CN108192838A - A kind of bacillus amyloliquefaciens with degradation Phos and diseases prevention double action - Google Patents
A kind of bacillus amyloliquefaciens with degradation Phos and diseases prevention double action Download PDFInfo
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- CN108192838A CN108192838A CN201711454747.4A CN201711454747A CN108192838A CN 108192838 A CN108192838 A CN 108192838A CN 201711454747 A CN201711454747 A CN 201711454747A CN 108192838 A CN108192838 A CN 108192838A
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- 230000015556 catabolic process Effects 0.000 title claims abstract description 23
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 23
- 230000002265 prevention Effects 0.000 title claims abstract description 16
- 230000009471 action Effects 0.000 title abstract description 6
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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Abstract
The present invention provides a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain WKPHO 12, the deposit number of the bacterial strain is CGMCC No.14954.The invention also discloses the microbial bacterial agents containing the bacterial strain.Bacterial strain WKPHO 12 of the present invention has degradation Inorganic Phosphorus Fractions in Soil and diseases prevention double action, i.e., can not only Phos in efficient degradation soil, improve the utilization rate of Phos in soil, reduce Phosphorus fertilizer usage；Also to the plant diseases such as eggplant verticillium wilt preventive effect height, average preventive effect is more than 70%；Secondly, 12 antimicrobial spectrums of bacterial strain WKPHO of the present invention are wide, and the bacterium that withers to eggplant Huang, cucumber Fusarium oxysporum, cotton wilt fusarium and potato black mole bacterium etc. have good inhibiting effect；In addition, 12 specializations of bacterial strain WKPHO of the present invention are strong, lasting medicine is good, it is environmentally friendly, moreover it is possible to substantially reduce production cost, promote the sustainable development of agricultural.
Description
Technical field
The invention belongs to field of agricultural microorganism, and in particular to a kind of solution with degradation Phos and diseases prevention double action
Bacillus amyloliquefaciens, further relate to the microbial bacterial agent containing the bacillus amyloliquefaciens and they prevention crop disease with
Promote the application in terms of plant growth.
Background technology
One of three big elements that phosphorus is not only in plant nutrient, while be also the important composition of organic compound in plant
Ingredient plays the growth and development of plant indispensable effect.Although the phosphorus content in soil is higher, wherein 95% is left
Right phosphorus is invalid form, and the 2~3% of full phosphorus amount can be only accounted for by the Inorganic phosphorus that plant is directly absorbed and utilized.China has 74%
Arable soil lack phosphorus, if chronic administration chemical fertilizer supplements the phosphorus in soil, can cause in soil nutrients ratio imbalance and
Soil physico-chemical property changes.The utilization rate for improving phosphorus in soil is the key that solve phosphorus element to lack.Phosphate solubilizing bacteria is that one kind can will be native
Slightly solubility or insoluble phosphorus are converted to the microorganism for being easy to that phosphorus is utilized by plant available in earth, and in addition to this, phosphate solubilizing bacteria is also
It can promote beneficial microorganism metabolic activity in soil, improve plant root nutrition, improve crop yield.
At present it is known that phosphate-solubilizing bacteria focus mostly at bacillus (Bacillus), such as bacillus amyloliquefaciens
(CN105385638A, CN105420156A), bacillus subtilis (Bacillus subtilis, CN104263679A,
CN103773709A), bacillus megaterium (Bacillus megaterium, Wu Haiyan etc., Jilin Auto Industry, 2014
(2):171-175；Zhang Weina etc., Jilin agricultural sciences 05 phase 2012 in 2012), bacillus laterosporus (Bacillus
Laterosporus, palace account for member etc., Heilongjiang Bayi Agricultural Reclamation University's journal, 2005,17 (5)：14~17) etc..Known to these
The bacteriostatic and disease prevention effect of phosphate solubilizing bacteria is not directed in phosphate solubilizing bacteria.
Eggplant verticillium wilt is a kind of soil-borne disease as caused by verticillium dahliae (Verticillium dahliae).In recent years
Come, as protection common pratia fruit cultivated area expands rapidly, the conditions such as special light temperature, liquid manure and continuous cropping make eggplant verticillium wilt by
Year aggravates.Bear fruit after eggplant is susceptible less, plant early ageing even death.15~20%, severe patient's incidence reaches general incidence
30~50%, the yield and quality of eggplant is influenced very big.
At present, the method that eggplant verticillium wilt is prevented in production mainly grafts prevention and chemical prevention.But grafting work amount
Greatly, Difficulty；And long-term a large amount of generations for leading to medicament residual and drug resistance using chemical pesticide.Therefore, using beneficial to micro-
Biological control eggplant verticillium wilt is a kind of control measure for having application prospect.Currently used for preventing the bacillus of eggplant verticillium wilt
Mainly have bacillus amyloliquefaciens (CN105238723A), bacillus subtilis (Bacillus subtilis,
CN102925394A；CN102154186A；Shandong studies of the Hongloumeng etc., hubei agricultural science, 2013,52 (21)：5199-5202；Cheng Lei etc.,
Changjiang University's journal, natural science edition, 06 phase in 2012；Sun Yi etc., Jiangsu's agriculture journal, 2008,24 (4)：425-430；Woods
Tinkling of pieces of jade etc., Chinese biological preventing and treating journal, 2010 (s1)：40-46), Paenibacillus polymyxa (Paenibacillus polymyxa,
CN103773708A；The Chinese Plants such as Zheng Yuanyuan pathology meeting Annual Conference, 2008) etc..
Above-mentioned known bacillus function is relatively single or only has phosphate solubilization or only has controlling disease effect；
Secondly, with the change of environmental condition or the evolution of pathogen, the function of bacillus may also evolve therewith, change
Become, therefore, from soil detach, screen there is efficient phosphorus-dissolution, and the bacillus that antimicrobial spectrum is wide, for adjusting soil phophorus
The imbalance between supply and demand of element is improved to plant disease preventive effect, is reduced production cost, is preserved the ecological environment, promotes the sustainable development of agricultural
Exhibition is of great significance.
Invention content
Present invention aims at a kind of Bacillus amyloliquefaciens strain is provided, which has degradation Phos and prevention eggplant
The double actions such as sub- verticillium wilt.
The present invention second is designed to provide the purposes of above-mentioned bacillus amyloliquefaciens.
Third of the present invention is designed to provide a kind of microbial bacterial agent containing above-mentioned bacillus amyloliquefaciens.
The present invention the 4th is designed to provide the preparation method of mentioned microorganism microbial inoculum.
The present invention the 5th is designed to provide the purposes of mentioned microorganism microbial inoculum.
To achieve the above object, the invention is realized by the following technical scheme：
A kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain WKPHO-12, deposit number
For CGMCC No.14954.
Applications of the above-mentioned bacterial strains WKPHO-12 on degradation Phos.
Applications of the above-mentioned bacterial strains WKPHO-12 on degradation Inorganic Phosphorus Fractions in Soil.
Applications of the above-mentioned bacterial strains WKPHO-12 on plant growth is promoted.
Applications of the above-mentioned bacterial strains WKPHO-12 in controlling plant diseases.
Plant disease described in above application refers to eggplant verticillium wilt (Verticillium dahliae Kleb), Huang
Cucurbit wilt (F.oxysporum f.sp.cucumebrium), cotton wilt (F.oxysporum
) or black scurf of potato (R.solani) etc. f.sp.vasinfectum.
Above-mentioned bacterial strains WKPHO-12 is in prevention eggplant verticillium wilt and the application degraded on Inorganic Phosphorus Fractions in Soil.
The present invention also provides the microbial bacterial agents containing above-mentioned Bacillus amyloliquefaciens strain WKPHO-12.
Mentioned microorganism microbial inoculum, dosage form are liquid preparation or solid pharmaceutical preparation.
The viable count of bacterial strain WKPHO-12 is 2.0 × 10 in mentioned microorganism microbial inoculum6~2.0 × 108Cfu/mL or 2.0 ×
106~2.0 × 108cfu/g。
Mentioned microorganism microbial inoculum, the preparation method of liquid preparation, includes the following steps：
(1) actication of culture：The WKPHO-12 bacterial strains of Cord blood are activated on LB plating mediums, picking single bacterium colony exists
It is cultivated 10~16 hours for 25~35 DEG C on LB slant mediums, obtains the bacterial strain of activation；
(2) prepared by seed liquor：With the inoculation that sterile oese one ring step (1) of scraping activates to 100mL LB liquid
In body culture medium, cultivated 10~16 hours under conditions of 25~35 DEG C, shaking speed is 150~220rpm, obtain seed liquor；
(3) fermented and cultured：The seed liquor of step (2) is linked into corn flour soya bean according to the ratio that volume ratio is 1~3%
In powder culture medium (pH value 7.2), the fermented and cultured under conditions of temperature is 25~35 DEG C, shaking speed is 150~220rpm
36~40h obtains zymotic fluid；Then every thalline and Number of spores in 30 minutes detection zymotic fluids, grown spore in zymotic fluid is treated
Account for gemma and thalline sum 90% when stop fermented and cultured；Gained is the liquid preparation of WKPHO-12 bacterial strains.
The constituent and its weight of LB plating mediums or LB slant mediums described in above-mentioned preparation method step (1)
Than for：8~12g of tryptone, 4~6g of yeast extract, 4~6g of sodium chloride, 12~18g of agar powder, water 1000mL.
The constituent of LB fluid nutrient mediums and its weight ratio described in above-mentioned preparation method step (2) are：Tryptone 8
~12g, 4~6g of yeast extract, 4~6g of sodium chloride, water 1000mL.
LB plating mediums, LB slant mediums or the LB fluid nutrient mediums is conventionally prepared.
Corn flour soybean powder medium described in above-mentioned preparation method step (3), constituent and its weight percent
Than for：Corn flour 1.0~3.0%, analysis for soybean powder 1.0~3.0%, NaCl 0.1~1.0%, MnSO4·H2O 0.5~1.0%,
Remaining is water.
The preparation method of the corn flour soybean powder medium, according to weight percent by corn flour, analysis for soybean powder, NaCl
And MnSO4·H2O is mixed, and is added water, and is adjusted pH and is stirred evenly.
Mentioned microorganism microbial inoculum is in the application on degradation Phos.
Application of the mentioned microorganism microbial inoculum on degradation Inorganic Phosphorus Fractions in Soil.
Application of the mentioned microorganism microbial inoculum on plant growth is promoted.
Mentioned microorganism microbial inoculum is in the application in controlling plant diseases.
Plant disease described in above application refers to eggplant verticillium wilt (Verticillium dahliae Kleb), Huang
Cucurbit wilt (F.oxysporum f.sp.cucumebrium), cotton wilt (F.oxysporum
) or black scurf of potato (R.solani) etc. f.sp.vasinfectum.
Mentioned microorganism microbial inoculum is in prevention eggplant verticillium wilt and the application degraded on Inorganic Phosphorus Fractions in Soil.
The application method of mentioned microorganism microbial inoculum：It is 10 that mentioned microorganism microbial inoculum is diluted with water to viable bacteria body number7cfu/
ML, the seed soaking half an hour before eggplant is sowed；Or adsorb mentioned microorganism microbial inoculum with calcium carbonate, WKPHO-12 pulvis is fabricated to,
According to pesticide-seeds ratio 1 before eggplant sowing：10 seed dressings.
The present invention also provides the genetic engineering bacteriums using above-mentioned bacterial strains WKPHO-12 as recipient bacterium.The genetic engineering bacterium carries
The high ability of degradation Phos improves the double of the preventive effect or degradation Phos to pathogen and the preventive effect to pathogen
Weight function is all improved.
The present invention has the advantage that and advantageous effect：(1) bacillus amyloliquefaciens WKPHO-12 of the present invention is that one kind can
Degradation Phos, and the microorganism with dual function of energy controlling plant diseases, can realize that a bacterium is mostly used.Using this bacterium, no
The utilization rate of Phos in soil can be only improved, reduces Phosphorus fertilizer usage, promotes plant growth, reduces production cost, and
To the higher preventive effect that has of the multiple diseases such as eggplant verticillium wilt, average preventive effect is more than 70.0%.(2) bacterial strain WKPHO- of the present invention
12 antimicrobial spectrums are wide, and the bacterium that not only withers to eggplant Huang is inhibited but also black to cucumber Fusarium oxysporum, cotton wilt fusarium and potato
Mole bacterium etc. also has good inhibiting effect.(3) bacterial strain WKPHO-12 of the present invention is strong to the specialization of pathogen, is not likely to produce anti-
Pharmacological property, lasting medicine is strong, reduces formulation rate and spraying times.(4) microbial bacterial agent of the present invention does not have people, animal safety
Environmental pollution；(5) microorganism formulation preparation method of the present invention it is simple, it is at low cost, using simple.
Biological deposits
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain WKPHO-12 of the present invention is the present invention
Inventor voluntarily screen to obtain, the bacterial strain oneself on November 22nd, 2017 be preserved in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center, preservation address are：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microbe research
Institute, deposit number are：CGMCC No.14954.
Description of the drawings
Fig. 1 are the WKPHO-12 bacterial strain systematic growth tree graphs obtained according to 16S rDNA sequences.
Fig. 2 are the WKPHO-12 bacterial strain systematic growth tree graphs obtained according to gyrB gene orders.
Specific embodiment
The present invention is further clearly explained with specific embodiment below, but is not formed in any way to the present invention
Limitation.Experimental method in following embodiments is conventional method unless otherwise instructed；Percentage in following embodiments contains
Amount, is weight percentage unless otherwise instructed.
Withering bacterium EVD-1 for examination cause of disease bacteria strain eggplant Huang involved in following embodiment, it is clear to be isolated from Baoding
Garden county Zhang Deng towns Zhang Dengdian villages eggplant disease fruit, isolates and purifies through Baoding micro-control bio tech ltd, through Agricultural University Of Hebei
Verticillium dahliae (Verticillium dahaliae) is accredited as, Pathogenic Tests show as High pathogenicity.
For examination Eggplant Varieties agricultural university 601 used in following embodiment, the kind being bred as Agricultural University Of Hebei.
The separation screening process and taxonomic identification of 1 bacterial strain WKPHO-12 of the present invention of embodiment
(1) the separation screening process of bacterial strain of the present invention:
In September, 2015 is tried to win the champion 5 points of county alum mountain phosphorus ore area, 5 parts of soil sample of acquisition in Hebei province's Zhangjiakou City, every part of 200g.
1.0g is weighed respectively to air-dry in triangular flask of the soil sample addition with sterilizing bead, is added 99mL sterile waters, is stood 20min,
30 DEG C on shaking table, 180r/min fully vibrate 30min, then carry out gradient dilutions by 10 times of dilution methods, take 10 respectively-3、10-4、
10-5100 μ L of dilution, be coated on solution Phos culture medium on, each 3 repetitions of concentration；It is stood in clean bench after coating
5-10min treats that bacterium solution is adsorbed into culture medium, in 35 DEG C of 5~7d of constant temperature incubation.It is sieved by transparent circle method, molybdenum antimony resistance colorimetric method
Bacterial strain of the choosing with degradation Phos ability, while using eggplant verticillium wilt as target, pass through tablet face-off method and pot experiment method
It is screened, the bacterial strain provided phosphorus decomposing and prevent eggplant verticillium wilt dual function is finally screened, by the strain was named
WKPHO-12。
(2) taxonomic identification of WKPHO-12 bacterial strains：
(1) identification by morphological characters
Thalline is rod-shaped on LB culture mediums, generates brood cell after cultivating 10h, is given birth in brood cell, and ellipse, cyst does not expand,
Acid-fast stain is negative, and no parasporal crystal can move, flagellum Zhousheng.On nutrient agar panel, Initial stage of culture bacterium colony is light milky white
Color, purulence shape is round, neat in edge, and bacterium colony protuberance is in steamed bun shape, surface wettability；Late stage of culture bacterium colony is faint yellow, and edge is not whole
Together, dry tack free has fold；It crosses and cultivates on nutrient agar slopes, it is linear；Static gas wave refrigerator in liquid medium, table
Face forms white mycoderm.These morphological features with《Common bacteria system identification handbook》(east show pearl etc. writes Science Presses
.2001 year) described in bacillus morphological feature it is basically identical, tentatively judge that WKPHO-12 bacterial strains belong to bacillus.
(2) classified using 16S rDNA Sequence Identifications
Using the genomic DNA of WKPHO-12 as template, PCR amplification, institute are carried out by primer of universal primer F27 and R1492
The primer sequence stated is：F27：5’-AGAGTTTGATCATGGCTCAG-3’；R1492：5’-GGCTACCTTGTTACGACTT-
3’；Wherein the reaction system of PCR (50 μ L) is:10×PCR Buffer(Mg2+) 5 μ L, dNTP Mixture (2.5mM) 5 μ L,
Taq (5U/ μ L) 1 μ L, F27 (10 μm of ol/L) 1 μ L, R1492 (10 μm of ol/L) 1 μ L, WKPHO-12 genomic DNA 50ng；
ddH2O complements to 50 μ L.The reaction condition of PCR is 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1.5min, 30 cycles；
72℃10min.Gained pcr amplification product is subjected to gel electrophoresis, the sequencing of Shanghai Sheng Gong bioengineering Co., Ltd is delivered, obtains
The 16S rDNA sequences of WKPHO-12 are (see SEQ ID No:1).By the 16S rDNA sequences of gained WKPHO-12 in Genbank
Tetraploid rice is carried out, as a result, it has been found that the 16S rDNA homologys of bacterial strain WKPHO-12 and bacillus reach 99%；Simultaneously
Using MEGA software building phylogenetic trees, as a result (see Fig. 1), WKPHO-12 is aggregated to together with bacillus, explanation
WKPHO-12 belongs to bacillus (Bacillus).
(3) it is identified and classified using gyrB gene orders
Using WKPHO-12 genomic DNAs template, using bacillus gyrB genes degenerate primer gyrB-F and gyrB-R as
Primer carries out PCR amplification, obtains pcr amplification product；The sequence of the wherein described gyrB-F and gyrB-R primers is：gyrB-F：5’-
TTGRCGGHRGYGGHTATAAAGT-3’；gyrB-R：5’-TCCDCCSTCAGARTCWCCCTC-3’；The PCR amplification of gyrB is anti-
It is 50 μ L to answer system:10×PCR Buffer(Mg2+) 5 μ L, dNTP Mixture (2.5mM) 5 μ L, Taq (5U/ μ L) 1 μ L,
GyrB-F (10 μm of ol/L) 1 μ L, gyrB-R (10 μm of ol/L) 1 μ L, WKPHO-12 genomic DNAs 50ng, ddH2O complements to 50 μ
L.The reaction condition of PCR is 95 DEG C of 5min；95 DEG C of 30s, 55 DEG C of 45s, 72 DEG C of 1min, 30 cycles；72℃10min.It will amplification
Product delivers the sequencing of Shanghai Sheng Gong bioengineering Co., Ltd, obtains the gyrB gene orders of WKPHO-12 bacterial strains (see SEQ ID
No:2).The gyrB gene orders of the WKPHO-12 bacterial strains of acquisition are subjected to tetraploid rice in Genbank, as a result, it has been found that
The gyrB gene homology highests of WKPHO-12 and bacillus amyloliquefaciens, reach 99%；MEGA software structures are utilized simultaneously
Phylogenetic tree is built, as a result WKPHO-12 bacterial strains are aggregated to together with bacillus amyloliquefaciens (see Fig. 2), illustrate that WKPHO-12 is
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and be a new strains.
In summary morphological feature, 16S rDNA and gyrB gene homology comparative analyses as a result, understanding
WKPHO-12 belongs to bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and solves starch gemma with existing
Bacillus strain is different, is a new Bacillus amyloliquefaciens strain.
The preparation of the microbial bacterial agent of the invention containing WKPHO-12 bacterial strains of embodiment 2
It carries out in accordance with the following steps：
(1) actication of culture：By the Bacillus amyloliquefaciens strain WKPHO-12 for being stored in -80 DEG C, (its deposit number is
CGMCC No.14954) (30 DEG C) are activated on LB plating mediums, picking single bacterium colony is on LB slant mediums 30
It is cultivated 12 hours at DEG C, obtains the bacterial strain of activation；The constituent and its weight ratio of LB plating mediums or LB slant mediums be：
Tryptone 10g, yeast extract 5g, sodium chloride 5g, agar powder 15g, water 1000mL；
(2) preparation of seed liquor：LB fluid nutrient medium (its constituent and its weight ratio are packed into 250mL triangular flasks
For：Tryptone 10g, yeast extract 5g, sodium chloride 5g, water 1000mL) 100mL, high pressure moist heat sterilization treats that temperature drops to room
Wen Hou accesses the bacterial strain activated in an oese step (1) in every bottle, under conditions of 30 DEG C, shaking speed 180rpm into
Row shaken cultivation 12 hours, obtains seed liquor；
(3) preparation of corn flour soybean powder medium：According to weight percent by corn flour 2.5%, analysis for soybean powder 2.5%,
NaCl 0.6%, MnSO4·H2O 0.6% is added to the water, and is uniformly mixed to get corn flour soybean powder medium；It is sub-packed in
In 500mL triangular flasks, every bottle of 200mL；Sterilizing 30 minutes is carried out to corn flour soybean powder medium, then cool to 30 at 121 DEG C
It is DEG C spare；
(4) fermented and cultured：Into every bottle of corn flour soybean powder medium 200mL obtained by step (3) obtained by inoculation step (2)
Seed liquor 2mL；Fermented and cultured 36 hours is carried out under the conditions of 30 DEG C, shaking speed 180rpm, later every 30 minutes from triangle
Bottle in sampling carry out microscopy, the gemma in the visual field and total thalline number are counted, and calculate gemma rate (gemma rate (%)=into
Ripe gemma number/(grown spore number+thalline number) × 100)；Gemma rate stops fermented and cultured when reaching 90%；Common fermentation culture 48
Hour obtains the liquid preparation of bacillus amyloliquefaciens WKPHO-12.
It is measured using the method for plate culture count, the viable bacteria content of gained WKPHO-12 liquid preparations is 2.3 × 108cfu/
mL。
3 bacterial strain WKPHO-12 of the present invention of embodiment degradation Phos ability qualitative determination experiments
It carries out as follows：
The WKPHO-12 bacterial strains point activated in 2 step of embodiment (1) is seeded in solution Phos with sterilizing toothpick to put down
(its constituent and its weight ratio are plate culture medium：Glucose 10.0g, (NH4)2SO40.5g, MgSO4·7H2O 0.5g,
NaCl 0.2g, Ca3(PO4) 2 5.0g, KCl 0.2g, MnSO40.03g, FeSO40.003g, agar 20.0g, distilled water
1000mL, pH：On 7.0-8.0), 30 DEG C of constant incubator cultures 5 days are subsequently placed in, measure the straight of transparent loop diameter and bacterium colony
Diameter.
As a result bacterial strain WKPHO-12 of the present invention is containing Ca3(PO4)2Phos plating medium on generate diameter 13.6
The transparent circle of millimeter illustrates that WKPHO-12 bacterial strains of the present invention can degrade Phos Ca very well3(PO4)2, have in degradation soil
The potentiality of Phos.
4 bacterial strain WKPHO-12 of the present invention of embodiment degradation Phos ability quantitative determination experiments
This experiment is carried out in early Febuary, 2016 in Baoding micro-control bio tech ltd artificial climate room.
It carries out as follows：
(1) prepared by fermentation medium：Proportionally by glucose 10.0g, (NH4)2SO40.5g, MgSO4·7H2O
0.5g, NaCl 0.2g, Ca3(PO4) 2 5.0g, KCl 0.2g, MnSO40.03g, FeSO40.003g is added to distilled water
In 1000mL, it is uniformly mixed to get fermentation medium (pH：7.0~8.0).Fermentation medium 100mL is packed into 300mL tapers
In bottle, autoclave sterilization, for use.
(2) prepared by zymotic fluid：By the WKPHO-12 bacterial strains seed liquor obtained by 2 step of embodiment (2) and blank control culture
Liquid (the LB fluid nutrient mediums for not being inoculated with WKPHO-12 bacterial strains) is inoculated into step according to the ratio that weight percent is 2% respectively
(1) in the fermentation medium prepared, every group of 3 repetitions in 30 DEG C, 180r/min culture 6d, obtain zymotic fluid.
(3) fermentation liquor treatment：Cultured zymotic fluid is transferred in sterile Centrifuge Cup, using KQ5200DE type numerical controls
It has children outside the state plan wave washer and carries out ultrasonic cell-break, broken condition：200-240V, 2A, 50/60Hz, time 20min.It is allowed to release
Release intracellular available phosphorus.2.5mL supernatants to be taken to add in 50mL colorimetric cylinders after the rotating speed centrifugation 10min of 8000r/min
2 drop 2,4-DNPs make indicator, and pH value is adjusted to solution just in yellowish with 10%NaOH and 5% dilution heat of sulfuric acid,
Add the anti-color developing agent 5mL of molybdenum antimony, constant volume, after reacting 30min.Supernatant is measured with T6 new centuries ultraviolet-uisible spectrophotometer to exist
OD values at 720nm.The available phosphorus content in supernatant is obtained according to standard curve.
(4) result calculates：The absorption value obtained by sample solution colorimetric calculates corresponding colorimetric solution on working curve
Phosphorus content (mg/L), then available phosphorus content in zymotic fluid is calculated as follows：
Phosphorus (mg/L) × extension rate of available phosphorus (mg/L)=color solution.
As a result (being shown in Table 1) is inoculated with titanium pigment content and blank control in the fermentation culture of WKPHO-12 bacterial strains of the present invention
Compared to 68.49mg/L is increased to, illustrate the ability that WKPHO-12 bacterial strains of the present invention have degradation Phos tricalcium phosphate.
1 WKPHO-12 strains for degrading Phos ability of the present invention of table quantitative determines result of the test
| Strain number | OD720(nm) | Titanium pigment content (mg/L) |
| WKBS-26 | 1.352 | 68.49 |
| CK | 0.203 | 10.27 |
5 bacterial strain WKPHO-12 of the present invention of embodiment tests the promotion growth of eggplant plant
(1) test material：
(1) matrix：Sand+substrate
Wherein：Sand is rinsed with water 3 times in advance, air-dries spare, pH6.0 or so；
Substrate：Tricalcium phosphate, 1g/kg matrix.
(2) test process：
(1) it handles：Tricalcium phosphate+WKPHO-12 zymotic fluids+scarce phosphorus nutrition liquid；
(2) it compares：Tricalcium phosphate+original fermentation medium+scarce phosphorus nutrition liquid.
(3) test method：This experiment is in early June, 2016 in Baoding micro-control bio tech ltd laboratory
It carries out.601 eggplant seedling of agricultural university is cultivated in nutritive cube, is transplanted when rough leaf is unfolded to the flowerpot of the husky amount 3.5kg/ basins of dress
In it is (high：20cm, basin mouth diameter：21cm, basin bottom diameter：14cm), it per 2 plants of basin, is placed in greenhouse and cultivates, start to try after slow seedling
It tests.Experiment setting：Handle the WKPHO-12 bacterial strain fermentation liquor dilution (concentration prepared to pour 4 step of 250mL embodiments (2)
It is 1 × 107CFU/mL) in crop root, blank control is to pour former fermented and cultured prepared by 4 step of embodiment (1) of equivalent
Base dilution.It is repeated 3 times, often repeatedly 3 basin.Period normal management, keeps the skin wet in due course, each 400mL/ basins；7d pours primary lack
Phosphorus nutrition liquid (constituent for lacking phosphorus nutrition liquid is shown in patent application 201110107663X), each 250mL/ basins.It is measured after 50d
The indexs such as phosphorus content in the plant height of eggplant, overground part fresh weight and available phosphorus in underground part fresh weight, matrix and eggplant plant body.
As a result the eggplant plant height for (being shown in Table 2) by bacillus amyloliquefaciens WKPHO-12 fermentation liquor treatments increases
3.17%, with compareing that there was no significant difference；Overground part, underground part fresh weight growth rate are respectively 24.76% and 35.55%, with sky
There are significant differences between white control.The above results illustrate that bacillus amyloliquefaciens WKPHO-12 bacterial strains of the present invention can be shown
Write the growth for promoting eggplant.
2 bacterial strain WKPHO-12 of the present invention of table is to the influence result of the test of potting eggplant plant height and fresh weight
From table 3 it is observed that after bacillus amyloliquefaciens WKPHO-12 of the present invention processing, the increasing of available phosphorus in matrix
Long rate is 10.31%, and available phosphorus growth rate is 107.05% in eggplant plant.Illustrate that bacterial strain WKPHO-12 of the present invention can have
The Phos in matrix degradation is imitated, and promotes the absorption of the available phosphorus after the degradation of eggplant plant pair, so as to promote eggplant plant
Growth.
3 bacterial strain WKPHO-12 of the present invention of table is to the influence result of the test of matrix and eggplant plant available phosphorus
6 bacterial strain WKPHO-12 of the present invention of embodiment tests the inhibiting effect of Verticillium Wilt Pathogen of Eggplant
It carries out as follows：
Eggplant Huang is withered bacterium EVD-1 activation culture 3-7 days on PDA plate first, then uses card punch
Bacterium piece is made in the punching of colony edge region；By the switching of bacterium piece in another PDA plate center, then will be living in 2 step of embodiment (1)
The bacillus amyloliquefaciens WKPHO-12 points of change are connected on away from 2.0 centimeters of indicator bacteria bacterium piece, if blank control (does not put and meets WKPHO-
12 bacterial strains).In 25 DEG C of constant temperature incubations 3-10 days, WKPHO-12 bacterial strains and the growing state for examination disease fungus were observed day by day, is treated
When blank control pathogen is grown to culture dish edge, the control increment (colony radius) of pathogen is measured and through solving starch gemma
The increment (the inhibition growth radius after inoculation WKPHO-12) of bacillus WKPHO-12 processing, antagonism is represented with bacteriostasis rate.
Bacteriostasis rate calculation formula is：Bacteriostasis rate (%)=(control increment-processing increment)/control increment × 100).
As a result the bacterial strain WKPHO-12 of the present invention that (is shown in Table 4) wither to eggplant Huang bacterium bacteriostasis rate up to 62.72%, illustrate bacterial strain
The WKPHO-12 bacterium that wither to eggplant Huang significantly inhibit, and have the Biocontrol Potential for the bacterium that withers to eggplant Huang.
4 WKPHO-12 bacterial strains of the present invention of table wither to eggplant Huang the bacteriostasis result of the test of bacterium
| Pathogen | Normal growth (mm) | Inhibit growth (mm) | Bacteriostasis rate (%) |
| Eggplant Huang withers bacterium (V.dahliae) | 33.8 | 12.6 | 62.72 |
7 WKPHO-12 liquid preparations of the present invention of embodiment test the control effect of eggplant verticillium wilt
(1) test process：
(1) WKPHO-12 liquid systems：100 times are diluted with the WKPHO-12 liquid preparations that water prepares embodiment 2.
(2) medicament compares：1000000000 living spores/gram bacillus amyloliquefaciens wettable powder (green rich biochemical section of Baoding section
Skill Co., Ltd)；It is diluted with water 100 times.
(3) blank control：Clear water
(2) test method：
This experiment is carried out in March, 2017 in Baoding micro-control bio tech ltd laboratory.Application is real before sowing
Apply 100 times of water diluent seed soaking half an hour of WKPHO-12 liquid preparations of the preparation of example 2；With " 1,000,000,000 living spores/gram solution starch gemma
100 times of water diluent seed soaking half an hour of bacillus wettable powder " compare as medicament；Using Seed soaking half an hour as blank pair
According to.After planting normal culture.When eggplant seedling grows the 4th~5 true leaf, withered bacterium EVD-1 using Qie Genfa inoculation eggplant Huangs
Spore suspension (107A spore/mL).Routine Management continues to investigate disease index when culture is fully fallen ill to blank control,
Calculate control effect.
As a result (table 5) WKPHO-12 of the present invention is 72.48% to the preventive effect of eggplant verticillium wilt, and prevention eggplant is compareed with medicament
The effect (69.58%) of verticillium wilt is similar.Illustrate that bacterial strain WKPHO-12 and its liquid preparation of the present invention have eggplant verticillium wilt
Good control effect.
5 WKPHO-12 liquid preparations of the present invention of table are to eggplant verticillium wilt efficiency test result
8 WKPHO-12 solid pharmaceutical preparations of the present invention of embodiment test the control effect of eggplant verticillium wilt
(1) test process：
(1) WKPHO-12 pulvis：According to 1：1 ratio adds carbonic acid into WKPHO-12 liquid preparations prepared by embodiment 2
WKPHO-12 pulvis is made in calcium.
(2) medicament compares：1000000000 living spores/gram bacillus amyloliquefaciens wettable powder (green rich biochemical section of Baoding section
Skill Co., Ltd).
(3) blank control：It is untreated
(2) test method：
This experiment is carried out in June, 2017 in Baoding micro-control bio tech ltd laboratory.By nursery soil high pressure
Steam sterilizing is put to room temperature for 2 hours, is paved with seedlings nursing plate, 1 plant of 601 eggplant seedling of agricultural university is colonized per cave in seedlings nursing plate, is cultivated to 4~5
It is spare during true leaf.Eggplant Huang is withered into bacterium EVD-1 inoculated by hypha block in PDB culture solutions, at 25 DEG C, 180rpm/min is cultivated 5 days,
It filters off mycelia and obtains conidium, conidium is mixed into sterile seedling medium, wherein Huang is made to wither the conidial concentration of bacterium
It is 106A/gram soil.The mixed seedling medium that carries disease germs is packed into flowerpot (diameter 15cm), the position for being colonized eggplant seedling wherein is applied
Enter 2.0 grams of WKPHO-12 pulvis, by (seedlings nursing plate is held up during transplanting makes the eggplant seedling root tip break in favor of disease in eggplant transplantation of seedlings to flowerpot
Opportunistic pathogen infects) and band soil bacteria is covered, it is colonized one plant of eggplant seedling in every basin.It can with 1,000,000,000 gemma of spreading manuer in holes/gram bacillus amyloliquefaciens
2.0 grams of wet powder is compareed for medicament, not process the eggplant seedling directly transplanted as blank control.Often processing is repeated 3 times, often
Repeat 5 basins.It is normally cultivated after transplanting, until investigating disease index when blank control is fully fallen ill, calculates control effect.
As a result (table 6) bacterial strain WKPHO-12 of the present invention is 70.74% to the preventive effect of eggplant verticillium wilt, and prevention is compareed with medicament
The effect (73.30%) of eggplant verticillium wilt is suitable.Illustrate bacterial strain WKPHO-12 and its solid pharmaceutical preparation of the present invention to eggplant verticillium wilt
With good control effect.
6 WKPHO-12 solid pharmaceutical preparations of the present invention of table are to eggplant verticillium wilt efficiency test result
| Processing | Disease index | Preventive effect (%) |
| WKPHO-12 pulvis | 24.94b | 70.74 |
| 1000000000 work brood cell/gram bacillus amyloliquefaciens wettable powders | 22.76b | 73.30 |
| Blank control | 85.23a | -- |
9 bacterial strain WKPHO-12 of the present invention of embodiment tests the inhibiting effect of three kinds of disease pathogens
(1) for examination cause of disease bacteria strain
(1)：Cucumber fusarium axysporum FOC-1：Dong Luobao townshiies of Baoding Dingxing County Dong Ce villages cucumber diseased plant is isolated from, through Hebei
Agriculture university is accredited as Fusarium oxysporum cucumber transformant (Fusarium oxysporum f.sp.cucumebrium).
(2)：Cotton-wilt fusarium FOV-7：Xingtai City Wei County cotton diseased plant is isolated from, point is accredited as through Agricultural University Of Hebei
Fusarium oxysporum wilting specialized form (Fusarium oxysporum f.sp.vasinfectum).
(3)：Black scurf of potato RS-3：It is isolated from the western poplar ditch village potato disease in Zhangjiakou City Shangyi County first stone river township
Strain, Rhizoctonia solani Kuhn (Rhizoctonia solani) is accredited as through Agricultural University Of Hebei.
Three above strain pathogenic strength measure shows as High pathogenicity.
(2) test method：
This experiment is carried out in early June, 2017 in Baoding micro-control bio tech ltd laboratory.It first will be for
Pathogen activation culture 4 days on PDA plate are tried, then use card punchIn colony edge region, bacterium is made in punching
Disk, then by bacterium disk transfer another PDA plate center, then by 2 step of embodiment (1) activate after bacillus amyloliquefaciens
WKPHO-12 points are connected on away from 2.0 centimeters of indicator bacteria bacterium disk, if blank control (do not put and connect WKPHO-12 bacterial strains).In 25 DEG C of constant temperature
Culture when blank control will cover with entire culture dish, measures control increment (colony radius) and the place of tomato gray mould bacterium
Increment (the inhibition growth radius after inoculation WKPHO-12) is managed, antagonism is represented with bacteriostasis rate.The calculation formula of bacteriostasis rate
For：
Bacteriostasis rate (%)=(control increment-processing increment)/control increment × 100.
7 bacterial strain WKPHO-12 of the present invention of table is to the inhibiting effect result of the test of various pathogenic bacteria
As a result the bacterial strain WKPHO-12 of the present invention that (is shown in Table 7) is 74.05% to the inhibiting rate of cucumber Fusarium oxysporum, is withered to cotton
The inhibiting rate of bacterium is 75.38%, and the inhibiting rate to black scurf of potato bacterium is 78.04%, illustrates bacillus amyloliquefaciens
WKPHO-12 significantly inhibits these three pathogens, has prevention cucumber fusarium axysporum, cotton wilt and Ma Ling
The Biocontrol Potential of potato tar spot.
Sequence table
<110>Baoding micro-control bio tech ltd
<120>A kind of bacillus amyloliquefaciens with degradation Phos and diseases prevention double action
<130> 2017S1137INH
<141> 2017-12-27
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1348
<212> DNA
<213> Bacillus amyloliquefaciens
<400> 1
agactgggat aactcaggga aaccggggct aataccggat ggttgtctga accgcatggt 60
tcagacataa aaggtggctt cggctaccac ttacagatgg acccgcgtcg cattagctag 120
ttggtgaggt aacggctcac caaggcgacg atgcgtagcc gacctgagag ggtgatcggc 180
cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg gactcttccg 240
caatggacga aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa 300
agctctgttg ttagggcaga acaagtgccg ttcaaatagg gcggcacctt gacggtacct 360
aaccagaaag ccacggctaa gtacgtgcca gcagccgcgg taatacgtag gtggcaagcg 420
ttgtccggaa ttattgggcg taaagggctc gcaggcggta tcttaagtct gatgtgaaag 480
cccccggctc aaccggggag ggtcattgga aactggggaa cttgagtgca gaagaggaga 540
gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag 600
ccgactctct ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg aacaggatta 660
gaaaccctgg tagtccacgc cgtaaacgat gagtgctaag tgttaggggg tttccgcccc 720
ttagtgctgc agctaacgca ttaagcactc cgcctgggga gtacggtcgc aagactgaaa 780
ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa 840
cgcgaagaac cttaccaggt cttgacatcc tctgacaatg ctagagatag gacgtcccct 900
tcgggggcag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgacgtg agatgttggg 960
ttaagtcccg caacgagcgc aacccttgat cttagttgcc agcattcagt tgggcactct 1020
aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc 1080
ttatgacctg ggctactcac gtgctacaat ggacagaaca aagggcagcg aaaccgcgag 1140
gttaagccaa tcacacaaat ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt 1200
gaagctggaa tcgctagtaa tcgcgaatca gcatgccgcg gtgaatacgt tcccgggcct 1260
tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt gaggtaacca 1320
ttatggagcc agccgccgaa gtgaacag 1348
<210> 2
<211> 828
<212> DNA
<213> Bacillus amyloliquefaciens
<400> 2
gacggaaaaa tccactatca ggcgtacgag tgcggtgtac ctgtggctga tcttgaagtg 60
atcggtgata ctgataagac cggaacgatt aagcacttcg ttccggatcc ggaaatcttc 120
aaagaaacaa tcgtatacga ctatgatctg ctttcaaacc gtgtccggga attggccttc 180
ctgactaaag gcgtaaacat cacgattgaa gacaaacgtg aaggacaaga acggaaaaac 240
gagtaccacg acgaaggcgg aatcaaaagc tatgttgagt acttaaaccg ttccaaagaa 300
gtcgttcatg aagagccgat ttatatcgaa ggcgagaaag acggcataac ggttgaagtt 360
gcgttgcaat acaacgacag ctatacaagc aacatttatt ctttcacaaa taacatcaac 420
acatacgaag gcgggacgca cgaagccgga tttgaaaccg gtctgacccg tgtcataaac 480
gactatgcaa gaagaaaagg gattttcaaa gaaaatgatc cgaatttaag cggagatgat 540
gtgagagaag ggctgactgc cattatttca attaagcacc ctgatccgca attcgaaggg 600
cagacgaaaa cgaagctcgg caactccgaa gcgagaacga tcactgatac gctgttttct 660
tctgcgctgg aaacatccct tcttgaaaat ccggactcag cccgcaaaat cgttgaaaaa 720
ggtttaatgg ccgcaagagc gcggatggca gcgaaaaaag cgcgggaatt gacccgccgc 780
aaaagtgcgc ttgagatttc caatctgccg gacaaactgg cggactgt 828
Claims (10)
1. a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) bacterial strain WKPHO-12, deposit number are
CGMCC No.14954。
2. applications of the bacterial strain WKPHO-12 described in claim 1 on degradation Phos.
3. applications of the bacterial strain WKPHO-12 described in claim 1 in controlling plant diseases；The wherein described plant disease is
Refer to eggplant verticillium wilt (Verticillium dahliae Kleb), cucumber fusarium axysporum (F.oxysporum
F.sp.cucumebrium), cotton wilt (F.oxysporum f.sp.vasinfectum) or black scurf of potato
(R.solani)。
4. bacterial strain WKPHO-12 described in claim 1 is in prevention eggplant verticillium wilt and the application degraded on Inorganic Phosphorus Fractions in Soil.
5. the microbial bacterial agent containing bacterial strain WKPHO-12 described in claim 1.
6. microbial bacterial agent according to claim 5, it is characterised in that bacterial strain WKPHO-12 in the microbial bacterial agent
Viable count be 2.0 × 106~2.0 × 108cfu/mL。
7. the microbial bacterial agent described in claim 5 is in the application on degradation Phos.
8. the microbial bacterial agent described in claim 5 is in the application in controlling plant diseases；The wherein described plant disease is
Refer to eggplant verticillium wilt (Verticillium dahliae Kleb), cucumber fusarium axysporum (F.oxysporum
F.sp.cucumebrium), cotton wilt (F.oxysporum f.sp.vasinfectum) or black scurf of potato
(R.solani)。
9. the microbial bacterial agent described in claim 5 is in prevention eggplant verticillium wilt and the application degraded on Inorganic Phosphorus Fractions in Soil.
10. using bacterial strain WKPHO-12 described in claim 1 as the genetic engineering bacterium of recipient bacterium.
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| CN109355233A (en) * | 2018-12-04 | 2019-02-19 | 沈阳化工研究院有限公司 | A kind of bacillus amyloliquefaciens and its application |
| CN110257309A (en) * | 2019-08-08 | 2019-09-20 | 广东省农业科学院蚕业与农产品加工研究所 | One bacillus SEM-2, the composite bacteria agent including SEM-2, composite microbe fertilizer and its application |
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| CN110699288A (en) * | 2019-10-31 | 2020-01-17 | 河北省农林科学院植物保护研究所 | Bacillus amyloliquefaciens strains and inoculants for the prevention and treatment of potato black nevus and their applications |
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| CN114027114A (en) * | 2021-10-27 | 2022-02-11 | 赤峰学院 | A kind of method for promoting the growth and development of eggplant to prevent and cure Verticillium wilt |
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