CN104073451B - Paenibacillus polymyxa and the application thereof of the multiple tree peony pathogen of one strain antagonism - Google Patents

Paenibacillus polymyxa and the application thereof of the multiple tree peony pathogen of one strain antagonism Download PDF

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CN104073451B
CN104073451B CN201410117673.5A CN201410117673A CN104073451B CN 104073451 B CN104073451 B CN 104073451B CN 201410117673 A CN201410117673 A CN 201410117673A CN 104073451 B CN104073451 B CN 104073451B
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tree peony
microbial inoculum
csm1101
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paenibacillus polymyxa
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CN104073451A (en
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韩继刚
王云山
胡永红
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SHANGHAI CHEN SHAN BOTANICAL GRADEN
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Abstract

The invention discloses Paenibacillus polymyxa and the application thereof of the multiple tree peony pathogen of a strain antagonism. Does is the Paenibacillus polymyxa of the multiple tree peony pathogen of this antagonism Paenibacillus polymyxa (Paenibacillus? polymyxa) CSM1101, does is its deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC? No.8527. Paenibacillus polymyxa (Paenibacillus? can polymyxa) CSM1101 significantly suppress tree peony botrytis (Botrytis? paeoniae), the mould (Cladosporium of tree peony branch spore? and the variegated tail spore of Chinese herbaceous peony mould (Cercospora paeoniae)? varricolor? Winter), and significantly promote the growth of tree peony.

Description

Paenibacillus polymyxa and the application thereof of the multiple tree peony pathogen of one strain antagonism
Technical field
The present invention relates to Paenibacillus polymyxa and the application thereof of the multiple tree peony pathogen of a strain antagonism.
Background technology
Tree peony returns Paeoniaceae (Paeoniaceae), Paeonia (Paeonia), is all Chinese most important sight all the timeOne of reward plant and medicinal plant, the history of existing more than 1600 year. At present, tree peony Main Cultivation place be at home Heze,Luoyang, Beijing, Linxia, day Peng, Tongling etc. In recent years, the oil of tree peony day by day highlights with being worth, and oil presents with the development of tree peonyThe situation of Rapid Expansion. For viewing and admiring tree peony, distinct issues are, due to different regions climatic environment and soilThe soil micro-ecosystem activity decreased that the difference of matter causes, be introduce a fine variety that regional culture tree peony upgrowth situation is bad, fancy points declinesOne of major reason. And for oil with tree peony, how to apply fertilizers scientifically and the output that significantly improves peony seeds is very urgentProblem. In the cultivation process of tree peony, be also faced with the threat of some fungal diseases, mainly contain gray mold (BotrytisPaeoniae), red spot disease (Cladosporiumaeoniae) and brown spot (Cercosporapaeoniae) etc. Therefore, grindSystem promotes the microbial bacterial agent of tree peony growth antagonism pathogen to have remarkable and urgent realistic meaning. Wherein, screening is to maleThe plant-growth promoting rhizobacteria microorganism resource that pellet has growth-promoting and antagonism pathogen function concurrently is the important bottle of restriction micro-organisms microbial inoculum developmentNeck factor.
Summary of the invention
A technical problem to be solved by this invention is to provide the multiple tree peony pathogen of a strain energy antagonism, promotes tree peony rawGrow and can produce taking maize straw as raw material the bacterial strain that promotes tree peony growth antagonism pathogen.
Bacterial strain provided by the present invention is Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101,Be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on December 09th, 2013,Preservation registration number is CGMCCNo.8527.
The morphological feature of described Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 is as follows: leatherThe Lan Shi positive, motion, it is shaft-like that thalline is, and diameter is 0.6-0.8 μ m, and long is 2.0-7.6 μ m. Gemma column, 1.6-3.5 × 1.2-1.5 μ m, middle life is raw to time end. Sporangium obviously enlarges into spindle-type or bar-shaped. The irregular splintery of its colony edge, translucentTo opaque. Rear its bacterium colony is coarse, shallow white, middle slightly projection. Bacterium colony adheres to media surface.
The physiological and biochemical property of described Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 is as follows:
Hydrolyzed starch: feminine gender;
Caseinhydrolysate: feminine gender;
Gelatin hydrolysate: feminine gender;
Utilize citrate: feminine gender;
Tyrosine decomposes: feminine gender;
PD experiment: feminine gender;
Yolk lecithin enzyme experiment: feminine gender;
Indoles produces: feminine gender;
Hippurate experiment: feminine gender;
Catalase experiment: the positive;
Nitrate reduction experiment: the positive;
Dihydroxyacetone produces: the positive;
Anti-lysozyme experiment: the positive;
Decomposition glucose produces sour aerogenesis: the positive;
Decompose arabinose and produce sour aerogenesis: the positive;
Decompose wood sugar and produce sour aerogenesis: the positive;
Decompose sweet mellow wine and produce sour aerogenesis: the positive;
Salt tolerance test: 7%NaCl growth;
In the nutrient broth medium (NB) of pH6.8 and the Sharpe glucose broth of pH5.7, all can grow. ?On MR-VP meat soup, produce acetyl methyl carbinol, pH6.0.
The 16SrDNA sequence of described Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 is as SEQShown in IDNo.1. Wherein, SEQIDNo.1 is made up of 1496 nucleotides.
Another object of the present invention is to provide a kind of microbial inoculum, and the active component of this microbial inoculum is described how sticky class gemma barBacterium (Paenibacilluspolymyxa) CSM1101.
This microbial inoculum is except the Paenibacillus polymyxa (Paenibacilluspolymyxa) comprising as active componentOutside CSM1101, also can comprise auxiliary material, as the stalk of the ight soil of the peat composed of rotten mosses, animal, all kinds of crops, loose shell, straw, peanut skin etc. InstituteState microbial inoculum and also can comprise carrier. Described carrier can be solid carrier or liquid-carrier. Described solid carrier is mineral material, plantMaterial or macromolecular compound; Described mineral material is clay, talcum, kaolin, montmorillonite, white carbon, zeolite, silica and diatomAt least one in soil; Described vegetable material is at least one in corn flour, bean powder and starch; Described macromolecular compound isPolyvinyl alcohol and/or polyglycols. Described liquid-carrier can be water. In described microbial inoculum, described active component can be to be cultivatedThe form of the mixture of the zymotic fluid of living cells, living cells, the filtrate of cell culture or cell and filtrate exists. Described combinationThe formulation of thing can be multiple formulation, as liquor, emulsion, suspending agent, pulvis, granule, wettable powder or water dispersible granules.
Described Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 can be following A 1)-A3) in appointA kind of microbial inoculum:
A1) microbial inoculum of the growth of promotion tree peony and antagonism pathogen;
Described pathogen is tree peony botrytis (Botrytispaeoniae), the mould (Cladosporium of tree peony branch sporePaeoniae) and in the variegated tail spore of Chinese herbaceous peony mould (CercosporavarricolorWinter) three kinds, any two or appointA kind of.
A2) microbial inoculum of antagonism pathogen; Described pathogen is tree peony botrytis (Botrytispaeoniae), tree peonyBranch spore mould (Cladosporiumpaeoniae) and the variegated tail spore of Chinese herbaceous peony mould (CercosporavarricolorWinter)In three kinds, any two or any.
A3) promote the microbial inoculum that tree peony grows.
Described Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 is at the described A1 of preparation)-A3)In any microbial inoculum in application also belong to protection scope of the present invention.
The following purposes of above-mentioned Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 or above-mentioned microbial inoculumAlso belong to protection scope of the present invention:
C1) promote tree peony growth and antagonism pathogen;
C2) described promotion tree peony growth;
C3) described antagonism pathogen.
The present invention also provides a kind of method that promotes tree peony growth.
The method of promotion tree peony provided by the present invention growth, is included in before taking root described in the Paeonia suffruticosa seed use after seed soakingThe step of culture of rootage processed 1-3 hour (as 1 hour) and carries out by microbial inoculum.
In said method, the physical state by the mode of the described microbial inoculum processing of Paeonia suffruticosa seed after soaking seed because of described microbial inoculumAnd different, if described microbial inoculum is liquid, directly soak the Paeonia suffruticosa seed after described seed soaking with described microbial inoculum, in described microbial inoculumThe content of described Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 can be 108cfu/ml-109cfu/Ml(is as 109Cfu/ml); If described microbial inoculum is solid-state, need to makes liquid bacterial agent to adding water in described microbial inoculum, then use this liquidBody microbial inoculum soaks the Paeonia suffruticosa seed after described seed soaking, the described Paenibacillus polymyxa in described liquid bacterial agent(Paenibacilluspolymyxa) content of CSM1101 can be 108cfu/ml-109Cfu/ml(is as 109cfu/ml）。
In a specific embodiment of the present invention, described tree peony is Feng Dan (Paeoniaostii).
In above-mentioned literary composition, the growth of described promotion tree peony can be presented as following B1)-B7) in any:
B1) shorten Paeonia suffruticosa seed rootage duration;
B2) improve Paeonia suffruticosa seed rooting rate;
B3) germinating time of shortening Paeonia suffruticosa seed;
B4) germination percentage of raising Paeonia suffruticosa seed;
B5) the main root length of raising tree peony;
B6) improve tree peony height of seedling;
B7) improve tree peony Seedling Biomass;
Described raising tree peony Seedling Biomass is presented as the fresh weight or the dry weight that improve tree peony seedling.
In said method, described pathogen can be tree peony botrytis (Botrytispaeoniae), tree peony branch spore is mould(Cladosporiumpaeoniae) three and in the variegated tail spore of Chinese herbaceous peony mould (CercosporavarricolorWinter)Kind, any two or any.
Another object of the present invention is to provide a kind of Paenibacillus polymyxa (Paenibacilluspolymyxa) of cultivatingThe method of CSM1101.
The side of cultivation Paenibacillus polymyxa provided by the present invention (Paenibacilluspolymyxa) CSM1101Method, comprises Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 is being glued to class gemma bars for cultivating moreThe step of cultivating in the culture medium of bacterium.
Another object of the present invention is to provide a kind of method of preparing described microbial inoculum.
The method of the described microbial inoculum of preparation provided by the present invention, comprises the steps: described Paenibacillus polymyxa(Paenibacilluspolymyxa) CSM1101, as active component, obtains described microbial inoculum.
Paenibacillus polymyxa of the present invention (Paenibacilluspolymyxa) CSM1101 can significantly suppress tree peony PortugalGrape spore bacterium (Botrytispaeoniae), tree peony branch spore mould (Cladosporiumpaeoniae) and the variegated tail spore of Chinese herbaceous peony mould(CercosporavarricolorWinter), their bacteriostasis rate has been reached respectively to 57.2%, 65.7% and 88.3%. ThisPaeonia suffruticosa seed (the microbial inoculum group lamination place that invention Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 processesReason) rootage duration shifted to an earlier date 10 days than the processing of control group lamination; The rooting rate of microbial inoculum group lamination processing reaches 95.0%, comparisonRooting rate according to group lamination processing (not using microbial inoculum processing) has improved 33.8%; The main root length of microbial inoculum group lamination processing is69.0mm is 1.68 times of control group lamination processing; The seed percentage of the main root length >=40mm of microbial inoculum group lamination processing reachesTo 90 ± 0.5%, be 1.3 times of control group lamination processing; The germinating time of microbial inoculum group lamination processing is processed contracting than control group laminationShort 10 days; The germination percentage of microbial inoculum group lamination processing reaches 99.0%, is 1.2 times of control group lamination processing; Microbial inoculum group lamination placeThe height of seedling of reason is 1.77 times of control group lamination processing, and the dry weight of microbial inoculum group lamination processing is 2.27 of control group lamination processingDoubly. Illustrate taking Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101CGMCCNo.8527 as active componentPromotion tree peony growth and the microbial inoculum of antagonism pathogen not only antagonism tree peony pathogen also promoted significantly the life of Paeonia suffruticosa seedThe growth of root (radicle sprouting) and germination (plumule sprouting) and tree peony seedling.
Preservation explanation
Strain name: Paenibacillus polymyxa
Latin name: Paenibacilluspolymyxa
Strain number: CSM1101
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on December 09th, 2013
Numbering: CGMCCNo.8527 registers on the books at preservation center
Brief description of the drawings
Fig. 1 is that microbial inoculum group lamination is processed and the control group lamination processing section seed culture of rootage photo of 60 days.
A is the processing of control group lamination, and b is the processing of microbial inoculum group lamination.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is further described in detail, the embodiment providing is only in order to explainBright the present invention, instead of in order to limit the scope of the invention. Experimental technique in following embodiment, if no special instructions, isConventional method. Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
Tree peony botrytis (Botrytispaeoniae) ACCC36053 and tree peony branch spore used in following embodimentMould (Cladosporiumpaeoniae) ACCC36063 was all concealed in Chinese microorganism strain and protects before the application's the applying dateHide administration committee agricultural microorganism center and (be called for short ACCC, address: No.12 ,zhongguancun south street,Haidian District, Beijing, Chinese agricultureIndustry academy of sciences agricultural resource and agricultural regionalization research institute, postcode 100081), the collection day of these two bacterial strains is in August, 200631, from collection, the public can obtain this bacterium from Chinese microorganism strain preservation administration committee agricultural microorganism center certainlyStrain.
In following embodiment, tree peony Pathogenic Bacteria Causing Brown Blotch Disease used is the variegated tail spore of Chinese herbaceous peony mould (CercosporaVarricolorWinter) (Zhang Jianguo, the Major Diseases of 2001. tree peonies and control. Chinese flower gardening, 23:32-34) public affairsCrowd can obtain from Shanghai Chenshan Botanical Garden, and this biomaterial related experiment of the present invention of only attaching most importance to is again used, not can be used as otherPurposes is used.
Tree peony in following embodiment is Feng Dan (Paeoniaostii) (JigangHan, YaoSong, ZhigangLiu,etc.2011.CulturablebacterialcommunityanalysisintherootdomainsofTwovarietiesoftreepeony (Paeoniaostii) .FEMSMicrobiolLett, 322:15 – 24) publicCan obtain from Shanghai Chenshan Botanical Garden, this biomaterial related experiment of the present invention of only attaching most importance to is again used, not can be used as other useWay is used.
Culture medium used in following embodiment is as follows:
Semi-solidNitrogen-free agar: sucrose, 10g; Malic acid, 5g; KH2PO4·H2O，0.4g；K2HPO4·H2O，0.1g；MgSO4·7H2O，0.2g；NaCl，0.1g；CaCl2·2H2O，0.02g；FeCl3，0.01g；Na2MoO4·2H2O, 0.02g; Agar, 3g; Be settled to 1000ml, pH7.0-7.2 with distilled water. 121 DEG C of steam sterilization 20min.
Nitrogen-free agar: sucrose, 10g; Malic acid, 5g; KH2PO4·H2O，0.4g；K2HPO4·H2O，0.1g；MgSO4·7H2O，0.2g；NaCl，0.1g；CaCl2·2H2O，0.02g；FeCl3，0.01g；Na2MoO4·2H2O，0.02g; Agar 18g; Be settled to 1000ml, pH7.0-7.2 with distilled water. 121 DEG C of steam sterilization 20min.
NB culture medium: glucose 5.0g, peptone 5.0g, beef extract 3.0g, is settled to 1000ml, pH7.0-with distilled water7.2,121 DEG C of sterilizing 20min.
NA culture medium: glucose 5.0g, peptone 5.0g, beef extract 3.0g, agar 18g, is settled to distilled water1000ml, pH7.0-7.2,121 DEG C of sterilizing 20min.
PDA culture medium: glucose 20g, agar 18g, potato 200g, the 1000mL that adds water boils 30min leaching juice, addsWater is supplied 1000ml, pH6.0-7.0,121 DEG C of sterilizing 20min.
Cellulase in following embodiment is purchased from Sangon Biotech (Shanghai) Co., Ltd.. In following embodimentCellulose enzyme unit of activity (FPU) be defined as 1g cellulase powder and at 55 DEG C, in 60min, be hydrolyzed filter paper gained glucose μMol number. The concrete assay method of cellulose enzyme vigor is as follows: extracting cellulose enzyme 10.00g, adds 100mlpH4.85's0.05mol/L acetic acid-sodium-acetate buffer fully dissolves, and obtains cellulase solution, by after cellulase solution dilution, draws0.5ml, adds 0.05mol/L acetic acid-sodium-acetate buffer 1.5ml, one of filter paper bar of 1 × 6cm Xinhua, hydrolysis at 55 DEG C60min. After hydrolysis, adopt the glucose content in following DNS method mensuration system, and calculate the enzyme activity of cellulase.
In following enforcement, in fermentation medium, the assay method of glucose content adopts DNS method, specific as follows:
(1) preparation of DNS reagent: 200g sodium potassium tartrate tetrahydrate, be dissolved in a certain amount of water, heating for dissolving, adds 10.0g3,5-dinitrosalicylic acid, 10gNaOH adds 2g phenol after dissolving, and 0.5g anhydrous sodium sulfite is all after heating for dissolving, coolingTo room temperature, be settled to 1000ml.
(2) drafting of glucose calibration curve: accurately take the analytically pure DEXTROSE ANHYDROUS of 100.0mg (in advance at 105 DEG CBe dried to constant weight), after dissolving with a small amount of distilled water, quantitatively transfer in 100ml volumetric flask, then constant volume to scale, shake up concentrationFor 1mg/mL. Get 10 test tubes, add respectively glucose standard liquid 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL,After 1.2mL, 1.6mL, to 2mL, then add 2.5mLDNS reagent mix even with distilled water diluting, in boiling water, heat 5min.After taking-up, with being water-cooled to room temperature, constant volume, to 25ml, shakes up. After 30min, under 520nm, survey OD value. With OD520Value is sat for verticalMark, glucose content is abscissa drawing standard curve, form is Y=aX+b.
(3) mensuration of concentration of glucose in zymotic fluid: get be diluted to certain density fermented supernatant fluid (4000r/min, fromHeart 20min), add 2.5mLDNS reagent mix even, in boiling water, heat 5min. Take out water cool to room temperature, be settled to25mL, shakes up, and after standing 30min, goes out to survey OD in 520nm520Value. OD per sample520Value is calculated in zymotic fluid with calibration curveGlucose content.
Separation and the qualification of embodiment 1, Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101
One, the separation of Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101
From the tree peony (Paeoniaostii, Feng Dan) of Kingsoft village, Shun An town, Tongling Anhui root bark of tree peony planting site (strain age 6Year) rhizosphere of plant gathers soil sample, soil sample put into the little triangular flask that sterilized water and sterilizing bead are housed, 200rpm/minVibration 20min; The suspension of getting after vibration does serial gradient dilution, 103、104The each 200 μ l that draw of dilution factor suspension are evenly coated withNitrogen-free agar, puts 30 DEG C and cultivates after 4d, picking list bacterium colony,Nitrogen-free agar solidOn culture medium flat plate, carry out purifying agaric with plate streak. By one of them bacterial strain called after tree peony root of separation and purification gainedBorder nitrogen-fixing bacteria CSM1101.
Two, the qualification of tree peony rhizosphere azotobacter CSM1101
1. morphological feature is observed and physio-biochemical characteristics mensuration
Morphology and physio-biochemical characteristics qualification are with reference to " uncle Jie Shi systematic bacteriology handbook " (Garrity, 2001) and " oneAs the conventional authentication method of bacteriology " (eastern elegant pearl etc., 2001), carry out according to conventional method.
The morphological feature of tree peony rhizosphere azotobacter CSM1101 is as follows: Gram-positive, and motion, it is shaft-like that thalline is, diameterFor 0.6-0.8 μ m, long is 2.0-7.6 μ m. Gemma column, 1.6-3.5 × 1.2-1.5 μ m, middle life is raw to time end. Sporangium is obviousEnlarge into spindle-type or bar-shaped. The irregular splintery of its colony edge, translucent to opaque. Rear its bacterium colony is coarse, shallow white,Middle slightly projection. Bacterium colony adheres to media surface.
The physiological and biochemical property measurement result of tree peony rhizosphere azotobacter CSM1101 is as follows:
Hydrolyzed starch: feminine gender;
Caseinhydrolysate: feminine gender;
Gelatin hydrolysate: feminine gender;
Utilize citrate: feminine gender;
Tyrosine decomposes: feminine gender;
PD experiment: feminine gender;
Yolk lecithin enzyme experiment: feminine gender;
Indoles produces: feminine gender;
Hippurate experiment: feminine gender;
Catalase experiment: the positive;
Nitrate reduction experiment: the positive;
Dihydroxyacetone produces: the positive;
Anti-lysozyme experiment: the positive;
Decomposition glucose produces sour aerogenesis: the positive;
Decompose arabinose and produce sour aerogenesis: the positive;
Decompose wood sugar and produce sour aerogenesis: the positive;
Decompose sweet mellow wine and produce sour aerogenesis: the positive;
Salt tolerance test: 7%NaCl growth;
In the nutrient broth medium (NB) of pH6.8 and the Sharpe glucose broth of pH5.7, all can grow. ?On MR-VP meat soup, produce acetyl methyl carbinol, pH6.0.
The sequence amplification of 2.16SrDNA and analysis
Adopt bacterial genomes DNA to extract kit (TIANGEN company) and extract the mould of genomic DNA as PCR reactionPlate. PCR primer (Pf5 '-CGGGATCCAGAGTTTGATCCTGGCTCAG-3 ' and Pr5 '-CGGGATCCAAGGAGGTGATCCAGCC-3 ') synthesized by Sangon Biotech (Shanghai) Co., Ltd.. Use Perkin-The 16SrDNA of ElmerGeneAmpPCRSystem9700PCR amplification bacterium. Reaction system is 10 × PCRbuffer,2.5uL; DNTP(2.5mM) (TaKaRa), 2uL, Pf(10pmol/uL) 0.1uL, Pr(10pmol/uL) 0.1uL, Taq polymerase(5U/uL) (TaKaRa), 0.125uL, genomic DNA 0.5uL, adds ddH2O to 25uL. PCR reaction condition is, 94 DEG C of denaturations5min, 94 DEG C of sex change 1min, 52 DEG C of renaturation 1min, 72 DEG C are extended 1min, 72 DEG C of last extension 10min, 30 circulations. PCR expandsVolume increase thing is by Sangon Biotech's purifying order-checking. The 16SrDNA sequence of bacterial strain is existedGenBank GenBank carries out sequence comparing analysis.
Result shows the tree peony rhizosphere azotobacter CSM110116SrDNA sequence total length 1496bp recording, as SEQIDNo.1. The result of carrying out blastn analysis at GENBANK shows, its sequence and PaenibacilluspolymyxastrainM-1(EF656457) similitude reaches 99%.
According to the morphological feature of tree peony rhizosphere azotobacter CSM1101, physiological and biochemical property and 16srDNA sequence homologyAnalysis result, is accredited as Paenibacillus polymyxa (Paenibacilluspolymyxa) by tree peony rhizosphere azotobacter CSM1101.Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 has been preserved in Chinese micro-life on December 09th, 2013(be called for short CGMCC, address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City at thing culture presevation administration committee's common micro-organisms centerNo. 3), deposit number is CGMCCNo.8527.
The biologically active of embodiment 2, Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101
One, the nitrogenase activity of Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 is measured
Nitrogenase activity is measured the Acetylene Reduction method that adopts. In 15mm × 15mm screw-cap test tube, add 2.5mL semisolidNitrogen-free agar, inoculation Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101CGMCCAfter No.8527, fill in tampon, cultivated 24h for 30 DEG C; Change again aseptic plug sealing, extract 10% gas out, inject the second of same volumeAlkynes, cultivates 24h for 30 DEG C. Extract gas sample and measure ARA(acetyiene on VarianVISTA6000 type gas chromatographReductionactivity, acetyiene reduction activity). Three repetitions are established in experiment, get its mean value and calculate variance to be not more than 5%.
Measurement result shows, CSM1101 existsIn nitrogen-free agar, show higher nitrogenase activity.Its acetyiene reduction activity is 12.6C2H4nmolmL-1h-1。
Two, the bacteriostatic activity of Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101
With tree peony botrytis (Botrytispaeoniae) ACCC36053, the mould (Cladosporium of tree peony branch sporePaeoniae) the arbitrary bacterial strain in ACCC36063, the variegated tail spore of Chinese herbaceous peony mould (CercosporavarricolorWinter)Be pathogen separately, all adopt dull and stereotyped face-off method to measure bacteriostasis rate, concrete grammar is as follows: pathogen bacterial strain is lived on PDA inclined-planeAfter change, add sterilized water to make pathogen suspension, pathogen suspension is added to the PDA training that is cooled to 45-50 DEG C after thawingSupport in base and mix and be down flat plate, cultivate 4 days at 30 DEG C, obtain pathogen flat board, beat and get pathogen bacterium with the card punch that diameter is 6mmCake.
Measure as follows Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 to every kind of diseaseThe bacteriostasis rate of former bacterium: experiment in triplicate, repeats pathogen at every turn and establishes two processing, i.e. CSM1101 group and control group. By 20Aseptic PDA flat board is divided into two groups, i.e. CSM1101 group and control group, every group of 10 flat boards, carry out following inoculation processing:The pathogen bacterium cake that the dull and stereotyped center inoculation of the aseptic PDA diameter of CSM1101 group is 6mm, by Paenibacillus polymyxa(Paenibacilluspolymyxa) CSM1101CGMCCNo.8527 slant activation bacterial classification is inoculated in apart from pathogen bacterium cake 3cmPlace. Meanwhile, only inoculate at the dull and stereotyped center of aseptic PDA of control group the pathogen bacterium cake that diameter is 6mm. Will be through above-mentioned inoculation placeCSM1101 group flat board and the control group flat board of reason are cultivated 5-7 days at 30 DEG C, treat that control group flat board (only inoculating pathogen) covers with wholeWhen individual culture dish, measure the pathogen colony diameter of control group and the pathogen colony diameter of CSM1101 group, calculate bacteriostasis rate.
Bacteriostasis rate (%)=(the pathogen colony diameter of pathogen colony diameter-CSM1101 group of control group)/control groupPathogen colony diameter × 100.
Result is as shown in table 1, shows Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101CGMCCNo.8527 is to tree peony botrytis (Botrytispaeoniae) ACCC36053, the mould (Cladosporium of tree peony branch sporePaeoniae) the variegated tail spore of ACCC36063 and Chinese herbaceous peony mould (CercosporavarricolorWinter) all has strongerDull and stereotyped bacteriostatic activity, its bacteriostasis rate has reached respectively 57.2%, 65.7% and 88.3%.
Table 1 Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101CGMCCNo.8527 is to pathogenBacteriostasis rate
The raw material that following embodiment 3-6 adopts is all identical, and just process conditions are variant, as shown in table 2.
The process conditions of table 2, embodiment 3-6
Embodiment 3, utilize maize straw enzymolysis liquid to produce the microbial inoculum A that promotes tree peony growth antagonism pathogen
1, the preparation of fermentation medium
Get maize straw 8000g(with dry weight basis), be cut into segment, drop into hydrothermal reaction kettle, at 180 DEG C of bars with 1.0MpaUnder part, carry out naturally drying after hydrothermal treatment consists 20min, obtain through pretreated maize straw; By the pretreated corn of this processAfter crushed stalk to 100 order, according to adding the ratio of 15FPU to add cellulase with every gram of stalk of dry weight basis, according to dry weightCount every 200 grams of dry straws and add the ratio of 1L distilled water to add distilled water, in 55 DEG C, 100rpm(radius of turn 20mm), pH=4.8 Water Under solution 48h after cooling centrifugal, collect respectively supernatant and precipitation, this supernatant is enzymolysis liquid, this precipitationFor enzymolysis residue. Measure the glucose content in enzymolysis liquid, result shows that the glucose content in enzymolysis liquid is 55g/L. Use enzymeSeparate liquid, (NH4)2SO4、MgSO4、CaCO3, corn steep liquor and water preparation fermentation medium (is that fermentation medium is by enzymolysis liquid, (NH4)2SO4、MgSO4、CaCO3, corn steep liquor and water composition), described in every liter of described fermentation medium, the content of enzymolysis liquid meets every literThe glucose content of described fermentation medium is 20g; In every liter of described fermentation medium, (NH4)2SO4Content be 10g,MgSO4Content be 0.4g, CaCO3Content be that the content of 6g, corn steep liquor is 2g.
2, fermented and cultured
2.1 seed culture
Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101CGMCCNo.8527 is cultivated at NAOn base inclined-plane, after activation culture, getting 2-3 ring is inoculated in the 50mlNB culture medium in 500ml triangular flask. 28 DEG C, 26mm amplitude,24h is as seed liquor for 200r/min shaken cultivation, prepares to cultivate for ferment tank.
2.2 fermented and cultured
The fermentation medium of getting 50L step 1 is placed in 100L automatic fermenter, in 121 DEG C of sterilizing 20min, cooling after,By the seed liquor access fermentation medium of 2.1 gained, inoculum concentration (V/V) is 10%. Cultivation temperature is 30 DEG C, ventilates by adjustingThe dissolved oxygen amount that amount and speed of agitator maintain in fermentation medium is 30%, and in sweat, automatically regulating pH is 6.8-7.2. Cultivate14 hours time, add the enzymolysis liquid of step 1, making the glucose content in fermentation medium is 15g/L, continues to cultivate 16 hours knotBundle fermentation. Altogether ferment 30 hours. Get zymotic fluid, measure the thalline content in zymotic fluid, in triplicate, result is averaged in experimentValue. Result shows the containing of Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 of embodiment 1 in zymotic fluidAmount is 2.58 × 1010Cfu/mL, gemma content is 85%. By mass mixings such as the enzymolysis residues in this zymotic fluid and step 1, sprayAfter mist is dry, (60 DEG C) granulate, and are promoted the microbial inoculum A of tree peony growth antagonism pathogen.
Embodiment 4, utilize maize straw enzymolysis liquid to produce the microbial inoculum B that promotes tree peony growth antagonism pathogen
1, the preparation of fermentation medium
Get maize straw 8000g(with dry weight basis), be cut into segment, drop into hydrothermal reaction kettle, at 180 DEG C of bars with 1.0MpaUnder part, carry out naturally drying after hydrothermal treatment consists 20min, obtain through pretreated maize straw; By the pretreated corn of this processAfter crushed stalk to 100 order, according to adding the ratio of 15FPU to add cellulase with every gram of stalk of dry weight basis, according to dry weightCount every 200 grams of dry straws and add the ratio of 1L distilled water to add distilled water, in 55 DEG C, 100rpm(radius of turn 20mm), pH=4.8 Water Under solution 48h after cooling centrifugal, collect respectively supernatant and precipitation, this supernatant is enzymolysis liquid, this precipitationFor enzymolysis residue. Measure the glucose content in enzymolysis liquid, result shows that the glucose content in enzymolysis liquid is 55g/L. Use enzymeSeparate liquid, (NH4)2SO4、MgSO4、CaCO3, corn steep liquor and water preparation fermentation medium (is that fermentation medium is by enzymolysis liquid, (NH4)2SO4、MgSO4、CaCO3, corn steep liquor and water composition), described in every liter of described fermentation medium, the content of enzymolysis liquid meets every literThe glucose content of described fermentation medium is 20g; In every liter of described fermentation medium, (NH4)2SO4Content be 10g,MgSO4Content be 0.4g, CaCO3Content be that the content of 6g, corn steep liquor is 2g.
2, fermented and cultured
2.1 seed culture
Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101CGMCCNo.8527 is cultivated at NAOn base inclined-plane, after activation culture, getting 2-3 ring is inoculated in the 50mlNB culture medium in 500ml triangular flask. 28 DEG C, 26mm amplitude,24h is as seed liquor for 200r/min shaken cultivation, prepares to cultivate for ferment tank.
2.2 fermented and cultured
The fermentation medium of getting 50L step 1 is placed in 100L automatic fermenter, in 121 DEG C of sterilizing 20min, cooling after,By the seed liquor access fermentation medium of 2.1 gained, inoculum concentration (V/V) is 10%. Cultivation temperature is 30 DEG C, ventilates by adjustingThe dissolved oxygen amount that amount and speed of agitator maintain in fermentation medium is 30%, and in sweat, automatically regulating pH is 6.8-7.2. Cultivate24 hours, finish fermentation. Get zymotic fluid, measure the thalline content in zymotic fluid, test in triplicate results averaged. KnotFruit shows that the content of Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 of embodiment 1 in zymotic fluid is1.86×108Cfu/mL, gemma content is 85%. By mass mixings such as the enzymolysis residues in zymotic fluid and step 1, spraying is dry(60 DEG C) granulate afterwards, are promoted the microbial inoculum B of tree peony growth antagonism pathogen.
Embodiment 5, utilize maize straw enzymolysis liquid to produce the microbial inoculum C that promotes tree peony growth antagonism pathogen
1, the preparation of fermentation medium
Get maize straw 8000g(with dry weight basis), be cut into segment, drop into hydrothermal reaction kettle, at 200 DEG C of bars with 2.0MpaUnder part, carry out naturally drying after hydrothermal treatment consists 20min, obtain through pretreated maize straw; By the pretreated corn of this processAfter crushed stalk to 100 order, according to adding the ratio of 15FPU to add cellulase with every gram of stalk of dry weight basis, according to dry weightCount every 200 grams of dry straws and add the ratio of 1L distilled water to add distilled water, in 55 DEG C, 100rpm(radius of turn 20mm), pH=4.8 Water Under solution 48h after cooling centrifugal, collect respectively supernatant and precipitation, this supernatant is enzymolysis liquid, this precipitationFor enzymolysis residue. Measure the glucose content in enzymolysis liquid, result shows that the glucose content in enzymolysis liquid is 60g/L. Use enzymeSeparate liquid, (NH4)2SO4、MgSO4、CaCO3, corn steep liquor and water preparation fermentation medium (is that fermentation medium is by enzymolysis liquid, (NH4)2SO4、MgSO4、CaCO3, corn steep liquor and water composition), described in every liter of described fermentation medium, the content of enzymolysis liquid meets every literThe glucose content of described fermentation medium is 20g; In every liter of described fermentation medium, (NH4)2SO4Content be 10g,MgSO4Content be 0.4g, CaCO3Content be that the content of 6g, corn steep liquor is 2g.
2, fermented and cultured
2.1 seed culture
Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101CGMCCNo.8527 is cultivated at NAOn base inclined-plane, after activation culture, getting 2-3 ring is inoculated in the 50mlNB culture medium in 500ml triangular flask. 28 DEG C, 26mm amplitude,24h is as seed liquor for 200r/min shaken cultivation, prepares to cultivate for ferment tank.
2.2 fermented and cultured
The fermentation medium of getting 50L step 1 is placed in 100L automatic fermenter, in 121 DEG C of sterilizing 20min, cooling after,By the seed liquor access fermentation medium of 2.1 gained, inoculum concentration (V/V) is 10%. Cultivation temperature is 30 DEG C, ventilates by adjustingThe dissolved oxygen amount that amount and speed of agitator maintain in fermentation medium is 30%, and in sweat, automatically regulating pH is 6.8-7.2. Cultivate24 hours, finish fermentation. Get zymotic fluid, measure the thalline content in zymotic fluid, test in triplicate results averaged. KnotFruit shows that the content of Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 of embodiment 1 in zymotic fluid is2.78×109Cfu/mL, gemma content is 85%. By mass mixings such as the enzymolysis residues in zymotic fluid and step 1, spraying is dry(60 DEG C) granulate afterwards, are promoted the microbial inoculum C of tree peony growth antagonism pathogen.
Embodiment 6, utilize maize straw enzymolysis liquid to produce the microbial inoculum D that promotes tree peony growth antagonism pathogen
1, the preparation of fermentation medium
Get maize straw 8000g(with dry weight basis), be cut into segment, drop into hydrothermal reaction kettle, at 200 DEG C of bars with 2.0MpaUnder part, carry out naturally drying after hydrothermal treatment consists 10min, obtain through pretreated maize straw; By the pretreated corn of this processAfter crushed stalk to 100 order, according to adding the ratio of 15FPU to add cellulase with every gram of stalk of dry weight basis, according to dry weightCount every 200 grams of dry straws and add the ratio of 1L distilled water to add distilled water, in 55 DEG C, 100rpm(radius of turn 20mm), pH=4.8 Water Under solution 48h after cooling centrifugal, collect respectively supernatant and precipitation, this supernatant is enzymolysis liquid, this precipitationFor enzymolysis residue. Measure the glucose content in enzymolysis liquid, result shows that the glucose content in enzymolysis liquid is 46g/L. Use enzymeSeparate liquid, (NH4)2SO4、MgSO4、CaCO3, corn steep liquor and water preparation fermentation medium (is that fermentation medium is by enzymolysis liquid, (NH4)2SO4、MgSO4、CaCO3, corn steep liquor and water composition), described in every liter of described fermentation medium, the content of enzymolysis liquid meets every literThe glucose content of described fermentation medium is 20g; In every liter of described fermentation medium, (NH4)2SO4Content be 10g,MgSO4Content be 0.4g, CaCO3Content be that the content of 6g, corn steep liquor is 2g.
2, fermented and cultured
2.1 seed culture
Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101CGMCCNo.8527 is cultivated at NAOn base inclined-plane, after activation culture, getting 2-3 ring is inoculated in the 50mlNB culture medium in 500ml triangular flask. 28 DEG C, 26mm amplitude,24h is as seed liquor for 200r/min shaken cultivation, prepares to cultivate for ferment tank.
2.2 fermented and cultured
The fermentation medium of getting 50L step 1 is placed in 100L automatic fermenter, in 121 DEG C of sterilizing 20min, cooling after,By the seed liquor access fermentation medium of 2.1 gained, inoculum concentration (V/V) is 10%. Cultivation temperature is 30 DEG C, ventilates by adjustingThe dissolved oxygen amount that amount and speed of agitator maintain in fermentation medium is 30%, and in sweat, automatically regulating pH is 6.8-7.2. Cultivate24 hours, finish fermentation. Get zymotic fluid, measure the thalline content in zymotic fluid, test in triplicate results averaged. KnotFruit shows that the content of Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 of embodiment 1 in zymotic fluid is4.42×108Cfu/mL, gemma content is 85%. By mass mixings such as the enzymolysis residues in zymotic fluid and step 1, spraying is dry(60 DEG C) granulate afterwards, are promoted the microbial inoculum D of tree peony growth antagonism pathogen.
Comparative example: utilize starchy material to produce and promote the also microbial inoculum E of antagonism pathogen of tree peony growth
1, fermentation medium preparation
Fermentation medium: with starch, (NH4)2SO4、MgSO4、CaCO3, (the i.e. fermentation of corn steep liquor and water preparation fermentation mediumCulture medium is by starch, (NH4)2SO4、MgSO4、CaCO3, corn steep liquor and water composition), starch described in every liter of described fermentation mediumThe content glucose content that meets every liter of described fermentation medium be 20g; In every liter of described fermentation medium, (NH4)2SO4'sContent is 10g, MgSO4Content be 0.4g, CaCO3Content be that the content of 6g, corn steep liquor is 2g. )
The component adopting in the fermentation medium of this comparative example is except maize straw enzymolysis liquid being replaced with etc. to glucose contentStarch outside, other is all identical with embodiment 3-6 for other component in fermentation medium.
2, fermented and cultured
In this fermented and cultured, the preparation method of zymotic fluid is identical with embodiment 4-6, specific as follows:
2.1 seed culture
Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101CGMCCNo.8527 is cultivated at NAOn base inclined-plane, after activation culture, getting 2-3 ring is inoculated in the 50mlNB culture medium in 500ml triangular flask. 28 DEG C, 26mm amplitude,24h is as seed liquor for 200r/min shaken cultivation, prepares to cultivate for ferment tank.
2.2 fermented and cultured
Get 50L fermentation medium and be placed in 100L automatic fermenter, in 121 DEG C of sterilizing 20min, cooling after, by 2.1The seed liquor access fermentation medium obtaining, inoculum concentration (V/V) is 10%. Cultivation temperature is 30 DEG C, by regulating throughput and stirringThe dissolved oxygen amount that rotating speed maintains in fermentation medium is 30%, and in sweat, automatically regulating pH is 6.8-7.2. Cultivate knot 24 hoursBundle fermentation. Get zymotic fluid, measure the thalline content in zymotic fluid, test in triplicate results averaged. Result shows fermentationIn liquid the content of the Paenibacillus polymyxa of embodiment 1 (Paenibacilluspolymyxa) CSM1101 be 3.66 ×109Cfu/mL, gemma content is 85%. (60 DEG C) after fermented liquid spray drying are granulated, be promoted tree peony growth antagonism diseaseThe microbial inoculum E of former bacterium.
The microbial inoculum of embodiment 7, the growth of promotion tree peony antagonism pathogen promotes Paeonia suffruticosa seed to sprout and tree peony growth of seedling
Seed collection: Feng Dan (Paeoniaostii) the Follicle radish fruit (Tongling, Anhui Province of gathering taking pericarp crab oil as standard JulyKingsoft village, Shun An town, city), put indoor drying in the shade and collect seed, 0.5%KMnO to pericarp cracking4Immersion 2h carries out disinfection and is disappearedFeng Dan (Paeoniaostii) seed after poison is for test. Test site is Shanghai Songjiang district Shanghai Chenshan Botanical Garden.
The system of Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101CGMCCNo.8527 bacteria suspensionStandby: the microbial inoculum A of the promotion tree peony growth of embodiment 3 antagonism pathogen is suspended in and in sterilized water, obtains Paenibacillus polymyxa(Paenibacilluspolymyxa) CSM1101CGMCCNo.8527 bacteria suspension, makes Paenibacillus polymyxa in bacteria suspension(Paenibacilluspolymyxa) content of CSM1101CGMCCNo.8527 is 109cfu/mL。
Cancel Feng Dan (Paeoniaostii) seed after poison, the Feng Dan after obtaining soaking seed with 50 DEG C of sterilized waters seed soaking 24h(Paeoniaostii) seed, hereinafter to be referred as seed soaking after Paeonia suffruticosa seed, carry out following seed take root (radicle sproutings) experiment withGermination (plumule sprouting) experiment. Take root (radicle sprouting) experiment and germinate (plumule sproutings) of seed tested all in triplicate, at every turnRepeat to arrange two kinds of processing, microbial inoculum group lamination is processed and the processing of control group lamination. Seed is taken root in (radicle sprouting) experiment, everyPlant the Paeonia suffruticosa seed of processing after 300 seed soaking. Germinate during (plumule sproutings) test, every kind of processing choose 50 main root length >=What 40mm and length were consistent take root seed. Specific experiment method is as follows:
1, seed take root (radicle sprouting) experiment
Microbial inoculum group lamination is processed with control group lamination and is processed in these two kinds of processing except the fore-pretreatment method of taking root is different, itsIts processing mode is identical. Before the taking root of microbial inoculum group lamination processing, pretreatment is as follows: the Paeonia suffruticosa seed after seed soaking is placed in manySticky series bacillus (Paenibacilluspolymyxa) CSM1101CGMCCNo.8527 bacteria suspension (109Cfu/mL) in25-30 DEG C soak 1 hour, complete take root before pretreatment. Before the taking root of control group lamination processing, pretreatment is as follows: by after seed soakingPaeonia suffruticosa seed is placed in sterilized water at 25-30 DEG C and soaks 1 hour, complete take root before pretreatment. Then will complete before taking root and locate in advanceThe seed of reason all carries out following culture of rootage: by complete take root before pretreated seed and the water content high-temperature sterilization that is 400%After perlite (lamination matrix) mixes according to the volume ratio of 1:3, pack polyethylene plastic bag into and be all placed in 10 DEG C-25 DEG C (daytime 20-25DEG C, night 10-15 DEG C) carry out culture of rootage. Observe every day 1 time, investigation rootage duration, culture of rootage 90d researching determining rooting rate,The seed percentage of main root length, main root length >=40mm. Data are carried out significance of difference analysis with SPSS software.
Wherein, the length of exposing seed coat taking radicle as seed length 1/2 for taking root.
Rooting rate=(L1/L0) × 100%;
L1: the seed grain number of taking root in the experiment seed of taking root; L0: the experiment seed grain number of taking root.
2, germination (plumule sprouting) experiment
The microbial inoculum group lamination of getting respectively step 1 is processed and control group lamination process the main root length that these two kinds of processing obtain >=What 40mm and length were consistent take root, and seed, is planted, in high 10cm, peat and perlitic cave dish is housed after 21 days 4 DEG C of storagesIn, be placed in 10-25 DEG C of greenhouse (daytime 20-25 DEG C, night 10-15 DEG C) and germinate and cultivate seedling, observe every day 1 time, measure and send outThe bud time, germinate and cultivate the dry weight of 90d mensuration germination percentage, height of seedling, whole tree peony seedling plant, calculate germination percentage. Data are usedSPSS software carries out significance of difference analysis.
Wherein, expose seed coat as germination taking plumule.
Germination percentage=(L1 '/L0 ') × 100%;
L1 ': chitting piece grain number in Seed germination seed; L0 ': Seed germination seed grain number.
Result is as shown in table 3-table 5, and the rootage duration of microbial inoculum group lamination processing has shifted to an earlier date 10 days than the processing of control group lamination;The rooting rate of microbial inoculum group lamination processing reaches 95.0%, has improved 33.8% than the rooting rate of control group lamination processing; Microbial inoculum group layerThe long-pending main root length of processing is 69.0mm, is 1.68 times of control group lamination processing; The main root length of microbial inoculum group lamination processing >=The seed percentage of 40mm reaches 90 ± 0.5%, is 1.3 times of control group lamination processing; The germinating time of microbial inoculum group lamination processing10 days are shortened than the processing of control group lamination; The germination percentage of microbial inoculum group lamination processing reaches 99.0%, is the processing of control group lamination1.2 doubly; The height of seedling of microbial inoculum group lamination processing is 1.77 times of control group lamination processing, and the dry weight of microbial inoculum group lamination processing is contrast2.27 times of group lamination processing. Illustrate with Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101CGMCCNo.8527 is that the promotion tree peony growth of active component the microbial inoculum of antagonism pathogen have promoted taking root of Paeonia suffruticosa seed significantlyThe growth of (radicle sprouting) and germination (plumule sprouting) and tree peony seedling. Fig. 1 has shown the processing of microbial inoculum group lamination and control group layerThe long-pending processing section seed culture of rootage situation of 60 days.
Table 3, microbial inoculum are processed the impact that Paeonia suffruticosa seed is taken root
Note: the different significance degree of The English alphabet differential of same column in table, between the processing of same letter in 0.05 level without aobviousWork difference, between alphabetical different processing, in 0.05 level, there were significant differences. Rootage duration refers to the 1st time that seed is taken root.
Table 4, microbial inoculum are processed the impact that Paeonia suffruticosa seed is germinateed
Process Germinating time (my god) Germination percentage (%)
The processing of microbial inoculum group lamination 26±1a 99.0±0.6a
The processing of control group lamination 36±2b 81.0±0.2b
Note: the different significance degree of The English alphabet differential of same column in table, between the processing of same letter in 0.05 level without aobviousWork difference, between alphabetical different processing, in 0.05 level, there were significant differences. Germinating time refers to the 1st main root length >=40mm'sTake root time of germination.
Table 5, microbial inoculum are processed the impact on tree peony growth of seedling
Process Height of seedling (mm) Dry weight (mg)
The processing of microbial inoculum group lamination 122.0±2.0a 501.0±5.0a
The processing of control group lamination 69.0±2.0b 221.0±3.0b
Note: the different significance degree of The English alphabet differential of same column in table, between the processing of same letter in 0.05 level without aobviousWork difference, between alphabetical different processing, in 0.05 level, there were significant differences. Height of seedling is the height of seedling aerial part, and dry weight is wholeThe dry weight of individual plant, comprises root and bud.

Claims (6)

1. Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101, it is at Chinese microorganism strain preservation pipeThe deposit number at reason committee common micro-organisms center is CGMCCNo.8527.
2. a microbial inoculum, its active component is Paenibacillus polymyxa (Paenibacillus claimed in claim 1polymyxa)CSM1101。
3. microbial inoculum according to claim 2, is characterized in that: described microbial inoculum is following A 1)-A3) in any microbial inoculum:
A1) microbial inoculum of the growth of promotion tree peony and antagonism pathogen;
Described pathogen is tree peony botrytis (Botrytispaeoniae), the mould (Cladosporium of tree peony branch sporePaeoniae) and in the variegated tail spore of Chinese herbaceous peony mould (CercosporavarricolorWinter) three kinds, any two or appointA kind of;
A2) microbial inoculum of antagonism pathogen; Described pathogen is tree peony botrytis (Botrytispaeoniae), tree peony branch sporeIn mould (Cladosporiumpaeoniae) and the variegated tail spore of Chinese herbaceous peony mould (CercosporavarricolorWinter)Three kinds, any two or any;
A3) promote the microbial inoculum that tree peony grows.
4. microbial inoculum according to claim 3, is characterized in that: described promotion tree peony growth is presented as following B1)-B7) inAny:
B1) shorten Paeonia suffruticosa seed rootage duration;
B2) improve Paeonia suffruticosa seed rooting rate;
B3) germinating time of shortening Paeonia suffruticosa seed;
B4) germination percentage of raising Paeonia suffruticosa seed;
B5) the main root length of raising tree peony;
B6) improve tree peony height of seedling;
B7) improve tree peony Seedling Biomass.
5. the method for Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 described in cultivation claim 1,Comprise Paenibacillus polymyxa (Paenibacilluspolymyxa) CSM1101 for cultivating Paenibacillus polymyxaCulture medium in the step of cultivating.
6. the preparation method of microbial inoculum described in claim 2,3 or 4, comprises the steps: many sticky class buds claimed in claim 1Spore bacillus (Paenibacilluspolymyxa) CSM1101, as active component, obtains described microbial inoculum.
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