
To press 25ml containing L-Trp (100mg/L) LB liquid nutrient medium, 50ml, 75ml, 100ml, 150ml are loaded in the triangular flask of 250mL, by 1%(v/v) inoculum concentration inoculation be in the HS2 of exponential phase after, be placed in 30 DEG C, 180rmin -124 h cultivated by shaking table, measure the amount of producing IAA by the method for quantitative assay.As shown in Figure 2, because bacterial strain HS2 is amphimicrobian metabolism, throughput affects the efficiency that bacterial strain produces IAA to result, and during 25mL liquid amount, bacterial strain produces IAA amount at most, and afterwards along with liquid amount increases, output is fewer.
LB medium containing L-Trp (100mg/L) is adjusted to respectively different pH(4,5,6,7,8,9,10), getting 50mL is loaded in the triangular flask of 250mL, by 1%(v/v) inoculum concentration inoculation be in the HS2 of exponential phase after, be placed in 30 DEG C, 180rmin -124h cultivated by shaking table, and measure the amount of producing IAA by the method for quantitative assay, result as shown in Figure 3, do not produce IAA when showing that pH is 10, in strong acid and strong base environment, thalline cannot carry out growth metabolism, bacterial classification produces IAA more than sour environment in micro-alkali environment, and the optimal pH of this bacterial classification high yield IAA is 6 ~ 9.
In containing L-Trp (100mg/L) minimal medium, add 1%(w/v respectively) carbon source, carbon source has glucose, wood sugar, sucrose, fructose, mannitol, lactose, maltose, getting 50ml is loaded in the triangular flask of 250ml, by 1%(v/v) inoculum concentration inoculation be in the HS2 of exponential phase after, be placed in 30 DEG C, 180 rmin -124 h cultivated by shaking table, measure the amount of producing IAA by the method for quantitative assay.As shown in Figure 4, this bacterial strain is when supplying maltose, and the ability of producing IAA is the strongest, and be secondly fructose, the availability of lactose is minimum for result.
In containing L-Trp (100mg/L) minimal medium (not comprising ammonium sulfate), add 0.1%(w/v respectively) nitrogenous source, nitrogenous source comprises ammonium nitrate, ammonium sulfate, potassium nitrate, peptone, dusty yeast, alanine, urea etc., get 50ml to be loaded in the triangular flask of 250ml by 1%(v/v) inoculum concentration inoculation be in the HS2 of exponential phase after, be placed in 30 DEG C, 180rmin -124 h cultivated by shaking table, measure the amount of producing IAA by the method for quantitative assay.As shown in Figure 5, when illustrating that getting dusty yeast is nitrogenous source, the amount of producing IAA is maximum, is secondly peptone for result.
Bacterial strain HS2 of the present invention has obvious growth promoting function to peanut, is described below by pot experiment.
The fresh soil of sandstone area 0 ~ 20cm soil layer under collection natural conditions, cross 5mm sieve, every basin fills native 700g, plantation peanut, regulate water content to 60% of maxmun field capacity, 30 days post-samplings, after obtaining root system image with root scanner (LA1600+ scanner, Canada) scanning, with root system analysis software (Winrhizo2003b, Canada) related root index analysis is carried out, measure soil IAA content by HPLC method, and measure soil water-storage, rapid available phosphorus, quick-acting potassium content, plant fresh weight, plant height and the full potassium of total nitrogen and total phosphor phosphorus.
Peanut seed: peanut seed carries out 20% hydrogen peroxide surface sterilization 20min, repeatedly, vernalization 2d, chooses the consistent seed that germinates for subsequent use to aseptic water washing.
Connect bacterium process: HS2 of the present invention is inoculated in LB liquid nutrient medium, 30 DEG C, 180rmin -1shaking table is cultivated, and cultivates bacterium and grows to exponential phase, then by bacteria suspension 10000rmin -1centrifugal 10min, then use sterile water resuspended, repeat three times, inoculum concentration is 10 8cFUg -1(i.e. every gram of dry ground inoculation 10 8cFUg -1hS2 bacterial classification).
Control treatment: in contrast, soil does not spray HS2 bacterium liquid, adds equivalent sterile water.
The results are shown in following each table:
Table 3 inoculating strain HS2 is on the impact of Peanut Root System
Process Root long (cm) Root surface area (cm 2） Root volume (cm 3） Tip of a root number (individual)
CK 931.72±198.45 147.66±15.68 1.84±0.49 3356.14±931.59
HS2 1498.83±372.96 ** 218.06±40.79 ** 2.75±0.27 ** 6186.25±1184.40 **
Note: in same row * indicate significant difference ( p<0.05), * * represent pole significant difference ( p<0.01); Lower same.
Table 4 inoculating strain HS2 is on the impact of peanut plant
Process 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
HS2 5.41±1.01 ** 25.10±1.10 ** 42.73±0.83 ** 2.22±0.18 * 2.63±0.71 ** 8.00±1.17 **
Table 5 inoculating strain HS2 is on the impact of soil water-storage, rapid available phosphorus and available potassium
Process Ammonium nitrogen (mg/kg) Nitrate nitrogen (mg/kg) Mineral nitrogen (mg/kg) Rapid available phosphorus (mg/kg) Available potassium (mg/kg)
CK 21.90±1.88 4.48±0.97 26.91±1.77 3.16±0.21 34.00±3.46
HS2 30.07±7.44 * 6.05±0.19 * 37.50±5.71 3.98±0.34 * 42.00±3.00 *
As can be seen from Table 4, be vaccinated with the overground part fresh weight of the peanut plant that HS2 soil-grown goes out, and plant height comparatively CK have obvious growth trend; Because HS2 has the effect of fixing nitrogen, dissolving phosphor and dissolving potassium, soil Mineral N, rapid available phosphorus and quick-acting potassium content is made to increase (table 5), thus facilitate the absorption (table 5) of plant to elements such as N, P, K, as can be seen from Table 3, inoculate HS2 process and do not connect bacterium process and contrast, Peanut Root System total length, root surface area, root volume and tip of a root number all significantly increase, and facilitate the growth of Peanut Root System; As can be seen from Figure 8, after connecing bacterium process, soil IAA content significantly increases, and exceeds about 3 times than control group.Can find out in conjunction with above result, peanut plant growth-promoting rhizobacteria HS2 of the present invention to the growth of root system, grow there is positive effect, IAA output is high, effectively can promote crop growth and improve output.Used at 3 mu of Peanut Fields continuously through 3 years, peanut yield all improves 10-15%, and the good of its effect was not expected.
The above is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (3)

1. a peanut plant growth-promoting rhizobacteria HS2, 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, and preserving number is CGMCC No.9888.
2. preserving number according to claim 1 is the application of peanut plant growth-promoting rhizobacteria HS2 in promotion peanut growth of CGMCC No.9888.
3. preserving number according to claim 1 is the application of peanut plant growth-promoting rhizobacteria HS2 in peanut cultivation of CGMCC No.9888.
CN201410761474.8A 2014-12-13 2014-12-13 Peanut growth promoting rhizobacteria HS2 and application thereof Pending CN104560789A (en)

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CN106011005A (en) * 2016-05-20 2016-10-12 东莞市保得生物工程有限公司 Bacillus amyloliquefaciens T600 and preparation method and application of microbial agent
CN106190887A (en) * 2016-05-20 2016-12-07 东莞市保得生物工程有限公司 A kind of bacillus subtilis T400 and bacterial preparation process thereof
CN115521884A (en) * 2022-07-20 2022-12-27 四川宏展致远环保科技有限公司 Paenibacillus HNP12 and application thereof
CN116286437A (en) * 2022-08-08 2023-06-23 河北农业大学 Bacillus subtilis 2-20 and application thereof

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CN105296381A (en) * 2015-08-31 2016-02-03 哈尔滨师范大学 Bacillus subtilis CYY-25 and application thereof
CN105296381B (en) * 2015-08-31 2018-11-30 哈尔滨师范大学 One bacillus subtilis CYY-25 and its application
CN106011005A (en) * 2016-05-20 2016-10-12 东莞市保得生物工程有限公司 Bacillus amyloliquefaciens T600 and preparation method and application of microbial agent
CN106190887A (en) * 2016-05-20 2016-12-07 东莞市保得生物工程有限公司 A kind of bacillus subtilis T400 and bacterial preparation process thereof
CN106011005B (en) * 2016-05-20 2020-01-17 东莞市保得生物工程有限公司 Bacillus amyloliquefaciens T600 and preparation method and application of microbial inoculum thereof
CN106190887B (en) * 2016-05-20 2020-01-17 东莞市保得生物工程有限公司 Bacillus subtilis T400 and preparation method of microbial inoculum thereof
CN115521884A (en) * 2022-07-20 2022-12-27 四川宏展致远环保科技有限公司 Paenibacillus HNP12 and application thereof
CN115521884B (en) * 2022-07-20 2023-12-29 四川宏展致远环保科技有限公司 Paenibacillus HNP12 and application thereof
CN116286437A (en) * 2022-08-08 2023-06-23 河北农业大学 Bacillus subtilis 2-20 and application thereof
CN116286437B (en) * 2022-08-08 2023-08-18 河北农业大学 Bacillus subtilis 2-20 and application thereof

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