CN109576164A - One plant of efficient phosphate-solubilizing fungi Rhizopus microsporus and its screening technique and application - Google Patents
One plant of efficient phosphate-solubilizing fungi Rhizopus microsporus and its screening technique and application Download PDFInfo
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Abstract
One plant of efficient phosphate-solubilizing fungi Rhizopus microsporus and its screening technique and application, belong to microbiological art.Aiming at the problem that Phos, promotion plant growth in soil of how degrading, the present invention provides one plant of efficient phosphate-solubilizing fungi Rhizopus microsporus (Rhizopus microsporus) NEAU-8, deposit numbers are as follows: CGMCC NO.14635.Its screening technique is as follows: 1) taking wheat rhizosphere soil, after the physiological saline enrichment culture that volume fraction is 0.85% is added, stand；2) it takes supernatant to carry out gradient dilution, takes each dilution, be respectively coated and cultivated on Phos solid medium；3) the microorganism picking for having transparent circle on each culture medium is gone out, is inoculated into new Phos solid medium culture；4) spore suspension is made in the thallus of culture respectively, is inoculated into Phos fluid nutrient medium and cultivates, measure the content of titanium pigment in bacterium solution, screening obtains the fungi Rhizopus microsporus of efficient phosphate-solubilizing.The present invention, which obtains fungi Rhizopus microsporus, can be used in agricultural production.
Description
Technical field
The invention belongs to microbiological arts, and in particular to one plant of efficient phosphate-solubilizing fungi Rhizopus microsporus and its screening technique with
Using.
Background technique
Phosphorus is one of three big nutrients necessary to plant growth and development, is inhereditary material and energy object in plant cell
The basic constitution element of the organic compounds such as matter, the photosynthesis of plant and intracorporal biochemical metabolism process must all have phosphorus ginseng
Add, phosphorus plays irreplaceable role in the entire life process of plant.It is reported that lacking in the arable soil in China 74%
Phosphorus, about 95% phosphorus exists in the form of invalid phosphorus in soil, cannot be absorbed and used by plants.Therefore, phosphorus becomes limitation crop
The one of the chief elements of growth and yield.In order to slow down influence of the phosphorus scarcity to plant growth, relies primarily on and apply in agricultural production
Increase phosphorus element supply with phosphate fertilizer.However, the phosphate fertilizer in being manured into soil largely can be with such as Al of the metal ion in soil3+、Fe3 +、Ca3+Insoluble phosphate is formed Deng combining, leading to the utilization rate of current year phosphorus is only 10-25%.To meet crops to phosphorus
Demand often largely applies phosphate fertilizer in agricultural production, and the excessive application of phosphate fertilizer not only results in serious economic loss, makes
The decline of Soil Microorganism diversity, also will cause soil hardening, Soil degradation, environmental pollution, ecological degeneration and agricultural product product
Therefore how the environmental problems such as matter decline efficiently use the unavailable phosphorus in soil, improve the utilization rate of phosphate fertilizer, to reduction agriculture
The use of chemical fertilizer and resource consumption in industry production, and preserve the ecological environment and have a very important significance.
Phosphate solubilizing microorganism can dissolve the insoluble phosphorus in soil, and invalid phosphorus is transformed into the absorbent available phosphorus of plant,
And the growth of plant can be stimulated by extracellular enzyme and organic acid.Phosphate solubilizing microorganism can be divided into phosphate-solubilizing bacteria, actinomyces and
Fungi, wherein the comparison of phosphate-solubilizing bacteria research is more, including enterobacteria category (Enterbacter), Agrobacterium
(Agrobacterium), Erwinia (Erwinia), pseudomonas (Pseudomonas), Serratia
(Serratia), Flavobacterium (Flavobacterium), bacillus (Bacillus), Micrococcus
(Micrococcus), azotobacter (Azotobacter), Chromobacterium (Chromabacterium), Salmonella
(Salmonella), Alcaligenes (Alcaligenes), Arthrobacter (Arthrobacter), Thiobacillus
(Thiobacillus), Escherichia (Escherichia)；Phosphorus decomposing actinomyces primarily focus on streptomyces
(Streptomyces)；Correlative study report in terms of phosphorus decomposing fungi is few, is mostly aspergillus (Aspergillus), head mold
Belong to (Rhizopus), Fusarium (Fusarium), Penicillium (Penicillium), Sclerotium (Sclerotium),
AM VA Mycorrhizal Fungi (Arbuscular mycorrhiza) etc..Many phosphate-solubilizing bacterias and actinomyces can lose it most after secondary culture
First dissolving P capacity, and this forfeiture cannot restore again.
Summary of the invention
Aiming at the problem that Phos, promotion plant growth in soil of how degrading, the present invention provides one plant of efficient phosphate-solubilizings
Fungi Rhizopus microsporus (Rhizopus microsporus) NEAU-8, deposit number are as follows: CGMCC NO.14635.
The present invention also provides the screening techniques of above-mentioned Rhizopus microsporus, the method is as follows:
1) wheat rhizosphere soil is taken, after 0.85% physiological saline enrichment culture is added, stands 15-30min；The soil
Ratio with physiological saline is 1:9 (g/mL)；
2) it then takes 1mL supernatant to carry out gradient dilution, takes the dilution of each dilution, be respectively coated in Phos
It is cultivated on solid medium；
3) the microorganism picking for having transparent circle on each culture medium is gone out, is inoculated into new Phos solid medium
Upper culture；
4) spore suspension then, is made in the thallus of culture respectively, is inoculated into Phos fluid nutrient medium and cultivates, pass through
The content of titanium pigment in bacterium solution is measured, screening obtains the fungi Rhizopus microsporus of efficient phosphate-solubilizing.
It further limits, the step 1) enrichment culture refers to 150r/min shaken cultivation 90- in 28 DEG C of shaking tables
120min。
It further limits, the step 2) gradient dilution refers to is diluted to supernatant the 10 of original volume respectively-1、10-2、10-3、 10-4、10-5、10-6。
It further limits, step 2) the Phos solid culture based formulas are as follows: glucose 10g, ammonium sulfate 0.5g, chlorine
Change sodium 0.3g, potassium chloride 0.3g, MgSO4.7H2O 0.3g, FeSO4.7H2O 0.03g, MnSO4.4H2O 0.03g, calcium phosphate
10g, agar 18g, deionized water 1000mL, pH are adjusted to 7.0.
Further limit, step 4) it is described inoculation refer to spore suspension is inoculated into according to 1% (volume fraction) it is inorganic
In phosphorus fluid nutrient medium；The culture, condition are 28 DEG C, 150r/min shaking table culture 7d.
The present invention also provides above-mentioned Rhizopus microsporus to inhibit the aborning application of disease fungus.
It further limits, the pathogenic fungi is withered pathogenic bacteria (Fusarium oxysporum) for cucumber, corn is big
Spot pathogenic bacteria (Exserohilum turcicum), cucumber foxiness pathogenic bacteria (Corynespora cassiicola), cucumber charcoal
Subcutaneous ulcer pathogenic bacteria (Colletotrichum orbiculare), corn stigma pathogenic bacteria (Helminthosporium maydis),
Rice banded sclerotial blight pathogenic bacteria (Rhizoctonia solani), Curvularia pathogenic bacteria (Curvularia lunata), Soybean Root
Rotten pathogenic bacteria (Rhizoctonia solani Kuhn) or soybean sclerotium pathogenic bacteria (Sclerotinia sclerotiorum).
The present invention also provides above-mentioned Rhizopus microsporus to promote the application in plant growth.
It further limits, after Rhizopus microsporus is mixed soil, is used for cultivated maize or wheat seed, the Rhizopus microsporus is in soil
Spore concentration in earth is 1.0 × 103Cfu/g~1.0 × 105Cfu/g, best spore concentration are 1.0 × 104cfu/g。
Beneficial effect
Present invention Rhizopus microsporus NEAU-8 isolated from wheat rhizosphere soil has calcium phosphate, trbasic zinc phosphate relatively strong
Dissolving P capacity, corresponding dissolving P capacity is respectively 403mg/L, 507mg/L.Pot experiment shows that the spore liquid of the bacterium can
Significantly to promote the growth of wheat and corn.Bacterial strain Rhizopus microsporus NEAU-8 and its microbial inoculum provided by the invention are in use process
In it is pollution-free, it is nuisanceless, the dosage of chemical fertilizer can be reduced, in addition, the stabilization characteristics of genetics of phosphorus decomposing fungi, secondary culture are general
It is not easy to lose dissolving P capacity, provides excellent strain resource for exploitation microbial-bacterial fertilizer, and there is good application and development prospect.
Bacterial strain Rhizopus microsporus (Rhizopus microsporus) NEAU-8 provided by the present invention, is from Hebei province Langfang
Separation obtains in city's wheat rhizosphere soil, is preserved in China General Microbiological culture presevation management on November 14th, 2017
Center (abbreviation CGMCC), preservation address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number CGMCC
NO.14635。
Detailed description of the invention
Fig. 1 is colony characteristics of the bacterial strain NEAU-8 in PDA culture medium.
Fig. 2 is that bacterial strain NEAU-8 bacterial strain cultivates time-division solution calcium phosphate effect picture in shaking flask.
Fig. 3 is phosphorus standard curve.
Fig. 4 is bacterial strain NEAU-8 to the antagonism of disease fungus, is compareed in plate for germ on the left of every picture, often
Picture right upper portion inoculating strain NEAU-8, lower part are inoculated with disease fungus.Disease fungus: a cucumber foxiness pathogenic bacteria
(Corynespora cassiicola), b cucumber anthracnose pathogenic bacteria (Colletotrichum orbiculare), c soybean sclerotium
Pathogenic bacteria (Sclerotinia sclerotiorum), d cucumber wither pathogenic bacteria (Fusarium oxysporum), and e corn is big
Pinta bacterium (Exserohilum turcicum), f soybean root rot pathogenic bacteria (Rhizoctonia solani Kuhn), g corn
Curved spore germ (Curvularia lunata), h rice banded sclerotial blight pathogenic bacteria (Rhizoctonia solani), i corn southern leaf blight
Bacterium (Helminthosporium maydis)
Fig. 5 is growth-promoting effect picture of the bacterial strain NEAU-8 to potting corn.
Fig. 6 is the result figure of influence of the bacterial strain NEAU-8 to potting wheat growth.
Specific embodiment
Below by way of specific embodiment, the present invention is described further, but not to limit the present invention, all in this hair
It is bright spirit and principle within, any modification, equivalent substitution, improvement and etc. done, should be included in protection scope of the present invention it
It is interior.
Rhizopus microsporus (Rhizopus microsporus) NEAU-8 of the present invention, in following embodiments or attached drawing
Referred to as bacterial strain NEAU-8, Rhizopus microsporus NEAU-8.
The plate refers to solid medium, is the common terminology of this field.
PDA culture medium, PDB culture medium and other chemical reagent or instrument and equipment, book, is normal unless otherwise specified
Reagent or instrument and equipment are advised, can be bought and be obtained by commercialization approach.
The separation of 1. Rhizopus microsporus of embodiment (Rhizopus microsporus) NEAU-8 is identified.
1. the separation and culture of bacterial strain
Rhizopus microsporus NEAU-8 is to screen to obtain from the wheat rhizosphere soil of Hebei province's Langfang City.Phosphate solubilizing bacteria screening uses
Phos solid medium: glucose 10g, ammonium sulfate 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, MgSO4.7H2O 0.3g,
FeSO4.7H2O 0.03g, MnSO4.4H2O 0.03g, calcium phosphate 10g, agar 18g, deionized water are settled to 1000mL, pH tune
To 7.0.
1) wheat rhizosphere soil is taken, after the physiological saline enrichment culture that volume fraction is 0.85% is added, stands 15-
30min；The ratio of the soil and physiological saline is 1:9 (g/mL).It is specific as follows: appropriate wheat rhizosphere soil is crossed into 20 mesh
Then sieve is added in mortar and is fully ground, the wheat rhizosphere soil after weighing 10g grinding pours into 250mL conical flask, adds simultaneously
Enter physiological saline 90mL, the 150r/min shaken cultivation 90-120min in 28 DEG C of shaking tables that volume fraction is 0.85%, with
120min is preferred, and is then taken out and is stood 30min in room temperature.
2) it then takes 1mL supernatant to carry out gradient dilution, takes the dilution of each dilution, be respectively coated in Phos
It is cultivated on solid medium；It is specific as follows: to take 1mL supernatant, according to dilution spread flat band method, respectively dilute supernatant
To the 10 of original volume-1、10-2、10-3、10-4、10-5、10-6, (can be diluted with sterile water) takes the dilution of each dilution
200 μ L of liquid, is uniformly coated on Phos solid medium, and 2 plates are repeated under each dilution.All plates are marked
It is sealed with preservative film, and is inverted in 28 DEG C of insulating boxs and cultivates.
3) in incubation, the microorganism picking that transparent circle is had on each plate is gone out, new Phos is incubated at
In solid medium, and carry out strain label, 4 DEG C of preservations.The bacterial strain with preferable dissolving P capacity that primary dcreening operation is obtained is lived
Change, culture 5 days is inverted in 28 DEG C of constant incubators.
4) spore suspension then, is made in the thallus of culture respectively, is inoculated into Phos fluid nutrient medium and cultivates, pass through
The content of titanium pigment in bacterium solution is measured, screening obtains the fungi Rhizopus microsporus of efficient phosphate-solubilizing.It is specific as follows: again will be activated
Bacterial strain is transferred on PDA Boiling tube inclined-plane and cultivates in 28 DEG C of constant incubators, and spore suspension is made in aseptic water washing after 7d, and
Spore concentration is counted with blood counting chamber.Spore suspension is accessed into Phos fluid nutrient medium according still further to the inoculum concentration of 1% (v/v)
In (formula are as follows: glucose 10g, ammonium sulfate 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, MgSO4.7H2O 0.3g,
FeSO4.7H2O 0.03g, MnSO4.4H27.0) O 0.03g, calcium phosphate 10g, deionized water 1000mL, pH are adjusted to, control group connects
The isometric sterile distilled water of kind, 28 DEG C, 150r/min shaking table culture 7d take a sample centrifugation every 1d, with the anti-colorimetric of molybdenum antimony
Method measures the content of titanium pigment in supernatant, and METHOD FOR CONTINUOUS DETERMINATION determines that test strain to the decomposition situation of indissoluble Phos, exceeds
Titanium pigment content is higher in control group, illustrates that bacterial strain is better to the discomposing effect of Phos, so that it is preferable to obtain phosphorus decomposing effect
Bacterial strain.It is NEAU-8 by the Strain Designation of purifying, is stored in -80 DEG C of ultra low temperature freezers with 20% glycerol.
2. the identification of bacterial strain.
(1) morphological feature: as shown in Figure 1, Rhizopus microsporus NEAU-8 is cultivated 5 days for 28 DEG C in PDA culture medium, bacterium colony is
Quickly sprawling is paved with entire plate, canescence is presented in mycelia, and flocculence, bacterium colony quality is loose, and surrounding, which has, to be arranged radially
Fluffy white mycelia, the culture medium back side are non-discolouring (non-pigment generation).Growth temperature range: 15-48 DEG C；Grow pH range:
3-8；Grow NaCl range: 3-10%.
(2) ITS rDNA sequence: Rhizopus microsporus NEAU-8 is inoculated on PDA plate in 28 DEG C of biochemical cultivation cases
Enough to hyphae length after cultivating 5-7d, scraping mycelia is in sterilizing mortar, being added liquid nitrogen grinding to powdered, using CTAB
(CetyLtriethyLamoium bromide) method extracts fungal DNA.
Sequence of the universal primer ITS1/ITS4 amplification for the examination area fungi 5.8Sr DNA-ITS.Its sequence is as follows:
ITS1 primer sequence are as follows: 5 '-TCCGTAGGTGAACCTGCGG-3 '
ITS4 primer sequence are as follows: 5 '-TCCTCCGCTTATTGATATGC-3 '
50 μ L reaction systems are selected, the ingredient of each component such as table 1:
Table 1PCR amplification system
React program thereby: 94 DEG C of initial denaturations 5min, 94 DEG C of denaturation 30s, 53 DEG C of annealing 30s, 72 DEG C of extension 1min, totally 28
A circulation, last 72 DEG C of extensions 5min.Amplified production is detected with 1% agarose gel electrophoresis, 1 × TAE electrophoretic buffer, on
5 μ L of sample amount.It is observed by gel imaging system.Amplified production carries out recovery purifying using gel reclaims kit.Concrete operations
Illustrate to carry out according to kit.
(Jilin provincial treasury U.S. biotechnology company) is sequenced by DNA sequencing company in gained glue recovery product.By the ITS of acquisition
RDNA sequence, input GenBank obtain accession number.Analysis is compared with Blast software.
For ITS rDNA sequence as shown in SEQ ID NO:1, which has logged in NCBI, accession number MF945552.Pass through
Blast program carries out sequence alignment analysis, the results showed that the ITS rDNA sequence and Rhizopus microsporus of the bacterial strain
CBS 130158 is like property up to 100%.
Comprehensive morphological features and ITS rDNA (as shown in SEQ ID No:3) sequence, isolated bacterial strain is reflected
It is set to Rhizopus microsporus NEAU-8.The bacterial strain is preserved in China General Microbiological bacterium on November 14th, 2017
Kind preservation administrative center (abbreviation CGMCC), deposit number are CGMCC NO.14635.
The measurement of 2. Rhizopus microsporus Rhizopus microsporus NEAU-8 dissolving P capacity of embodiment.
With the solution of Phos Liquid Culture based assays phosphorus decomposing fungi Rhizopus microsporus Rhizopus microsporus NEAU-8
The Rhizopus microsporus NEAU-8 cultivated on inclined-plane is first inoculated into PDB fluid nutrient medium and (the potato culture of agar is not added by phosphorus ability
Base) in be used as seed liquor, the shaken cultivation 12h in 28 DEG C, 200r/min constant-temperature table, then by 1% inoculum concentration (v/v) will
Cultured bacterium solution is inoculated into Phos fluid nutrient medium triangular flask, and control group does not connect bacterium by 1% inoculum concentration (v/v) inoculation
PDB Liquid Culture be based on Phos fluid nutrient medium in, the shaken cultivation 7d in 28 DEG C, 200r/min constant-temperature table.Phosphorus decomposing
Effect is shown in Fig. 2, is inoculated with the fluid nutrient medium clear of Rhizopus microsporus NEAU-8 as seen from the figure, precipitating completely disappears, and compares
Fluid nutrient medium is muddy in group, and has no that precipitating dissolves, and the Rhizopus microsporus NEAU-8 for illustrating that present invention screening obtains has very by force
Phosphorus decomposing effect.
Molybdenum antimony resistance colorimetric method surveys the available phosphorus content in supernatant.The 100mg/L of respective volume is separately added into volumetric flask
Standard phosphorus solution, add 2 drops 2,6- dinitrophenol makees indicator, adjust pH value with dilute sulfuric acid and 10%NaOH (g/v) solution,
Add the anti-color developing agent 5ml of molybdenum antimony, be settled to scale, making standard phosphorus concentration is respectively 0,0.2,0.4,0.6,0.8 and 1.0mg/L, is shaken
It is even, UV-1601PC ultraviolet-visible spectrophotometer colorimetric at 720nm is used after (25 DEG C or so) reaction 30min at room temperature,
Standard curve is drawn according to result, corresponding available phosphorus content is calculated according to standard curve.By fermentation liquid 8000r/min from
Heart 10min takes 2.5~5.0ml of supernatant into 50ml volumetric flask, carries out colorimetric according to the above method.
Phosphorus standard curve is shown in Fig. 3, we can obtain phosphorus calibration curve equation are as follows: y=0.4405x+0.0484, related coefficient
Up to 0.9993.Wherein y is OD720, x is that standard phosphorus solution concentration (mg/L) according to the calibration curve equation can calculate solution
The dissolving P capacity of phosphorus bacterium.Available phosphate concentration in placebo solution is 87.4mg/L, pH 5.82, in the solution of Rhizopus microsporus
Available phosphorus content is 403.2mg/L, pH 2.08, is somebody's turn to do experiments have shown that Rhizopus microsporus is one plant of efficient phosphate-solubilizing fungi.
Disease fungus is aborning answers inhibiting by 3. Rhizopus microsporus of embodiment (Rhizopus microspores) NEAU-8
With.
Using the culture dish center containing PDA solid medium as origin, cultured bacterium bacteria cake to be measured is upside down in culture
It is upside down in the symmetrical position in the origin other side at 1/2 radius of origin, while by the bacteria cake of fungi in ware, it is to be measured not to be inoculated with
The culture dish of bacterium is control, and each combination in triplicate, is cultivated in 28 DEG C of incubators, and separated in time observes disease fungus
The presence or absence of distance change situation, antibacterial band and its size between fungus colony size to be measured, two bacterium colonies, and with marking pen flat
It is marked on plate, observes situation of change.The distance between antibacterial bandwidth and two bacteria cakes are measured, bacteriostasis rate is calculated.
In opposite culture, Rhizopus microsporus NEAU-8 generates inhibiting effect to a variety of disease fungus in laboratory, shows good
Good bacteriostatic activity, it is seen that the broad spectrum activity of its antimicrobial spectrum.Wherein, maximum to wither pathogenic bacteria inhibiting rate of cucumber, up to 100%,
Bacteriostasis rate sequence to 9 kinds of disease fungus is the withered big spot pathogenic bacteria of the pathogenic bacteria > corn > cucumber foxiness pathogenic bacteria > cucumber of cucumber
Anthrax pathogenic bacteria > corn stigma pathogenic bacteria > rice banded sclerotial blight pathogenic bacteria > Curvularia pathogenic bacteria > soybean root rot pathogenic bacteria > soybean bacterium
Core pathogenic bacteria (table 2).Rhizopus microsporus NEAU-8 is extended by largely generating spore, is inhibited by substrate competition for examination
Disease fungus grows (Fig. 4).Rhizopus microsporus NEAU-8 shows the Antagonism to multiple diseases fungi on plate antagonistic experiment,
In consideration of it, can be applied to soil, inhibiting effect is played to disease fungies certain in soil, is mentioned during plant growth for crop
It is protected for biological and ecological methods to prevent plant disease, pests, and erosion.
2 disease fungus inhibiting rate % of table
4. Rhizopus microsporus of embodiment (Rhizopus microsporus) NEAU-8 is promoting the application in plant growth.
One, growth-promoting functions of Rhizopus microsporus (Rhizopus microsporus) NEAU-8 to corn.
Rhizopus microsporus NEAU-8 is activated in PDA culture medium is inoculated in 28 DEG C of cultures, 7d on PDA Boiling tube inclined-plane afterwards twice
Sterile water collects mycelia and pityrosporion ovale filament afterwards, after being smashed with magnetic stirring apparatus, is diluted to 1.0 × 10 with sterile water4cfu/mL
It is spare.Every gram of soil of processing group is added 1mL spore suspension and is uniformly mixed, and final spore concentration is 1.0 × 104Cfu/g, with sterile
Water is as control.It selects intact full corn seed to be wrapped with wet towel, 28 DEG C of temperature moisturizing vernalization, until when 80% seed shows money or valuables one carries unintentionally
Sowing.It selects the consistent corn seed to show money or valuables one carries unintentionally of growing way gently to be pressed from both sides out with tweezers in the basin for being placed in and filling 1kg soil, every basin is put
3 seeds are set, 4 repetitions are arranged in each processing.After 30 days, the plant height of corn is measured respectively, root long, the fresh weight of Miao Hegen and dry
Weight.As a result see Fig. 5 and table 3.
Influence of the 3 bacterial strain NEAU-8 spore liquid of table to potting corn growth
As it can be seen that compared with the control, fungi Rhizopus microsporus NEAU-8 spore concentration is 1.0 × 104When cfu/g, have to corn
There is significant growth-promoting effect, plant height increases by 24.1%, and root long increases by 34.8%, and seedling fresh weight increases by 67.1%, and seedling stem increases again
38.4%, root fresh weight increases by 69.0%, and root dry weight increases by 71.1%.
Two, growth-promoting functions of Rhizopus microsporus (Rhizopus microsporus) NEAU-8 to wheat.
Rhizopus microsporus NEAU-8 is activated in PDA culture medium is inoculated in 28 DEG C of culture 7d on PDA Boiling tube inclined-plane afterwards twice
Afterwards, sterile water is diluted to 1.0 × 104Cfu/mL is spare.Every gram of soil of processing group is added 1mL spore suspension and is uniformly mixed, finally
Spore concentration is 1.0 × 104Cfu/g, using sterile water as control.It selects intact full wheat seed to be wrapped with wet towel, 28
DEG C temperature moisturizing vernalization, until sowing when 80% seed shows money or valuables one carries unintentionally.The consistent wheat seed to show money or valuables one carries unintentionally of growing way is selected gently to be pressed from both sides with tweezers
It is placed in the basin for filling 1kg soil out, every basin places 10 seeds, and 4 repetitions are arranged in each processing.After 30 days, survey respectively
Measure the plant height of wheat, root long, the fresh weight and dry weight of Miao Hegen.As a result see Fig. 6 and table 4.
Influence of the 4 bacterial strain NEAU-8 spore liquid of table to potting wheat growth
| Plant height (cm) | Root long (cm) | Seedling fresh weight (g) | Seedling stem weight (g) | Root fresh weight (g) | Root dry weight (g) | |
| CK | 46.03±2.86 | 18.53±1.95 | 1.662±0.203 | 0.149±0.014 | 0.695±0.022 | 0.053±0.012 |
| Bacterial strain NEAU-8 | 57.45±2.83 | 24.51±2.46 | 2.366±0.311 | 0.197±0.023 | 0.996±0.036 | 0.095±0.021 |
As it can be seen that compared with the control, the concentration of bacterial strain is 1.0 × 104When cfu/g, there is significant growth-promoting effect to wheat,
Plant height increases by 24.8%, and root long increases by 32.3%, and seedling fresh weight increases by 42.4%, and seedling stem increases by 32.2% again, and root fresh weight increases
43.3%, root dry weight increases by 79.2%.
Nucleotides sequence list
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<120>one plants of efficient phosphate-solubilizing fungi Rhizopus microsporus and its screening technique and applications
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ggcctgttct ggggtttgat cgatgccaat caggattacc tttcttcctt tgggaaggaa 180
ggtgcctggt accctttacc atataccatg aattcagaat tgaaagtata atataataac 240
aacttttaac aatggatctc ttggttctcg catcgatgaa gaacgtagca aagtgcgata 300
actagtgtga attgcatatt cgtgaatcat cgagtctttg aacgcagctt gcactctatg 360
gatcttctat agagtacgct tgcttcagta tcataaccaa cccacacata aaatttattt 420
tatgtggtga tggacaagct cggttaaatt taattattat accgattgtc taaaatacag 480
cctctttgta attttcatta aattacgaac tacctagcca tcgtgctttt ttggtccaac 540
caaaaaacat ataatctagg ggttctgcta gccagcagat attttaatga tctttaacta 600
tgatctgaag tcaagtggga ctacccgctg aacttaagca tatcaaa 647
Claims (10)
1. one plant of efficient phosphate-solubilizing fungi Rhizopus microsporus (Rhizopus microsporus) NEAU-8, deposit number are as follows: CGMCC
NO.14635。
2. the screening technique of Rhizopus microsporus described in claim 1, which is characterized in that method is as follows:
1) wheat rhizosphere soil is taken, after the physiological saline enrichment culture that volume fraction is 0.85% is added, stands 15-30min；Institute
The ratio for stating soil and physiological saline is 1:9 (g/mL)；
2) it then takes 1mL supernatant to carry out gradient dilution, takes the dilution of each dilution, be respectively coated in Phos solid
It is cultivated on culture medium；
3) the microorganism picking for having transparent circle on each culture medium is gone out, is inoculated into new Phos solid medium training
It supports；
4) spore suspension then, is made in the thallus of culture respectively, is inoculated into Phos fluid nutrient medium and cultivates, passes through measurement
The content of titanium pigment in bacterium solution, screening obtain the fungi Rhizopus microsporus of efficient phosphate-solubilizing.
3. screening technique according to claim 2, which is characterized in that the step 1) enrichment culture refers in 28 DEG C of shaking tables
150r/min shaken cultivation 90-120min.
4. screening technique according to claim 2, which is characterized in that the step 2) gradient dilution refer to supernatant is distinguished it is dilute
Release the 10 of original volume-1、10-2、10-3、10-4、10-5、10-6。
5. screening technique according to claim 2, which is characterized in that step 2) the Phos solid culture based formulas are as follows: Portugal
Grape sugar 10g, ammonium sulfate 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, MgSO4.7H2O 0.3g, FeSO4.7H2O 0.03g,
MnSO4.4H2O 0.03g, calcium phosphate 10g, agar 18g, deionized water 1000mL, pH are adjusted to 7.0.
6. screening technique according to claim 2, which is characterized in that the step 4) inoculation refers to spore suspension according to 1%
(volume fraction) is inoculated into Phos fluid nutrient medium；The culture, condition are 28 DEG C, 150r/min shaking table culture 7d.
7. Rhizopus microsporus described in claim 1 is inhibiting the aborning application of disease fungus.
8. application according to claim 8, which is characterized in that the pathogenic fungi is the withered pathogenic bacteria of cucumber
(Fusarium oxysporum), the big spot pathogenic bacteria of corn (Exserohilum turcicum), cucumber foxiness pathogenic bacteria
(Corynespora cassiicola), cucumber anthracnose pathogenic bacteria (Colletotrichum orbiculare), corn stigma cause
Germ (Helminthosporium maydis), rice banded sclerotial blight pathogenic bacteria (Rhizoctonia solani), Curvularia cause a disease
Bacterium (Curvularia lunata), soybean root rot pathogenic bacteria (Rhizoctonia solani Kuhn) or soybean sclerotium pathogenic bacteria
(Sclerotinia sclerotiorum)。
9. Rhizopus microsporus described in claim 1 is promoting the application in plant growth.
10. application according to claim 9, which is characterized in that after Rhizopus microsporus is mixed soil, be used for cultivated maize or wheat
Seed, the spore concentration of the Rhizopus microsporus in the soil are 1.0 × 103Cfu/g~1.0 × 105cfu/g。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101439996A (en) * | 2008-12-18 | 2009-05-27 | 杨雪枣 | Flower-protecting and sweet-enhancing composite biological-organic liquid fertilizer |
| CN104774784A (en) * | 2015-03-02 | 2015-07-15 | 河南科技大学 | Phosphate dissolving bacterium and acquisition method thereof |
| CN107858296A (en) * | 2017-12-15 | 2018-03-30 | 山东农业大学 | The preparation and application of the aspergillus niger and its microbial inoculum of one plant of phosphorus decomposing, potassium decomposing and degraded cellulose |
-
2019
- 2019-01-23 CN CN201910064466.0A patent/CN109576164B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101439996A (en) * | 2008-12-18 | 2009-05-27 | 杨雪枣 | Flower-protecting and sweet-enhancing composite biological-organic liquid fertilizer |
| CN104774784A (en) * | 2015-03-02 | 2015-07-15 | 河南科技大学 | Phosphate dissolving bacterium and acquisition method thereof |
| CN107858296A (en) * | 2017-12-15 | 2018-03-30 | 山东农业大学 | The preparation and application of the aspergillus niger and its microbial inoculum of one plant of phosphorus decomposing, potassium decomposing and degraded cellulose |
Non-Patent Citations (2)
| Title |
|---|
| SAOHARIT NITAYAVARDHANA等: "Innovative biorefinery concept for sugar-based ethanol industries: production of protein-rich fungal biomass on vinasse as an aquaculture feed ingredient", 《BIORESOUR TECHNOL.》 * |
| VANESSA SAYURI SATO等: "Characterization of a Thermotolerant Phytase Produced by Rhizopus microsporus var. microspores Biofilm on an Inert Support Using Sugarcane Bagasse as Carbon Source", 《APPLIED BIOCHEMISTY AND BIOTECHNOLOGY》 * |
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