CN108624528A - A kind of composite bacteria agent and its application to legume with growth-promoting production-increasing function - Google Patents
A kind of composite bacteria agent and its application to legume with growth-promoting production-increasing function Download PDFInfo
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Abstract
The invention discloses a kind of composite bacteria agent and its applications to legume with growth-promoting production-increasing function.Complex micro organism fungicide provided by the invention, including bacillus megaterium and rhizobium, the bacillus megaterium are bacillus megateriumBacillus megaterium HZP1, deposit number are CCTCC NO：M 2018061.The bacterial strain has the biological characteristics such as growing nitrogen-fixing, Soluble phosphorus, production auxin, with rhizobium with the use of in the soil to legume growth with good facilitation effect；Plant growth of the legume under the nutrients restrictive condition such as nitrogen phosphorus can be improved, improve crop yield, reduce the nitrogen application in agricultural production, tremendous economic and ecological benefits will be generated.The novel Inoculant of the present invention has considerable market value in microbial manure field.
Description
Technical field
The invention belongs to agricultural microbial agent technical fields, and in particular to a kind of to there is growth-promoting volume increase to make legume
Composite bacteria agent and its application.
Background technology
Microorganism group (microbiome) refers to whole microorganisms and its heredity in a specific environment or the ecosystem
Information, including its cell colony and quantity, whole inhereditary materials (genome), it, which is defined, covers micropopulation and its all loses
It passes and physiological function, intension includes the interaction of microorganism and its environment and host, it is micro- with the interdependent interpromoting relation in five elements of crops
Biological group is an important factor for influencing plant growth, yield and quality.It is multiplied jointly in legume rhizosphere microecological system
A large amount of bacillus and rhizobium, they form symbiosis during long-term evolution with legume.2016
Zgadzaj etc. reports the achievement in research in pattern legume crowtoe rhizosphere, it is found that nodule formation also determines symbiosis
The foundation (Zgadzaj, et al.PNAS, 2016) of related, unique rhizosphere, root and rhizobia structure of community.This result
Show symbiotic nitrogen fixation for legume rhizosphere microorganism group foundation have key regulatory effect (Science Daily,
2016).These important research achievements have expanded field and the thought of traditional symbiotic nitrogen fixation basic research.From apply angle,
It is inadequate in legume inoculation rhizobium that researcher, which further infers that only, and should be inoculated with the micro- life of legume rhizosphere simultaneously
Related Bacteria in object group is to improve nitrogen-fixing efficiency.The novel inoculation of structure is combined with rhizobium using root nodule symbiosis Related Bacteria
Agent improves plant growth of the legume under the nutrients restrictive condition such as nitrogen phosphorus, crop yield is improved, to reduce agricultural production
In nitrogen application.
Through retrieval, the complex microorganism seed dressing of the researchs such as Chen Long men in 2014 has well non-leguminous plant as base manure
Effect of increasing production.There is the azotobacteria fertilizer of effect of increasing production that there is preferable nitrogen fixing capacity in alfalfa disclosed in Zhu Ruifen in 2017
With growth-promoting effect.But the double inoculations of rhizobium+bacillus megaterium are as dedicated legume inoculation agent without report.
Invention content
It is described the object of the present invention is to provide a kind of composite bacteria agent to legume with growth-promoting production-increasing function
Composite bacteria agent includes rhizobium and bacillus megaterium (Bacillus megaterium) HZP1, the bacillus megaterium
Deposit number be CCTCC NO：M 2018061, the bacterial strain have growing nitrogen-fixing, the secretion work(such as heteroauxin and efficient phosphate-solubilizing
Energy.
It is another object of the present invention to provide to legume have growth-promoting production-increasing function composite bacteria agent application,
The composite bacteria agent is particluarly suitable for low-phosphorous soil application；The microbial inoculum can be used for preparing seed dressing or legume increasing agent,
Or nitrogenous fertilizer partial alternative agent.
In order to achieve the above object, the present invention is achieved by the following technical solutions：
Applicant is from legume nodule microorganism group, the list for obtaining having efficient phosphate-solubilizing ability through separation screening culture medium
Bacterium colony, the bacterial strain are sent on January 26th, 2018 to China typical culture collection center preservation, Classification And Nomenclature：Huge gemma
Bacillus (Bacillus megaterium) HZP1, address：Wuhan, China Wuhan University, deposit number：CCTCC NO：M
2018061。
The morphological feature of the bacillus megaterium HZP1 includes：Rod-short, Gram-positive, bacterium colony are round, edge is whole
Together, gemma is produced.
The cultural character of the bacillus megaterium HZP1 includes：PH is 5.0-9.0, optimal pH 6.0-7.0；Temperature is
22-48 DEG C, optimum temperature is 37-39 DEG C；Salt tolerance reaches 8%NaCl.
The growth-promoting characteristic of the bacillus megaterium HZP1 includes：The functions such as growing nitrogen-fixing, production growth hormone, Soluble phosphorus.
The growing nitrogen-fixing enzymatic activity of the bacillus megaterium HZP1 is 208.5nmol/ (mLh), point of heteroauxin
The amount of secreting is 214.84mg/L, amount of phosphorus dissolved 195.80mg/L.
A kind of composite bacteria agent to legume with growth-promoting production-increasing function, the composite bacteria agent includes rhizobium and huge
Bacterium anthracoides (Bacillus megaterium) HZP1, current published rhizobium can complete the present invention；
In above-described composite bacteria agent, it is preferred that the rhizobium are Rhizobium of Milk Vetch 7653R or Fei Shi China
Rhizobium strains HH103.
In above-described composite bacteria agent, it is preferred that the rhizobium and bacillus megaterium (Bacillus
Megaterium) effective bacteria concentration ratio of HZP1 is 10~5:1.
The application to legume with the composite bacteria agent of growth-promoting production-increasing function, including the use of microbial inoculum system provided by the invention
For at seed dressing, Inoculant；Or legume increasing agent or nitrogenous fertilizer partial alternative agent.
In above-described application, it is preferred that when being prepared as seed dressing, formula includes：
The effective bacterial content of the rhizobium is 20 × 108-50×108cfu/ml；The bacillus megaterium HZP1 bacterium solutions
Effective bacterial content is 5 × 108-10×108cfu/ml；
The formula of the liquid microelement is：H3BO35g, Na2MoO45g, water 1000ml.
Compared with prior art, the present invention has following advantageous effect：
The present invention pointedly detaches legume nodule symbiosis correlation PGPR bacteriums, with the long-term symbiosis of rhizobium.Profit
The novel Inoculant of structure is combined with rhizobium with root nodule symbiosis Related Bacteria, has the function of nitrogen phosphorus synergistic effect, pulse family can be improved
Plant growth, nitrogen-fixing efficiency of the crop under the nutrients restrictive condition such as nitrogen phosphorus, to improve crop yield.It is novel in the present invention
Inoculant is suitble to low-phosphorous soil, application that Chinese milk vetch ground biomass is made to significantly improve, soybean increases mainly for legume
Production, is good legume Inoculant.
Description of the drawings
Fig. 1 is bacterial strain HZP1 phosphorus decomposing effect diagrams.
Fig. 2 is bacterial strain HZP1 Gram's staining microscopically observation (× 1000) schematic diagram.
Fig. 3 is bacterial strain HZP1 spore stainings microscopically observation (× 400) schematic diagram.
Fig. 4 is bacterial strain HZP1 16S rDNA fragments gel electrophoresis detection schematic diagrames.
Fig. 5 is the influence schematic diagram that pH grows bacterial strain HZP1.
Fig. 6 is the influence schematic diagram that salt stress grows bacterial strain HZP1.
Fig. 7 is phosphorus solution standard curve schematic diagram.
Fig. 8 is that bacterial strain HZP1 secretes heteroauxin (IAA) measurement schematic diagram.
Fig. 9 significantly improves Chinese milk vetch ground biomass schematic diagram after being bacillus HZP1 and the double inoculations of rhizobium.
Figure 10 is that composite bacteria agent remarkably promotes soybean root system development and nodulation and nitrogen fixation schematic diagram in Field information.
Specific implementation mode
The invention will be further described with the following Examples.It should be noted that following embodiment contributes to this field
Technical staff further understands the present invention, under the premise of not departing from present invention conception, can make improvement and extension.These are all
It belongs to the scope of protection of the present invention.
Embodiment 1：
Bacillus megaterium HZP1 separation and screening：
From legume nodule microorganism group, the bacterium colony for through separation screening culture medium obtaining that there is dissolving P capacity.It will smash to pieces
90ml sterile waters are added in root nodule sample 10g afterwards, and various concentration gradient is diluted to after fully shaking mixing contains bacteria suspension, takes
100ul is applied on NBRIY culture mediums, is crossed repeatedly after growing bacterium colony until isolating single bacterium colony.Obtained new isolate
Sterile circular filter paper piece is immersed bacterium solution and is then labelled to NBRIY solid mediums by shaking flask LB cultures, and lawn to be grown observes phosphorus decomposing
Size is enclosed, there is the comparison of dissolving P capacity bacterial strain with existing, be finally obtained the bacterial strain (Fig. 1) of one plant of phosphorus decomposing best results.
Separation screening culture medium described above is a kind of phosphorus decomposing culture medium-NBRIY culture mediums, and formula is as follows:Glucose,
10g；Ca3(PO4)2, 5g；(NH4)2SO4, 0.5g；NaCl, 0.2g；MgSO4·7H2O, 0.1g；KCl,0.2g；MnSO4·H2O,
0.002g；FeSO4·7H2O, 0.002g, dH2O to 1000mL, pH 7.0.
Obtained new isolate is subjected to Gram's staining (Fig. 2) and spore staining (Fig. 3), the results showed that it is gram
Positive bacteria；The 16S rDNA of PCR amplification bacterial strain, detected through gel electrophoresis (Fig. 4) and be sequenced identified, through BLAST analysis and
Structure chadogram show that the bacterial strain is Bacillus megaterium.
The bacterial strain is sent on January 26th, 2018 to China typical culture collection center preservation, Classification And Nomenclature：It is huge
Bacillus (Bacillus megaterium) HZP1, address：Wuhan, China Wuhan University, deposit number：CCTCC NO：M
2018061。
Growth characteristics of the HZP1 at different pH and salinity：The bacillus carries out acid-base property and salt tolerance
It measures, and draws growth tendency figure, it is found that its optimal pH is 6.0-7.0 (see Fig. 5), resistance to salinity reaches 8%NaCl (see figure
6).The specific method is as follows：
5ml LB liquid mediums are inoculated in from picking bacillus megaterium HZP1 single bacterium colonies on activating solid culture medium,
After 12h by 1% inoculum concentration be inoculated into containing different pH (4.0,5.0,6.0,7.0,8.0,9.0) and NaCl (4%, 5%, 6%,
7%, 8%) LB liquid medium 250ml, 200rpm, 37 DEG C shaking table culture measures an OD every 6h600, not connect bacterium culture
Base compares, in triplicate.
Measurement result 1：In 4~9 range of pH value, bacillus megaterium HZP1 is grown most preferably at 6~7.
Measurement result 2：Bacillus megaterium HZP1 declines as NaCl concentration increases increment, and 8% when remains to grow.
The measurement of amount of phosphorus dissolved：Molybdenum antimony resistance colorimetric method measures bacillus megaterium (Bacillus megaterium) HZP1
Amount of phosphorus dissolved is 195.80mg/L, and determination step is as follows：
One ring bacterium of picking is in 5ml beef-protein mediums, 37 degree, 12h is cultivated, as primary seed solution.It takes
0.5ml primary seed solutions are in 5ml beef-protein mediums, 37 degree, 12h cultivated, as secondary seed solution.It is connect with 4%
Kind amount is inoculated in the 500ml triangular flasks equipped with 100ml beef-protein mediums, 37 degree, 220r/min, culture 72h (with
The beef-protein medium that equivalent sterile water is added is blank control).
Phosphorus standard solution 0ml, 1ml, 2ml, 3ml, 4ml, 5ml, 6ml is drawn respectively in 50ml volumetric flasks, adds distilled water
It is diluted to 30ml, dinitrophenol dinitrophenolate indicator 2 is added to drip, it has been in just micro- Huang to adjust pH to solution with sodium hydroxide solution or dilution heat of sulfuric acid
Color.Be added the anti-color developing agent 5ml of molybdenum antimony, shake up, constant volume to get phosphorus concentration 0.0mg/L, 0.1mg/L, 0.2mg/L, 0.3mg/L,
The standard solution of 0.4mg/L, 0.5mg/L, 0.6mg/L.30min is stood at room temperature, under 700nm wavelength, with 0.0mg/L standards
Solution is blank control zeroising, measures absorbance.Using absorbance as ordinate, phosphorus concentration is abscissa, draws standard curve
(Fig. 7).
Sample 4000r/min is centrifuged into 20min, 5~10ml of Aspirate supernatant is diluted with water in 50ml volumetric flasks
30ml adds dinitrophenol dinitrophenolate indicator 2 to drip, and it has been in just yellowish to adjust pH to solution with sodium hydroxide solution or dilution heat of sulfuric acid.So
The anti-color developing agent 5ml of molybdenum antimony is added afterwards, shakes up, constant volume.30min is stood at room temperature, under 700nm wavelength, is joined with blank test solution work
Than zeroising, absorbance is measured.
As a result it calculates
Content (mg/L)=(P × V of phosphorus in culture solution1×K)/V2
P-standard curve checks in the concentration (mg/L) of phosphorus
K-extension rate
V1- developing solution volume (ml)
V2- Aspirate supernatant volume (ml)
Measurement result：Water-soluble phosphorus content in bacillus megaterium HZP1 culture solutions is 10.70 times of blank test solution.
Phosphorus decomposing rate measures under different phosphorus concentrations：In different initial phosphorus concentration medium culture bacillus megateriums
Its phosphorus decomposing rate is measured after (Bacillus megaterium) HZP1, steps are as follows：
One ring bacterium of picking is in 5ml beef-protein mediums, 37 degree, cultivates 12h, and 4000r/min centrifuges 10min,
Go after supernatant to be inoculated in different initial phosphorus concentrations (0mg/L, 5mg/L, 10mg/L, 50mg/L, 100mg/L, 500mg/L,
NBRIY Liquid Cultures 1000mg/L) are based in 500ml triangular flasks, 37 degree, 220r/min, cultivate 72h.
Phosphorus content in culture solution is measured with reference to molybdenum antimony resistance colorimetric method in embodiment 1, then calculating maximum phosphorus decomposing rate, (phosphorus is reduced
Amount accounts for the percentage of initial phosphate concentrations), it is as a result as follows：
Conclusion：Bacillus megaterium HZP1 phosphorus decomposing efficiency in low-phosphorous culture solution is up to 71.3%, shows that HZP1 is suitble to
Soluble phosphorus content is compared with performance phosphorus decomposing effect in low environment.
Generate IAA quantitative determinations：Bacillus megaterium (Bacillus megaterium) HZP1 is measured into production IAA (figures
7), as a result, it has been found that in the LB culture solutions containing 200mg/L Trp, the secretory volume of the bacillus megaterium 12h heteroauxins is
214.84mg/L determination step is as follows：
1) preparation of Salkowski color solutions：10g FeCl3·6H2O is dissolved in 7.9mol/LH2SO4In 500ml.
2) selection standard product IAA prepares two groups of IAA standard solution, one group of configuration concentration is 2.5,5,7.5,10,12.5,
15,17.5,20 μ g/ml, another group is 25,50,75,100,125,150,175,200 μ g/ml.Every part takes 4ml, respectively enters 4ml ratios
Color liquid, in triplicate, dark stand 30min, measure OD530, make standard curve.
The 6000r/min centrifuging and taking supernatant 4ml after the LB culture solutions of the Trp containing 200mg/L shake inoculation HZP1,12h, add
Enter isometric color solution, dark stands 30min, measures OD530, with the standard items OD on standard curve530Estimate IAA contents.With
Blank cultures CK and the HZP1 bacterium solutions for being not added with Trp handle (Fig. 8) as a contrast, as a result measure such as following table：
Nitrogenase activity determination：Using acetylene reduction method, the growing nitrogen-fixing enzymatic activity of bacterial strain HZP1 is measured, as a result
It was found that reaching 208.5nmol/ (mLh) after the growing nitrogen-fixing enzymatic activity 12h of the bacterial strain, it is as follows：
Bacillus megaterium (Bacillus megaterium) HZP1 is activated on LB solid plates, is inoculated in 4ml
K.oxytoca has nitrogen fluid nutrient medium test tube, cultivates 12h, being forwarded to 50ml K.oxytoca has in nitrogen fluid nutrient medium shaking flask
30 DEG C, 200rpm cultivates 15h, and nitrogen source exhausts substantially at this time, and whole culture solutions, centrifugation is taken to remove supernatant.With 50ml K.oxytoca
Nitrogen-free fluid nutrient medium is resuspended, and is seeded to nitrogen-free semisolid K.oxytoca culture mediums, 30 DEG C of shake cultures measure after 4h
It restores the ability of acetylene.Ensure OD in bottle600There is preferable measurement effect at 0.2.Cell azotase is counted as follows than work
It calculates：
Cell restores ethyne reactive (nmolC2H2·ml-1·h-1)=ethylene peak area × 90/ (1nmol ethylene base peaks
Area × 1ml × 0.5h)
Above-mentioned K.oxytoca has nitrogen culture medium prescription as follows：Sucrose 40g, NaCl 2g, MgSO4·7H2O 0.2g, NH4Ac
1.54g, ironic citrate 36mg, Na2HPO4·12H2O 9.84g, Na2MoO4·2H2O 7.6mg, KH2PO41.7g.Wherein
Na2HPO4·12H2O and KH2PO4Individually sterilizing, NH4Ac individually sterilizes.
There is nitrogen K.oxytoca to remove NH4Ac is nitrogen-free agar.
Different vaccination time bacillus megaterium HZP1 restores acetylene data after being inoculated with nitrogen-free K.oxytoca：
Embodiment 2：
The preparation of complex microorganism seed dressing：
Activated bacillus megaterium HZP1 and rhizobium, which are inoculated in respectively in NB culture mediums and YMA medium, obtains seed
Liquid, then be switched to fermentation medium BSE progress fermented and cultured and obtain zymotic fluid.
The NB culture mediums, formula are as follows：Beef extract 3.0g, peptone 10.0g, NaCl 5.0g, water 1000ml, pH
7.4-7.6。
The YMA medium, formula are as follows：Sucrose 10.0g, MgSO4·7H2O 0.2g, K2HPO40.5g, NaCl
0.1g, CaCl2·5H2O 0.1g, Rh liquid microelements 4ml, dH2O1000ml。
The BSE culture mediums, formula are as follows：Sucrose 10.0g, MgSO4·7H2O 0.2g, K2HPO40.5g, NaCl
0.1g, CaCl2·5H2O 0.1g, Rh liquid microelement 4ml, bean sprout juice the 1000ml (bean sprouts 500g+1500ml H2O boils 30 points
Zhong Houyong filtered through gauze is twice), pH 6.8-7.0.
50ml bacillus megateriums (Bacillus megaterium) HZP1 zymotic fluids and 50ml root nodule fermented liquids is mixed
The sterile bag for attaching together sterilized 450g turfs and 50g ground phosphate rock adds 1ml sterile micro element liquid and is prepared into composite solid
Body microbial inoculum.Place at room temperature, according to national standards about《The technical indicator of agricultural microbial agent product》Prescriptive procedure is surveyed
Determining viable count is：Hundred million/g of rhizobium hundred million/g of 7.55-8.16, bacillus megaterium 0.82-0.86.Viable count and miscellaneous Counting alive microbial knot
Fruit is as follows：
The microbial inoculum 1 is mixed for bacillus megaterium (Bacillus megaterium) HZP1 and Rhizobium of Milk Vetch 7653R
Composite bacteria agent after conjunction, microbial inoculum 2 are bacillus megaterium (Bacillus megaterium) HZP1 and Sinorhizobium fredii bacterium
The strain mixed composite bacteria agents of HH103.
The effective bacterial content of Sinorhizobium fredii bacterial strain HH103 bacterium solutions is 20 × 108-50×108cfu/ml；Institute
It is 5 × 10 to state the effective bacterial content of bacillus megaterium bacterium solution8-10×108Cfu/ml, the two bacterium number ratio is about 10:1；
The effective bacterial content of Rhizobium of Milk Vetch 7653R bacterium solutions is 20 × 108-50×108cfu/ml；It is described huge
The effective bacterial content of bacillus bacterium solution is 5 × 108-10×108Cfu/ml, the two bacterium number ratio is about 10:1；
The humic acid is marketable material, also known as turf, and water content 12%, K contents are 13.18mg/kg, P content
For 0.94mg/kg, organic matter 428.12g/kg, total nitrogen content 0.52%, pH 5.30.
The ground phosphate rock is commercial reagent, and also known as apatite, slightly solubility superphosphate, main component are Ca5(PO4)3Cl contains 3% effective phosphorus composition.
The formula of the liquid microelement is：H3BO35g, Na2MoO45g, water 1000ml.
Embodiment 3：
For complex micro organism fungicide to the Field information in terms of legume growth-promoting, volume increase, application process is as follows：
Earth culture and plot experiment are same low-phosphorous soil for examination soil, derive from Hubei Shishou crop field.Its physics and chemistry
Matter measures as follows：Organic matter 12.66g/kg, full nitrogen 0.87g/kg, available potassium 101.34mg/kg, rapid available phosphorus 6.36mg/kg, pH
6.70, Native Sheep skin quantity 7.9 × 103cfu/g。
Measurement of the complex micro organism fungicide to Chinese milk vetch growgh promoting effects：
(1) select full grains, size uniform Chinese Milk Vetch Seeds, with 75% alcohol treatment 5min, then use sodium hypochlorite
Sterilize 3min, then with sterile water washing 8-10 times.It is uniformly laid on 1% water agar plate, 28 DEG C are just being set vernalization 2-3d.
(2) seed after vernalization is seeded in the native alms bowl containing nitrogen-free plant nutrition liquid, per 2,1 week, alms bowl after kick Miao Liuchang
The similar seedling of gesture.After plant grows first compound leaf, rhizobium and gemma bar when root system inoculation 2ml bacterium solutions, double inoculations
Each 1ml of bacterium；Distilled water pours once a day, and the later stage once two days, keeps water-retaining quantity among field of soil.And at seeding time and after planting
Pour standard liquid nutrient, each 10ml within 3rd week.Dross situation is observed after 25-30d is cultivated in illumination room, and measures Plant aboveground
The indexs such as part fresh weight, under ground portion fresh weight, root nodule number, root nodule fresh weight, fixed nitrogen enzyme activity.
Processing scheme is as follows：
CK：Bacterium is not connect, sterile water/2ml is met；
Processing 1：Meet Rhizobium of Milk Vetch 7653R/2ml；
Processing 2：Meet bacillus megaterium HZP1/2ml；
Processing 3：Double inoculation (7653R+HZP1)/2ml；
Bacillus megaterium HZP1 bacterial concentrations OD600It is 0.2, Rhizobium of Milk Vetch 7653R bacterium solutions OD600It is 0.5.
Measurement result is as follows：
Conclusion：Double inoculation comparisons singly meet bacillus megaterium HZP1 or singly connect the parts on the ground Rhizobium of Milk Vetch 7653R
(Fig. 9) and Underground biomass, fixed nitrogen enzyme activity, knurl weight are increased.Composite bacteria agent is significant to Chinese milk vetch biomass
(different letters are indicated in p<0.05 is horizontal lower with conspicuousness) effect of increasing production.
Complex microorganism seed dressing is in field to the measurement of soybean yield-increasing effect：Complex microorganism prepared by embodiment 2
Seed dressing (bacillus megaterium Bacillus megateriumHZP1 and Sinorhizobium fredii HH103) is mixed with soya seeds
It is sowed after kind, the florescence carries out character observation, and the maturity period completes cell production and measures.It specifically designs and implements as follows：
The plot experiment is that (preceding crop of field soil is small in the Hubei Shishou big towns Wan locality institute of agricultural sciences sample plot
Wheat) it carries out and completes.4.8 meters wide per cell, 5 meters long, 0.4 meter of long aisle, experimental plot surrounding at least 1 meter of wide guarantor are stayed in minizone
Shield row, experimental plot real area：4.8 meters * 5 meters * 6 cell=144 square metre.This time sow a kind：Middle yellow 13；Per cell
200g is sowed, after emerging, by the standard thinning for retaining one plant per 10CM.It is handled according to following 2, each handles 3 cells.
By the staff of the local institute of agricultural sciences, field management is carried out in strict accordance with each processing scheme, carries out florescence investigation and maturity period
Survey production.
Field plot processing is as follows：
Processing 1：It does not dress seed with microbial inoculum, middle yellow 13 conventional seed planting broadcasts 200 grams/cell；
Processing 2：Microbial inoculum is dressed seed, middle yellow 13 conventional seed planting, 200 grams of soybean seed dressing 2000g composite bacteria agents/cells；
Seed dressing step include：The Arabic gum aqueous solution of 20ml 20% is added in every 100 grams of complex microorganism seed dressings, instead
It stirs evenly again, is tuned into paste.If microbial inoculum water content itself is smaller, can not adjust paste, can slightly increase Arabic gum aqueous solution
Amount.After being tuned into paste, the amount addition beans kind for 1000 grams of soybean of dressing seed according still further to 100 grams of original complex microorganism seed dressings, repeatedly
Stirring, makes turf microbial inoculum uniform adhesion in the surface of the seed.The soybean mixed is spread out on newspaper or plastic cloth, about 30-60 minutes
It can dry.
It is as follows that florescence field plot investigation and maturity period survey production result：
It can be seen that：2 comparison 1 ground fresh weight of processing of processing, underground root nodule numbers (Figure 10) and nodule weight increase.Production
Comparative measurements is measured the results show that the soybean yields of inoculating compound bacterium agent improves 16.9%.
Embodiment 4：
For complex micro organism fungicide in the Field information for reducing nitrogen application, application process is as follows：
Plot experiment base is located at suburb of Harbin academy of agricultural sciences Experimental Base.Plot experiment ground area comes to 1400m2,
Each cell 39m2, 6 ridge area of cell goes for 10 meters and grows, altogether 36 cell.This time Quarter Design includes two soybean varieties (dragon
Breathe out 10-4139 and Hei Long 61) and composite bacteria agent (microbial inoculum 2 in embodiment 2), random alignment.6 kinds of different disposals, each
Processing, using 3 repetitions.Soybean is sowed in May, 2017, the harvest species test of in September, 2017 and survey production.
Test process designs：
Processing 1：Conventional fertilizer application does not apply composite bacteria agent.
Processing 2：50% nitrogen fertilizer application, does not apply composite bacteria agent.
Processing 3：Not nitrogen fertilizer application completely, does not apply composite bacteria agent.
Processing 4：Conventional fertilizer application+inoculating compound bacterium agent.
Processing 5：50% nitrogen fertilizer application+inoculating compound bacterium agent.
Processing 6：Not nitrogen fertilizer application+inoculating compound bacterium agent completely.
Soybean dresses seed step referring to embodiment 3 with microbial inoculum.The Fertilizer application of conventional fertilizer application is as follows：Every 38 grams of cell urea+
58 grams of 97 grams+potassium sulfate of double superhosphate, 50% nitrogen fertilizer application amount are that urea reduces 70%, and nitrogen fertilizer application is not apply urea completely.
Dragon breathes out 10-4139 species tests and survey production analysis result is as follows:
Black imperial 61 species test and survey production analysis result are as follows：
It can be seen that：Two soybean varieties species tests are analysis shows that processing 4,5,6 is saved relative to 1,2,3 soybean pod numbers of processing
Number, single-strain grain weight, 100-grain weight increased, and show that add composite bacterial fertilizer produces good effect to Soybean Growth Characters growth.
Two kinds of inoculating compound bacterium agent have effect of increasing production in the case of only applying 50% nitrogenous fertilizer：Total nitrogenous fertilizer 50% is being reduced, is being equivalent to
Under conditions of reducing urea 70%, dragon can be made to breathe out 10-4139 volume increase 14.0%, black imperial 61 volume increase 21.6%.
Claims (9)
1. the bacillus megaterium of one plant of separation, which is characterized in that the bacillus megaterium is bacillus megateriumBacillus megaterium HZP1, deposit number are CCTCC NO：M 2018061.
2. a kind of complex micro organism fungicide, including bacillus megaterium and rhizobium, the bacillus megaterium is huge bud
Spore bacillusBacillus megaterium HZP1, deposit number are CCTCC NO：M 2018061.
3. complex micro organism fungicide according to claim 2, the rhizobium are Rhizobium of Milk Vetch 7653R or Fei Shi
S. meliloti strains HH103.
4. complex micro organism fungicide according to claim 2, the rhizobium and bacillus megaterium（Bacillus megaterium）Effective bacteria concentration ratio of HZP1 is 10 ~ 5:1.
5. application of the composite bacteria agent described in claim 2 in preparing seed dressing.
6. the application according to 5, the formula of the seed dressing：
Constituent content
50 ~ 60 ml of rhizobium
50 ~ 60 ml of bacillus megaterium HZP1
440 ~ 460 g of humic acid
45 ~ 55g of ground phosphate rock
0.1 ~ 1 ml of liquid microelement
The effective bacterial content of the rhizobium is 20 × 108 - 50×108cfu/ml；The bacillus megaterium HZP1 bacterium solutions
Effective bacterial content is 5 × 108 - 10×108cfu/ml；
The formula of the liquid microelement is：H3BO35 g, Na2MoO45 g, 1000 ml of water.
7. application of the composite bacteria agent described in claim 2 in preparing Inoculant.
8. application of the composite bacteria agent in preparing legume increasing agent described in claim 2.
9. application of the composite bacteria agent in preparing nitrogenous fertilizer partial alternative agent described in claim 2.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109769615A (en) * | 2019-01-25 | 2019-05-21 | 广西壮族自治区农业科学院 | A method of promoting peanut nodule fixed nitrogen |
| CN113693082A (en) * | 2021-08-30 | 2021-11-26 | 深圳市芭田生态工程股份有限公司 | Seed coating agent for promoting leguminous seeds to germinate into seedlings in saline-alkali soil and preparation method thereof |
| CN114009449A (en) * | 2021-10-19 | 2022-02-08 | 中国农业科学院油料作物研究所 | Method for increasing number of root nodules with nitrogenase activity on roots of leguminous crops and application of root nodules on leguminous crops |
| CN114015629A (en) * | 2021-12-20 | 2022-02-08 | 南京林业大学 | Complex microbial inoculum for promoting growth of soybeans and improving abundance of probiotic microbial populations |
| CN115287227A (en) * | 2022-07-26 | 2022-11-04 | 中国农业大学 | Bacillus for promoting nitrogen nutrition and growth of plants and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1757716A (en) * | 2005-07-25 | 2006-04-12 | 中国林业科学研究院热带林业研究所 | Coculturing fermentation method of root nodule bacterial and Bacillus phosphorus |
| CN102826895A (en) * | 2012-08-01 | 2012-12-19 | 邓振山 | Multi-element composite microorganism bacterial manure and preparation method thereof |
| CN104263677A (en) * | 2014-09-02 | 2015-01-07 | 华中农业大学 | Composite microbial seed dressing agent and preparation method and application of composite microbial seed dressing agent |
| CN104609992A (en) * | 2014-12-31 | 2015-05-13 | 天津北洋百川生物技术有限公司 | Dedicated composite bacterial fertilizer for saline-alkali soil and preparation method thereof |
| CN105950520A (en) * | 2016-07-18 | 2016-09-21 | 武汉市农业科学技术研究院作物科学研究所 | Rhizobium capable of efficiently solubilizing phosphorus and application of rhizobium |
| CN106011004A (en) * | 2016-05-20 | 2016-10-12 | 东莞市保得生物工程有限公司 | Nitrogen-fixing microorganism G96 as well as bacterium agent preparation method and application thereof |
| EP3105198A1 (en) * | 2014-02-10 | 2016-12-21 | Biofil Mikrobiológiai, Géntechnológiai És Biokémiai KFT. | Soil bacteria for inoculating stress soils |
| CN107881130A (en) * | 2017-11-10 | 2018-04-06 | 黑龙江省农业科学院草业研究所 | Rhizobium and its application with efficient phosphate-solubilizing ability |
-
2018
- 2018-05-14 CN CN201810454089.7A patent/CN108624528B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1757716A (en) * | 2005-07-25 | 2006-04-12 | 中国林业科学研究院热带林业研究所 | Coculturing fermentation method of root nodule bacterial and Bacillus phosphorus |
| CN102826895A (en) * | 2012-08-01 | 2012-12-19 | 邓振山 | Multi-element composite microorganism bacterial manure and preparation method thereof |
| EP3105198A1 (en) * | 2014-02-10 | 2016-12-21 | Biofil Mikrobiológiai, Géntechnológiai És Biokémiai KFT. | Soil bacteria for inoculating stress soils |
| CN104263677A (en) * | 2014-09-02 | 2015-01-07 | 华中农业大学 | Composite microbial seed dressing agent and preparation method and application of composite microbial seed dressing agent |
| CN104609992A (en) * | 2014-12-31 | 2015-05-13 | 天津北洋百川生物技术有限公司 | Dedicated composite bacterial fertilizer for saline-alkali soil and preparation method thereof |
| CN106011004A (en) * | 2016-05-20 | 2016-10-12 | 东莞市保得生物工程有限公司 | Nitrogen-fixing microorganism G96 as well as bacterium agent preparation method and application thereof |
| CN105950520A (en) * | 2016-07-18 | 2016-09-21 | 武汉市农业科学技术研究院作物科学研究所 | Rhizobium capable of efficiently solubilizing phosphorus and application of rhizobium |
| CN107881130A (en) * | 2017-11-10 | 2018-04-06 | 黑龙江省农业科学院草业研究所 | Rhizobium and its application with efficient phosphate-solubilizing ability |
Non-Patent Citations (9)
| Title |
|---|
| P. MEHTA等: "Phosphate solubilisation and plant growth promoting potential by stress tolerant Bacillus sp. isolated from rhizosphere of apple orchards in trans Himalayan region of Himachal Pradesh", 《ANNALS OF APPLIED BIOLOGY》 * |
| XU, MINGSHUANG等: "Bacterial community compositions of tomato ( Lycopersicum esculentum Mill.) seeds and plant growth promoting activity of ACC deaminase producing Bacillus subtilis (HYT-12-1) on tomato seedlings", 《WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY》 * |
| 刘青海: "六株溶磷菌与四株固氮菌互作效应及其菌剂对苜蓿促生效果研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 康慧颖等: "两株具促生作用的苜蓿内生菌的分离纯化与鉴定", 《微生物学通报》 * |
| 李建宏: "优良植物根际促生菌Bacillus mycoides Gnyt1特性研究及全基因组测序分析", 《中国博士学位论文全文数据库 基础科学辑》 * |
| 李艳萍等: "根瘤菌和复合促生菌对大豆结瘤和生长的影响", 《大豆科学》 * |
| 沈其荣主编: "《土壤肥料学通论》", 28 February 2001, 高等教育出版社 * |
| 潘春梅主编: "《微生态制剂生产及应用》", 30 September 2014, 中国农业大学出版社 * |
| 邱立友编著: "《固态发酵工程原理及应用》", 31 August 2008, 中国轻工业出版社 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109769615A (en) * | 2019-01-25 | 2019-05-21 | 广西壮族自治区农业科学院 | A method of promoting peanut nodule fixed nitrogen |
| CN113693082A (en) * | 2021-08-30 | 2021-11-26 | 深圳市芭田生态工程股份有限公司 | Seed coating agent for promoting leguminous seeds to germinate into seedlings in saline-alkali soil and preparation method thereof |
| CN114009449A (en) * | 2021-10-19 | 2022-02-08 | 中国农业科学院油料作物研究所 | Method for increasing number of root nodules with nitrogenase activity on roots of leguminous crops and application of root nodules on leguminous crops |
| CN114015629A (en) * | 2021-12-20 | 2022-02-08 | 南京林业大学 | Complex microbial inoculum for promoting growth of soybeans and improving abundance of probiotic microbial populations |
| CN114015629B (en) * | 2021-12-20 | 2022-04-29 | 南京林业大学 | Complex microbial inoculum for promoting growth of soybeans and improving abundance of probiotic microbial populations |
| CN115287227A (en) * | 2022-07-26 | 2022-11-04 | 中国农业大学 | Bacillus for promoting nitrogen nutrition and growth of plants and application thereof |
| CN115287227B (en) * | 2022-07-26 | 2023-11-10 | 中国农业大学 | Bacillus for promoting nitrogen nutrition and growth of plants and application thereof |
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