CN101967455B - Bacillus amyloliquefaciens EA19 for controlling wheat root diseases and preparation thereof - Google Patents
Bacillus amyloliquefaciens EA19 for controlling wheat root diseases and preparation thereof Download PDFInfo
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- CN101967455B CN101967455B CN2009102734988A CN200910273498A CN101967455B CN 101967455 B CN101967455 B CN 101967455B CN 2009102734988 A CN2009102734988 A CN 2009102734988A CN 200910273498 A CN200910273498 A CN 200910273498A CN 101967455 B CN101967455 B CN 101967455B
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Abstract
The invention provides a bacillus amyloliquefaciens EA19 which is preserved in a China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC M209316, the EA19 strain is an endophytic bacterium obtained by the processing steps of separating and purifying health leaves of a plant of annual fleabane herb picked up in a Wuhan botanical garden of the Chinese Academy of Sciences by adopting a flat screening separating method. The bacillus amyloliquefaciens EA19 has remarkable bacteriostatic activity on wheat root rot bipolaris sorokiniana, wheat root rot leaf phoma tracheiphila, gaeumannomyces graminis, wheat sclerotium rolfsii sacc and rhizoctonia cerealis. A particle bactericide, a wettable powder bactericide and a water bactericide prepared from the bacillus amyloliquefaciens EA19 can be used for controlling the wheat root rot bipolaris sorokiniana, the wheat root rot leaf phoma tracheiphila, the gaeumannomyces graminis, the wheat sclerotium rolfsii sacc and the rhizoctonia cerealis. Compared with the adopted chemical agents, the bacillus amyloliquefaciens EA19 has the advantages of simple use, little pollution and low cost, and has better agricultural application prospect and organism development value.
Description
Technical field
The present invention relates to a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and utilize this bacillus amyloliquefaciens microbial inoculum of producing and the application that prevents and treats the wheat root disease.This bacterial strain has been preserved in Chinese typical culture collection center, and preserving number is CCTCC NO:M 209316, and bacterial strain is called bacillus amyloliquefaciens (Bacillus amyloliquefaciens) EA19, belongs to biological technical field.
Background technology
Along with the extensive concern of the whole world to resource and environment, utilize endophyte of plant or endophyte of plant excretory secondary substance to come antagonism or kill harmful pathogen of plant, be a new direction of current biological pesticide development field.Root rotof flax, gaeumannomyces graminis disease, southern blight and hypochnus; In Henan, Hebei, Shandong, Jiangsu, Hubei and other wheat main producing regions all have big area to take place; Be that harm China topmost soil of Wheat Production passes root disease; It can cause the general field piece underproduction 10%～30%, and the piece underproduction of grave illness field is more than 50%, even No kernels or seeds are gathered, as in a year of scarcity.In recent years the conservation tillage system of advocating has further increased the weight of the harm of wheat soil biography root disease, and this has become the outstanding problem in some Mai Qu productions.The plantation disease-resistant variety is the most economical effective meanss of a kind of these diseases of control, yet does not still have the disease-resistant variety of control wheat root disease at present.Therefore, currently use the main method that chemical pesticide is still these diseases of control, but that it exists is seriously polluted, poor with Environmental compatibility, pathogenic bacteria is easy to generate problems such as resistance, and these problems become increasingly conspicuous.The public nuisance-free agricultural chemicals of development of new is to solve pressing for of wheat soil-borne disease.
Bacillus, it distributes extensively, and aerobic or amphimicrobian can form gemma (statospore), is soil or plant surface and inner one type of important microbial population, is generally Gram-positive.The gemma that genus bacillus forms all has very strong resistibility to heat, ultraviolet ray, radioactive rays and chemical substance etc., the survival in environment of the production of the gemma biological prevention and control agent that therefore is highly advantageous to, formulation processing and genus bacillus, grows and breeding surely.Simultaneously; A kind of plant endogenesis non-pathogenic bacteria that genus bacillus extensively exists as nature; To person poultry harmless, environmentally safe, be easy to breeding and fermentation, accommodative ability of environment is strong; And various active materials such as the subtilyne that produces in its process of growth, polymyxin, nystatin, linear gramicidins have the obvious suppression effect to the pathogenic bacterium of plant or the pathogenic bacterium of autogenous infection, and therefore, genus bacillus has important exploitation valency and plants in the biological control Plant diseases.At present, the genus bacillus that is used for controlling plant diseases of registration mainly contains in the production: subtilis (B.subtilis), Bacillus cereus (B.cereus), bacillus pumilus (B.pumillus) and bacillus polymyxa (B.polymyma) etc.
Bacillus amyloliquefaciens is the kind of genus bacillus.In enzyme industry, this bacterium is the main bacteria seed that is used to produce AMS; In foodstuffs industry, genus bacillus can suppress to cause the growth of pathogenic bacterium such as the septic sickle-like bacteria of numerous food, aspergillus, mould and Mucor well, also has broad application prospects.In agricultural, although existing bacillus amyloliquefaciens has active report to the several diseases fungal pathogens, so far, bacillus amyloliquefaciens is processed biological prevention and control agent, and to be used for the report of controlling plant diseases less.Li Hongyu etc. (patent publication No. CN1952116A) disclose a kind of from soil isolating bacillus amyloliquefaciens I7 and the anthracnose of peach bacterium; The pears alternaria; The apple brown rot bacterium; Fusarium acuminatum; Fusarium oxysporum; Fusarium semitectum; The capsicum black rot; Cucumber fusarium axysporum; Tomato early blight bacterium; Eggplant early blight bacterium; Dry thread Pyrenomycetes; The root rotof flax bacterium; The julienne potatoes pyrenomycetes; The target bacterium; Phytophthora infestans; Application in the disease controls such as watermelon crystal mildew.Ji Guanghai etc. (patent publication No. CN1792167A) disclose a kind of bacillus amyloliquefaciens (preserving number CGMCC NO.1553) and the application in control konjaku bacterial soft rot thereof.Disclose a kind of bacillus amyloliquefaciens that obtains that from Taiwan lily ground soil, separates in the U.S. Pat 6960342, its biotechnological formulation of processing can be used for the control of the grape grey mould that oval grape spore causes.About research, still there is not report both at home and abroad with the concrete disease of bacillus amyloliquefaciens control wheat root.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) EA19 and preparation method thereof; And the microbial inoculum that utilizes bacillus amyloliquefaciens EA19 to process, the application of bacillus amyloliquefaciens EA19 in the wheat root disease control.
By China's typical culture collection center preservation, preserving number is CCTCC NO.M 209316, and this bacterial strain is a kind of endophyte of plant.
For solving technical problem of the present invention, the technical scheme that is provided is:
A kind of bacillus amyloliquefaciens EA19 is provided, is preserved in Chinese typical culture collection center on December 25th, 2009, the preservation address is a Wuhan City, Hubei Province Wuhan University, and preserving number is CCTCC NO:M 209316.
Described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) EA19 system from Botanical gardens, Chinese Academy of Sciences Wuhan adopt " Herba Erigerontis Annui " (Erigeron annuus (L.) Pers) plant health blade; Employing is organized grinding flat plate screening and separating method to separate and is obtained, be the endogenetic bacteria of a kind of plant.
The preparation process of bacillus amyloliquefaciens EA19 bacterial strain is following:
(1) preparation plant tissue diluent: take by weighing " Herba Erigerontis Annui " plant health blade 1 gram of harvesting, alcohol surface sterilization 30-45s, Youxiaolin sterilization 60-90s with 70% (weight percentage) put into sterilized mortar after then it being shredded; The sterilized water that adds 5 milliliters; On Bechtop, fully grind, again lapping liquid is transferred in the EP pipe, with the centrifugal 5-10 of 8000 rev/mins centrifugation rate minute; Draw out supernatant, using sterilized water that supernatant is diluted respectively is 10
-1, 10
-2, 10
-3, 10
-4, 10
-5With 10
-6Isoconcentration gradient liquid;
(2) be coated with bacterium: draw 1 milliliter of each concentration dilution liquid, add in the NA culture medium flat plate, smoothen, in 30 ℃ of thermostat containers, cultivated 24-36 hour with the sterilization cotton swab.Described NA culture medium flat plate consists of: beef extract 3 grams, and peptone 5 grams, glucose 10 grams, yeast extract 1 gram, agar 15 grams, 1000 milliliters of zero(ppm) water, PH 7.0;
(3) choose bacterium: after treating that the NA culture medium flat plate grows bacterium colony, according to form picking list bacterium colony in 12 milliliters of NA liquid nutrient mediums, 180 rev/mins, 30 ℃ constant-temperature shaking 24-36 hour;
(4) purifying: on the NA solid plate, adopt plate streak that nutrient solution is rule repeatedly, until obtaining pure culture, bacterial strain called after EA19;
(5) bacterial strain is preserved: the bacterial strain that purifying is obtained adds-20 ℃ of preservations in the test tube that fills glycerine of sterilizing, and the final concentration of glycerine is 15%.
The characteristic of bacillus amyloliquefaciens EA19 bacterial strain:
(1) morphological feature
The EA19 inoculation on the NA liquid nutrient medium, behind the shaking culture 48h, is dyeed with methylene blue staining, gram staining method (Viola crystallina acid amide staining) and spore staining method.Observe: the EA19 bacterial strain is a rod-short, Gram-positive, and peritrichous, interior production gemma forms oval, opaque oyster white bacterium colony on the NA substratum, surface irregularity, slightly depression.
(2) physiological and biochemical property
Analyze through VITEK 2 Systems Version:03.01 full automatic microorganism identification systems, get Physiology and biochemistry and measure the result, see table 1.Identify through preliminary Physiology and biochemistry: the EA19 bacterial strain is a kind of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) or subtilis (Bacillus subtilis subsp.subtilis).
The Physiology and biochemistry of table 1EA19 is measured the result
Annotate :+expression positive reaction ,-expression negative reaction
(3) Molecular Identification characteristic
(1) adopt the Auele Specific Primer pcr amplification to identify
In order to identify that further bacterial strain EA19 is bacillus amyloliquefaciens (Bacillus amyloliquefaciens); Still with the nearer subtilis subspecies (B subtilis subsp.subtilis) of bacillus amyloliquefaciens sibship; According to gene yyaR and the yyaO of the gene TetB-tetL upstream and downstream both sides of coding translocator position different in the genome of above-mentioned two kinds of bacteriums, employing Auele Specific Primer (yyaR-F/TetB-R and yyaO-F/TetB-R; Primer sequence: YyaO-F:5 ' GGAACCAGTCCACAGGGTTGTGG 3 '; YyaR-F:5 ' CGATTGAGTGGGCRAAGGAGAATCATTTWTGYGGT 3 '; TetB-R:5 ' CCATATAGAGCTGTTCCAATGGAGAAG 3 ') amplification of the method for PCR is identified; Be that primer yyaO-F/TetB-R can amplify band (stripe size possibly not wait) in the bacillus amyloliquefaciens genome, and in subtilis, can not amplify band; And primer yyaR-F/TetB-R can not amplify band in the bacillus amyloliquefaciens genome, but in subtilis, can amplify band.In view of the above, verified further that from the molecule angle EA19 bacterial strain is a kind of bacillus amyloliquefaciens, sees Fig. 1.
(2) to 16S rRNA gene sequencing
Beijing ancient cooking vessel state bio tech ltd Wuhan Company is carried out gene sequencing to the 16S rRNA of EA19; Sequencing primer is: 27bf:5 ' AGAGTTTGATCCTGGCTCAG 3 ' and 1492br:5 ' GGTTACCTTGTTACGACTT 3 '; Obtaining length is the gene fragment of 1511bp, and the result sees sequence table.16rRNA gene order contrast with this sequence and the registered bacillus amyloliquefaciens of U.S. biotechnology center NCBI (Bacillus amyloliquefaciens); The homology that obtains both is up to 99%; And be the fundamental construction phylogenetic tree with the 16rRNA gene order, see Fig. 2.With this sequence and (patent publication No. CN1952116A) disclosed bacillus amyloliquefaciens (B.amyloliquefaciens) I7 sequence alignments such as bacillus amyloliquefaciens (B.amyloliquefaciens) dhs-28 that has reported and Li Hongyu; The result shows; EA19 is a kind of new bacillus amyloliquefaciens dhs-28 that promptly is different from; Also be different from the bacillus amyloliquefaciens of bacillus amyloliquefaciens I7, see Fig. 3.
Utilize the microbial inoculum of bacillus amyloliquefaciens EA19 bacterial strain preparation.
Described microbial inoculum can be the granular bacteria preparation type, wettable powder microbial inoculum type or water microbial inoculum type.
The application of bacillus amyloliquefaciens EA19 of the present invention in the wheat diseases control, described wheat diseases is the wheat root disease, comprises root rotof flax evil, wheat root rotted leaf rot evil, take-all evil, wheat southern blight evil or wheat hypochnus evil.
The EA19 bacterial strain all has good bacteriostatic activity to root rotof flax bacterium, wheat root rotted leaf rot bacterium, gaeumannomyces graminis, wheat southern blight bacterium and rhizoctonia cerealis.
The method of use of microbial inoculum of the present invention:
Turn green the jointing stage at wheat, spread fertilizer over the fields the EA19 granular bacteria preparation, perhaps spray EA19 water microbial inoculum or wettable powder microbial inoculum in the wheat stalk base portion in wheat root.
Beneficial effect of the present invention:
Bacillus amyloliquefaciens provided by the present invention (Bacillus amyloliquefaciens) EA19 all has tangible bacteriostatic activity to root rotof flax bacterium, wheat root rotted leaf rot bacterium, gaeumannomyces graminis, wheat southern blight bacterium and rhizoctonia cerealis; Granular bacteria preparation, wettable powder microbial inoculum and the water microbial inoculum of this bacterial strain preparation can be used for the control of root rotof flax, root-rot leaf blight, gaeumannomyces graminis disease, southern blight and sheath blight fungus.Microbial inoculum of the present invention is easy to use, pollution is little, cost is low, has agricultural application prospect and good biology exploitation value widely.
Description of drawings
Fig. 1 identifies figure for adopting the Auele Specific Primer pcr amplification.The M:100bp molecular weight standard; 1 and 4: subtilis; 2 and 5: bacillus amyloliquefaciens; 3 and 6:EA19.1,2,3: primer yyaR-F/TetB-R amplified production; 4,5,6: primer yyaO-F/TetB-R amplified production.
Fig. 2 is the bacillus amyloliquefaciens EA19 phylogenetic tree that obtains according to 16S rRNA sequential analysis.
Fig. 3 compares for the 16S rRNA gene order homology of bacillus amyloliquefaciens EA19 and bacillus amyloliquefaciens (B.amyloliquefaciens) dhs-28 and bacillus amyloliquefaciens (Bacillus amyloliquefaciens) I7.Among the figure, EA19 is bacillus amyloliquefaciens EA19, and dhs-28 is for separating starch gemma bar dhs-28, and I7 is bacillus amyloliquefaciens I7.Wherein in dhs-28 and I7 gene order, "-" representes that its base with EA19 is identical; In EA19 and dhs-28 gene order, ". " represented it and compares with I7, do not have this corresponding base.In the I7 gene order, ". " represented it and compares with dhs-28 with EA19, do not have this corresponding base.
Embodiment
In order to understand the present invention better, below in conjunction with embodiments of the invention, essentiality content of the present invention is described further, but content of the present invention is not limited thereto.
The preparation of embodiment 1:EA19 bacterial strain fermentation liquor
(1) culture of seed liquid:
Scrape the bacterium liquid that takes a morsel from the bacterial classification surface of having preserved, line on NA substratum (Carnis Bovis seu Bubali cream 3 grams, peptone 10 grams, sodium-chlor 5 grams, agar 20 grams, 1000 milliliters in water, pH value 7.0) flat board was cultivated 24 hours for 30 ℃ then; Picking list bacterium colony to 2 milliliter BPY substratum (glucose 5 grams, peptone 1 gram, Carnis Bovis seu Bubali cream 5 grams; Yeast powder 5 grams, NaCl 5 grams, 1000 milliliters in water; PH value 7.0) in, 200 rev/mins, cultivated 24 hours for 30 ℃; Again in itself and BPY substratum be 10% ratio (V/V) to insert BPY substratum loadings be in 250 milliliters of triangular flasks of 80 milliliters, 200 rev/mins, cultivated 48 hours for 30 ℃.
(2) enlarge fermentation:
The seed liquor that step (1) is obtained is inoculated in the 5L automatic fermenter by the inoculative proportion of 3.5% (V/V), drops into 3.5L fermention medium (glucose 20 grams, Carnis Bovis seu Bubali cream 8 grams, L-Sodium Glutamate 5 grams, MgSO then
40.5 gram, KCl0.5 gram, KH
2PO
41 gram, FeSO
46H
20.15 milligram of O, MnSO
4H
25 milligrams of O, CuSO
45H
20.16 milligram of O, 1000 milliliters in water), fermentation condition is: 30 ℃ of temperature, 120 rev/mins, initial p H is 7.0, ferments 3 days.
The preparation of embodiment 2:EA19 wettable powder microbial inoculum
In the fermented liquid that EA19 ferments, add weighting agent, Plate Filtration, making beating, drying, pulverizing, process the wettable powder microbial inoculum, concrete making processes is following:
(1) adds weighting agent: in the storage tank of storing fermented liquid, add weighting agent; Stir 30min then; Described weighting agent is a zeyssatite, the remnant (kilogram) in diatomaceous add-on (kilogram)=(fermented liquid bacterium number (hundred million/milliliter) * hold tank volume (liter) * yield)/finished product bacterium number-fermented liquid;
(2) Plate Filtration: the material that step (1) is obtained is pressed into sheet frame with 2 kilograms/square centimeter pressure, through No. 7 filtered through gauze, and regulates institute's applied pressure at any time, makes the content bacterium in the filtered liq be no more than 0.2 hundred million/milliliter;
(3) making beating: step (2) is filtered the filter cake that obtains be discharged into the making beating jar; Dense newborn No. 100 of the tensio-active agent that adds weighting agent weight 8% then; Or the sodium lauryl sulphate of weighting agent weight 3% (SDS), add the filtrating of an amount of step (2) again, at the uniform velocity stirred 30 minutes;
(4) mixing: the bacterium zeyssatite that contains that will obtain after will pulling an oar is pulverized, and dries in the shade, and adds the water dilution of 5 times of volumes again, stirring and evenly mixing.
(5) drying: air seasoning to water cut is 5～8% in temperature is the baking room below 60 ℃.
(6) pulverize
The drop temperature of pulverizing back EA19 wettable powder microbial inoculum can not surpass 60 ℃, in case the thalline inactivation.
The determination of quality index of finished product: with reference to national enterprise's mark (Q/KWL02-2003), bacteria containing amount, water cut, suspensibility and fineness are measured, got bacteria containing amount 3.6 * 10
10CFU/g, all other index conformance with standard.
The preparation of embodiment 3:EA19 granular bacteria preparation
The fermented liquid that EA19 is fermented is processed into granular bacteria preparation according to following method:
(1) fermented liquid and lactose is even according to the mixed of 1: 4 (milliliter/gram), get mixture 1;
(2) with mixture 1, gather ethylbenzene pyrrolidone K30, tartrate and sodium hydrogencarbonate according to 12: 1: 3: 4 weight ratio is mixed, mixture 2;
(3) mixture 2 is crossed 16 mesh sieves, granulation on tablets press;
(4) the 60 ℃ of vapour-liquid oven dry of particle that step (3) obtained, and its moisture content of The real time measure make moisture controlled 3%～5%, and baking 2h packs through aseptic aluminium tinfoil paper again, promptly obtains the EA19 granule.
Through measuring, the viable bacteria total concn is 5 * 10 in this EA19 granular bacteria preparation
9CFU/g.
The preparation of embodiment 4:EA19 water microbial inoculum
The fermented liquid that EA19 is fermented is processed into the water microbial inoculum according to following method: at first above-mentioned fermented liquid is removed residue through deposition, filtration; Add stablizer styroyl Soxylat A 25-7, sodium-acetate and acetic acid then; Stir, dissolve fully to sodium-acetate, pack product.The weight ratio of said fermented liquid, styroyl Soxylat A 25-7, sodium-acetate and acetic acid is 186: 12: 1: 1.
Through measuring, the viable bacteria total concn is 5 * 10 in this EA19 water microbial inoculum
9CFU/ml.
Embodiment 5:EA19 thalline is measured the bacteriostatic activity of root rotof flax bacterium, wheat root rotted leaf rot bacterium, gaeumannomyces graminis, wheat southern blight bacterium and rhizoctonia cerealis
TP: with the EA19 bacterial strain with NA liquid nutrient medium shaking culture 5 days in 30 ℃ of thermostat containers; After centrifugal, get bacterial sediment and be resuspended on the saline water, get 1 milliliter of suspension-s then; Use the sterilization toothpick evenly to be applied on the NA solid plate and connect the confession examination germ bacterium cake of diameter as 6mm in dull and stereotyped central authorities; Cultivate after 3～5 days for 26 ℃, the growth diameter with right-angled intersection method measurement bacterium colony calculates bacteriostatic activity.
Described confession examination germ is any in root rotof flax bacterium (Fusarium spp.), wheat root rotted leaf rot bacterium (Biqolarls sorokinlana), gaeumannomyces graminis (Gaeumannomyces graminis (Sacc.) Arx et Oliver var.tritici (Sacc.) Walker), wheat southern blight bacterium (Sclerotium rolfsii) and the rhizoctonia cerealis (Rhizoctonia cerealis Vander Hoeven).
Do not use the EA19 bacterial strain in the controlled trial on the NA liquid nutrient medium.
Suppress active=(contrast diameter-processing diameter)/(contrast diameter-bacterium cake diameter) * 100%
In this formula, the contrast diameter is to supply examination pathogen growth diameter in the controlled trial, and handling diameter is to supply examination pathogen growth diameter in the Processing Test, and bacterium cake diameter is for supplying the colony diameter of examination germ, i.e. 6mm.
Table 2EA19 thalline is to the bacteriostatic activity of 5 kinds of pathogenic bacterias
The result shows: the EA19 thalline all has good bacteriostatic activity to root rotof flax bacterium, wheat root rotted leaf rot, gaeumannomyces graminis, wheat southern blight bacterium and rhizoctonia cerealis.
Among the following embodiment 6-8,
Diseased plant rate (the %)=morbidity strain number/total strain number of investigation * 100
Disease index=∑ (diseased plant numbers at different levels * this rank is represented numerical value)/(total strain number * highest level typical value) * 100
Preventive effect=(blank test disease index-Processing Test disease index)/blank test disease index * 100
Embodiment 6: pot-culture method is measured the efficiency test of EA19 water microbial inoculum to the wheat southern blight
TP: with wheat vernalization 2 days under indoor 17 ℃ condition; The seed that obtains after the vernalization is soaked 12h in 10 times of diluents of EA19 water microbial inoculum, to be sowed at the bore of sterilizing and being rich in detritus be in the flowerpot of 30cm to pendulum then, 15 in every alms bowl; Sprinkle one deck previously prepd wheat southern blight germ bacterium cake then on the surface; Every alms bowl 100g covers sterile soil, places 18 ± 1 ℃ interior the cultivation 50 days of illumination box.
Replace EA19 water microbial inoculum to soak chitting piece with sterilized water in the blank test.
More than test all repeats 3 times.
Result's investigation: all stem and leaf of Wheat are washed root, and investigation morbidity strain number and severity calculate diseased plant rate, disease index then, preventive effect and average preventive effect, and the result sees table 3.
Table 3 pot-culture method is measured the efficiency test of EA19 water microbial inoculum to the wheat southern blight
The result shows: after EA19 water microbial inoculum soaked the wheat germination seed, the diseased plant rate of wheat southern blight obviously reduced, and prevention rate can reach 56.6%.
Embodiment 7:EA19 granular bacteria preparation is to the field efficiency test of root rotof flax, gaeumannomyces graminis disease and hypochnus
TP: at 10m
2The wheat paddock district, dial the joint phase turning green, in wheat clump base portion, spread fertilizer over the fields or spray medicament, spread fertilizer over the fields again after 14 days or spray medicament 1 time, each test repetition 3 times.
Test is provided with: specifically test institute's with medicament, dosage and insecticide-applying way and see table 4.
Table 4 test institute with medicament, dosage and insecticide-applying way
Result investigation: press the pesticide field efficacy medicine test criterion, after spreading fertilizer over the fields agricultural chemicals the 2nd time, state of an illness result was investigated in the 14th day, calculate disease index, preventive effect and average preventive effect, the result sees table 5.
The field efficiency test of table 5EA19 granular bacteria preparation root rotof flax, gaeumannomyces graminis disease and hypochnus
The result shows: the EA19 granular bacteria preparation is respectively 76.3%, 63.6% and 72.9% to root rotof flax, gaeumannomyces graminis disease and hypochnus preventive effect, wherein to the preventive effect of take-all apparently higher than contrast medicament trazodone wettable powder.
Embodiment 8:EA19 wettable powder microbial inoculum is to root rotof flax, gaeumannomyces graminis disease, the field efficiency test of hypochnus
TP: identical with embodiment 7.
Test is provided with: specifically test institute's with medicament, dosage and insecticide-applying way and see table 6.
Result's investigation: press the pesticide field efficacy medicine test criterion, behind the 2nd spraying pesticide, carried out state of an illness investigation on the 14th day, calculate disease index, preventive effect and average preventive effect, the result sees table 7.
Table 6 test institute with medicament, dosage and insecticide-applying way
Table 7EA19 wettable powder microbial inoculum is to the field efficiency test of root rotof flax, gaeumannomyces graminis disease and hypochnus
The result shows: EA19 wettable powder microbial inoculum has 71.4%, 63.8% and 62.5% preventive effect respectively to root rotof flax, gaeumannomyces graminis disease and hypochnus, wherein to the preventive effect of take-all apparently higher than contrast medicament trazodone wettable powder.
The sequence table of bacillus amyloliquefaciens EA19
< 110>Hubei Academy of Agricultural Science, Plant Protection and Soil Fertilizer Inst
< 120>the bacillus amyloliquefaciens strain EA19 and the preparation thereof of control wheat root disease
<160>1511
<210>1
<211>1511bp
<212>RNA
< 213>bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
<400>1
AGAGTTTGAT?CCTGGCTCAG?GACGAACGCT?GGCGGCGTGC?CTAATACATG?CAAGTCGAGC 60
GGACAGATGG?GAGCTTGCTC?CCTGATGTTA?GCGGCGGACG?GGTGAGTAAC?ACGTGGGTAA 120
CCTGCCTGTA?AGACTGGGAT?AACTCCGGGA?AACCGGGGCT?AATACCGGAT?GGTTGTCTGA 180
ACCGCATGGT?TCAGACATAA?AAGGTGGCTT?CGGCTACCAC?TTACAGATGG?ACCCGCGGCG 240
CATTAGCTAG?TTGGTGAGGT?AACGGCTCAC?CAAGGCGACG?ATGCGTAGCC?GACCTGAGAG 300
GGTGATCGGC?CACACTGGGA?CTGAGACACG?GCCCAGACTC?CTACGGGAGG?CAGCAGTAGG 360
GAATCTTCCG?CAATGGACGA?AAGTCTGACG?GAGCAACGCC?GCGTGAGTGA?TGAAGGTTTT 420
CGGATCGTAA?AGCTCTGTTG?TTAGGGAAGA?ACAAGTGCCG?TTCAAATAGG?GCGGCACCTT 480
GACGGTACCT?AACCAGAAAG?CCACGGCTAA?CTACGTGCCA?GCAGCCGCGG?TAATACGTAG 540
GTGGCAAGCG?TTGTCCGGAA?TTATTGGGCG?TAAAGGGCTC?GCAGGCGGTT?TCTTAAGTCT 600
GATGTGAAAG?CCCCCGGCTC?AACCGGGGAG?GGTCATTGGA?AACTGGGGAA?CTTGAGTGCA 660
GAAGAGGAGA?GTGGAATTCC?ACGTGTAGCG?GTGAAATGCG?TAGAGATGTG?GAGGAACACC 720
AGTGGCGAAG?GCGACTCTCT?GGTCTGTAAC?TGACGCTGAG?GAGCGAAAGC?GTGGGGAGCG 780
AACAGGATTA?GATACCCTGG?TAGTCCACGC?CGTAAACGAT?GAGTGCTAAG?TGTTAGGGGG 840
TTTCCGCCCC?TTAGTGCTGC?AGCTAACGCA?TTAAGCACTC?CGCCTGGGGA?GTACGGTCGC 900
AAGACTGAAA?CTCAAAGGAA?TTGACGGGGG?CCCGCACAAG?CGGTGGAGCA?TGTGGTTTAA 960
TTCGAAGCAA?CGCGAAGAAC?CTTACCAGGT?CTTGACATCC?TCTGACAATC?CTAGAGATAG 1020
GACGTCCCCT?TCGGGGGCAG?AGTGACAGGT?GGTGCATGGT?TGTCGTCAGC?TCGTGTCGTG 1080
AGATGTTGGG?TTAAGTCCCG?CAACGAGCGC?AACCCTTGAT?CTTAGTTGCC?AGCATTCAGT 1140
TGGGCACTCT?AAGGTGACTG?CCGGTGACAA?ACCGGAGGAA?GGTGGGGATG?ACGTCAAATC 1200
ATCATGCCCC?TTATGACCTG?GGCTACACAC?GTGCTACAAT?GGACAGAACA?AAGGGCAGCG 1260
AAACCGCGAG?GTTAAGCCAA?TCCCACAAAT?CTGTTCTCAG?TTCGGATCGC?AGTCTGCAAC 1320
TCGACTGCGT?GAAGCTGGAA?TCGCTAGTAA?TCGCGGATCA?GCATGCCGCG?GTGAATACGT 1380
TCCCGGGCCT?TGTACACACC?GCCCGTCACA?CCACGAGAGT?TTGTAACACC?CGAAGTCGGT 1440
GAGGTAACCT?TTATGGAGCC?AGCCGCCGAA?GGTGGGACAG?ATGATTGGGG?TGAAGTCGTA 1500
ACAAGGTAAC?C 1511
Claims (4)
1. a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) EA19 has been preserved in Chinese typical culture collection center, and deposit number is CCTCC NO.M 209316.
2. microbial inoculum that bacillus amyloliquefaciens EA19 according to claim 1 processes.
3. microbial inoculum according to claim 2 is characterized in that: described microbial inoculum is granular bacteria preparation type, wettable powder microbial inoculum type or water microbial inoculum type.
4. the application of bacillus amyloliquefaciens EA19 according to claim 1 in the wheat diseases control; It is characterized in that described wheat diseases is the wheat root disease, described wheat root disease is root rotof flax evil, wheat root rotted leaf rot evil, take-all evil, wheat southern blight evil or wheat hypochnus evil.
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| CN103159570A (en) * | 2011-12-12 | 2013-06-19 | 河北农业大学 | Solid microbial fertilizer and preparation method thereof |
| CN102776146B (en) * | 2012-08-19 | 2013-09-11 | 南京农业大学 | Bacillus amyloliquefaciens and application thereof |
| CN103173397B (en) * | 2013-04-02 | 2015-02-04 | 山东省农业科学院农产品研究所 | Broad-spectrum antibacterial bacillus amyloliquefaciens strain and application thereof |
| CN104164382B (en) * | 2014-06-05 | 2016-06-15 | 广东省农业科学院农业资源与环境研究所 | Biocontrol bacterial strain G58 and bacterial preparation process and the application of control fungal diseases of plants |
| CN104195072A (en) * | 2014-08-06 | 2014-12-10 | 江苏农林职业技术学院 | Bacillus amyloliquefaciens subsp.plantarum B232 and application thereof |
| CN105340883B (en) * | 2015-11-26 | 2018-01-30 | 贵州大学 | A kind of aspergillus niger micropill |
| CN105316266A (en) * | 2015-12-08 | 2016-02-10 | 领先生物农业股份有限公司 | Bacillus amyloliquefaciens LX-J1 and application thereof |
| CN106591185B (en) * | 2016-12-13 | 2020-09-29 | 山东农业大学 | Bacillus amyloliquefaciens plant subspecies and preparation and application of microbial inoculum thereof |
| CN107347919A (en) * | 2017-08-14 | 2017-11-17 | 南阳市农业科学院 | A kind of composite bactericide for preventing and treating wheat scab |
| CN110791445B (en) * | 2019-10-29 | 2022-09-02 | 西北农林科技大学 | Bacillus amyloliquefaciens and biological pesticide fertilizer |
| CN110896966A (en) * | 2019-12-19 | 2020-03-24 | 河南农贝得农业科技有限公司 | Microbial agent for crop take-all and preparation method thereof |
| CN114806966B (en) * | 2022-05-20 | 2023-02-28 | 河北农业大学 | Pesticide-resistant Kulas-resistant wheat holoetch disease biological control bacillus subtilis and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1952116A (en) * | 2006-04-18 | 2007-04-25 | 兰州大学 | Bacillusamyloliquefaciens strain and application thereof |
| CN1972167A (en) * | 2005-11-23 | 2007-05-30 | 华为技术有限公司 | Point-to-multipoint access network and method to enhance security and branch/coupler |
| CN101423812A (en) * | 2008-12-17 | 2009-05-06 | 河南省农业科学院 | Bacillus amyloliquefaciens and microbiological preparation and preparation method thereof |
-
2009
- 2009-12-31 CN CN2009102734988A patent/CN101967455B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1972167A (en) * | 2005-11-23 | 2007-05-30 | 华为技术有限公司 | Point-to-multipoint access network and method to enhance security and branch/coupler |
| CN1952116A (en) * | 2006-04-18 | 2007-04-25 | 兰州大学 | Bacillusamyloliquefaciens strain and application thereof |
| CN101423812A (en) * | 2008-12-17 | 2009-05-06 | 河南省农业科学院 | Bacillus amyloliquefaciens and microbiological preparation and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 张淑梅 等.大豆内生细菌的分离及根腐病拮抗菌的筛选鉴定.《微生物学通报》.2008,第35卷(第10期),1593-1599. * |
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