CN104988098A - Bacillus stain for prevention and control of sugarbeet root rot and promotion of sugarbeet growth - Google Patents
Bacillus stain for prevention and control of sugarbeet root rot and promotion of sugarbeet growth Download PDFInfo
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Abstract
The invention discloses a bacillus stain for prevention and control of sugarbeet root rot and promotion of sugarbeet growth. The bacillus stain for prevention and control of sugarbeet root rot and promotion of sugarbeet growth is bacillus cereus LHTYBA, and has the preservation number of CGMCC No.7566 in China General Microbiological Culture Collection Center. Field experiments prove that the bacillus cereus LHTYBA enables the sugarbeet root rot prevention and control effect to reach 86.7%, and can make sugarbeet production to be increased by 46.00%. The bacillus cereus LHTYBA provided by the invention can perform colonization in rhizosphere of crops, has a remarkable effect for use in increasing sugarbeet production, and has broad prospects in application of production of microbial agents and biological organic fertilizers.
Description
Technical field
The present invention relates to a strain and prevent and treat root rot of beets and the Bacillus strain promoting Sugarbeet Growth.
Background technology
Root rot of beets is that the upper serious plant disease occurred produced by beet, by multiple fungus and bacterium respectively or mixed infection cause.Root rot of beets all has generation in China's beet main producing region, and the morbidity of continuous cropping plot is heavier, and the general underproduction 10% ~ 40% seriously can cause total crop failure.The Prominent pathogen of China's root rot of beets is sickle-like bacteria (Fusarium), is secondly rhizoctonia (Rhizoctonia) pathogenic bacterium.Domestic and international research shows, the Breeding and application of disease-resistant variety is the most effective way of preventing and treating root rot of beets, when China there is no at present high resistance root rot of beets kind can for production application, biological control and chemical prevention are all the effective technology measures controlling root rot of beets occurrence and harm, domestic chemical prevention mainly applies thiram, hymexazol etc., and biological control has nontoxic, free of contamination feature, receives extensive attention.
Gemma Pseudomonas (Bacillus sp.) is the important component part of Plant diseases Biocontrol microorganism, there is significant Biocontrol Potential, the gemma that heat-resistant is inverse can be produced, be beneficial to the production of biocontrol fungicide, formulation and survive in the environment, surely grow and breeding.Stability, with the consistency of chemical pesticide etc. in, be obviously better than non-genus bacillus and fungi biocontrol fungicide, genus bacillus has one to participate in several mechanism in the process of antagonism pathogenic bacteria.Wherein, mainly comprise antagonistic action, Competition, inducing plant obtains system resistant, Promoting plant growth etc.Antimicrobial substance mainly comprises microbiotic or bacteriocin class, antibacterial peptide class and generation chitinase.
Although chemical pesticide is still occupied an leading position in controlling plant diseases at present, but genus bacillus sterilant has very strong antagonistic action to various plants pathogenic bacteria, there is environment compatibility good, strong stress resistance, to person poultry safety, the advantage such as not easily develop immunity to drugs, and more meets the demand of modern society to agriculture production and integrated pest management.Genus bacillus not only can be used alone, can also with chemical pesticide or botanical fungicide composite, realize have complementary advantages, reduce consumption, strengthen preventive effect, also can be used in combination with other Antagonistic Fungis, realize multiple antibiotic bacteria to have complementary functions, anti-multiple diseases of holding concurrently, the collaborative prevention effect of persistent.Therefore, genus bacillus as biocontrol fungicide, the development potentiality larger than showing with other microbials and more wide application prospect.
Summary of the invention
Technical problem to be solved by this invention is to provide a strain and can be used for preventing and treating root rot of beets and the genus bacillus can improving beet root output.
Genus bacillus provided by the present invention is bacillus cereus (Bacillus cereus) LHTYBA, and it is CGMCC No.7566 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.This bacterial strain is called for short LHTYBA in this application.
Microbial inoculum containing described bacillus cereus (Bacillus cereus) LHTYBA also belongs to protection scope of the present invention.
Described microbial inoculum can be following 1)-3) in arbitrary microbial inoculum:
1) for preventing and treating the microbial inoculum of root rot of beets and raising Sugarbeet Yield;
2) for preventing and treating the microbial inoculum of root rot of beets;
3) for improving the microbial inoculum of Sugarbeet Yield.
Described bacillus cereus (Bacillus cereus) LHTYBA is in preparation following 1)-3) in application in arbitrary microbial inoculum also belong to protection scope of the present invention:
1) for preventing and treating the microbial inoculum of root rot of beets and raising Sugarbeet Yield;
2) for preventing and treating the microbial inoculum of root rot of beets;
3) for improving the microbial inoculum of Sugarbeet Yield.
Biological organic fertilizer containing described bacillus cereus (Bacillus cereus) LHTYBA or described microbial inoculum also belongs to protection scope of the present invention.
Wherein, the active ingredient of described microbial inoculum can be described bacillus cereus (Bacillus cereus) LHTYBA, still can contain other materials, as the microbial inoculum bacterial strain that other are prevented and treated root rot of beets and/or improve Sugarbeet Yield.Described microbial inoculum also can comprise carrier.Described carrier can be solid carrier or liquid vehicle.Described solid carrier is mineral material, biomaterial or macromolecular compound; Described mineral material is at least one in clay, talcum, kaolin, montmorillonite, white carbon, zeolite, silica, turfy soil and diatomite; Described biomaterial is at least one in the ight soil of the stalk of all kinds of crop, loose shell, straw, Pericarppium arachidis hypogaeae, Semen Maydis powder, bean powder, starch, the peat composed of rotten mosses and animal; Described macromolecular compound is polyvinyl alcohol and/or polyglycol.Described liquid vehicle can be organic solvent, vegetables oil, mineral oil or water; Described organic solvent is decane and/or dodecane.In described microbial inoculum, described bacillus cereus (Bacillus cereus) LHTYBA can with by cultivate viable cell, the fermented liquid of viable cell, the filtrate of cell culture or cell and filtrate the form of mixture exist.The formulation of described microbial inoculum can be multiple formulation, as liquor, emulsion, suspension agent, pulvis, granule, wettable powder or water dispersible granules.
The method of the method and the described microbial inoculum of preparation of cultivating described bacillus cereus (Bacillus cereus) LHTYBA also belongs to protection scope of the present invention.
The method of the described bacillus cereus of cultivation provided by the present invention (Bacillus cereus) LHTYBA, comprises described bacillus cereus (Bacillus cereus) LHTYBA for cultivating the step of cultivating in the substratum of genus bacillus.
The preparation method of described microbial inoculum provided by the present invention, comprises the steps: described bacillus cereus (Bacillus cereus) LHTYBA as activeconstituents, to obtain described microbial inoculum.
Described bacillus cereus (Bacillus cereus) LHTYBA or the application of described microbial inoculum in cultivated beet also belong to protection scope of the present invention.
The method of described bacillus cereus (Bacillus cereus) LHTYBA cultivated beet is utilized also to belong to protection scope of the present invention.
The method of cultivated beet provided by the present invention, comprises and apply described bacillus cereus (Bacillus cereus) LHTYBA or described microbial inoculum in the soil of cultivated beet.
In the method for above-mentioned cultivated beet, the consumption of described bacillus cereus (Bacillus cereus) LHTYBA or described microbial inoculum is every mu and applies 10
11cfu-10
12cfu is (as 5 × 10
11cfu) described bacillus cereus (Bacilluscereus) LHTYBA.
Field experiment proves, bacillus cereus (Bacillus cereus) LHTYBA can reach 86.7% to the prevention effect of root rot of beets, can make beet production-increasing 46.00%.Bacillus cereus provided by the present invention (Bacilluscereus) LHTYBA, can grow surely at crop rhizosphere, for beet production obvious effect of increasing production, in microbiobacterial agent and biological organic fertilizer production application, has bright prospects.
preservation explanation
Strain name: bacillus cereus
Latin name: (Bacillus cereus)
Strain number: LHTYBA
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on May 7th, 2013
Register on the books numbering in preservation center: CGMCC No.7566
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Root rot of beets bacterium-dry thread Pyrenomycetes (Rhizoctonia solani) (preliminary study of China root rot of beets cause of disease dry thread Pyrenomycetes in following embodiment, Chinese Plants pathology meeting Annual Conference collection of thesis in 2011) provided by plant pathology system of China Agricultural University Wu Xuehong professor laboratory, the public can obtain from China Agricultural University, this biomaterial related experiment of the present invention of only attaching most importance to again is used, not can be used as other purposes and uses.
Bacillus thuringiensis (Bacillus thuringiensis) bacterial strain LHTXYB in following embodiment (being called for short LHTXYB) (Du Juan. the screening of root rot of beets biocontrol bacteria and mechanism of action pre-test. China Agricultural University master thesis .2013 June) public can obtain from China Agricultural University, this biomaterial related experiment of the present invention of only attaching most importance to again is used, not can be used as other purposes and uses.
Substratum in following embodiment is as follows:
LB liquid nutrient medium: 10g Tryptone (Oxoid Products), 10g NaCl, 5g Yeast Extract (Oxoid Products), be settled to 1L with water, pH 7.2 ~ 7.4, autoclaving 20min.
LB solid medium: add 15g agar powder in often liter of LB liquid nutrient medium, autoclaving 20min.
Extractum carnis solid medium (NA): soy peptone 7g, NaCl 5g, extractum carnis 3g, agar 20g, is settled to 1L with water, pH 7.2 ~ 7.4, autoclaving 20min.
Extractum carnis liquid nutrient medium: soy peptone 7g, NaCl 5g, extractum carnis 3g, is settled to 1L with water, pH 7.2 ~ 7.4, autoclaving 20min.
PDA substratum: potato 200g, glucose 20g, 1L H
2o, agar 16g, autoclaving 20min.
In following embodiment, root rot of beets is investigated in units of whole strain, and disease scale standard is as follows:
0 grade: block root growth is normal, intact;
L level: block tail and root table tissue have Minimal change, pathology is not yet invaded and root in-vivo tissue;
2 grades: root tail and root table tissue have obvious pathology, and vascular bundle is brown, but not yet lignifying;
3 grades: vascular bundle is Vandyke brown, conduit lignifying, fertility is seriously obstructed, and old complaint has part early to start to decay, and decayed portion accounts for less than 10% of block root;
4 grades: decayed portion accounts for 10% ~ 30% (not comprising 10%) of block root;
5 grades: block root rot part account for more than 30% (not comprising 30%) of block root or complete stool due to illness withered.
Disease index=[Σ (old complaint number × corresponding value of series)/(investigating total radical × superlative degree)] × 100
Prevention effect (%)=[(check plot disease index-treatment zone disease index)/check plot disease index] × 100
The isolation identification of embodiment 1, bacillus cereus (Bacillus cereus) LHTYBA CGMCC No.7566
1, sample collecting: collected specimens is in beet tails and rhizosphere soil, and place is Laune R. city of Heilongjiang Province.
2, separation method: soil most of on beet root is shaken off, get with hairbrush the rhizosphere soil that 1g sticks to beet surface and put into the sterilized test tube that 9mL sterilized water is housed, abundant vibration 2min, gets in the test tube that 1mL diluent adds containing 9mL sterilized water, mixes rear gradient dilution to 10
-5concentration, gets 10 respectively
-3, 10
-4with 10
-5the diluent 100uL painting extractum carnis of concentration is dull and stereotyped, TSA is dull and stereotyped and PDA is dull and stereotyped, and cultivate after 3 days at 30 DEG C, single bacterium colony that picking is different, carries out tube after purifying and be stored in 4 DEG C.
3. the biological activity determination of biocontrol microorganisms and biological and ecological methods to prevent plant disease, pests, and erosion related substances detect:
The dull and stereotyped antagonistic experiment of 3.1 biocontrol microorganisms
PDA flat board activates, using root rot of beets bacterium as target bacterium, on pine root fungus flat board, breaks into the bacterium cake of 5mm with the punch tool of 5mm.The antagonistic effect of isolated strains is detected by flat board face-off method, at the root rot of beets bacterium bacterium cake that PDA plate center inoculation diameter is 5mm, test strains is inoculated in apart from target bacterium cake 3cm place, cultivate 4 ~ 5 days in 28 DEG C of thermostat containers, treat that blank (does not inoculate test strains, only inoculate target bacterium) when covering with whole culture dish, the contrast increment of measurement target drone bacterium (representing with the colony radius of target bacterium in the flat board not inoculating test strains) and process increment (representing with the colony radius of target bacterium in the flat board inoculating test strains), represent with inhibiting rate, select the obvious bacterium of antagonistic effect and carry out growth-promoting test and greenhouse diseases prevention test.Each process three repetition, each repetition 3 flat boards.
Inhibiting rate (%)=(contrast increment-process increment)/contrast increment × 100%
The detection of 3.2 biological and ecological methods to prevent plant disease, pests, and erosion related substanceses
3.2.1 the detection of proteolytic enzyme
By the test strains percutaneous puncture-inoculation of activation on 1% skimmed milk agar plate, 30 DEG C cultivate 24,48, observe the generation of the peripheral transparent circle of bacterium colony after 72h, occur that transparent circle shows to have the generation of proteolytic enzyme.Each process three repetition (Schwyn et al., 1987).
3.2.2 the detection of chitinase
Chitinase tobacco brown spot pathogen substratum detects, the test strains of activation being inoculated in chitinase detects on culture medium flat plate, 30 DEG C cultivate 24,48, observe the production of the peripheral transparent circle of bacterium colony after 72h, if there is transparent circle, show the generation having chitinase, each process three repetition (Reimmann et al., 1997).
3.2.3 dextranase detects
Test strains is inoculated in containing ABP substratum (ABP substratum: with mortar, block Poria cocos is ground powdering, make it by 120 object sieves, get KH
2pO4 6.8g, K
2hPO412H
2o 17.9g, yeast extract 6.7g, Poria powder or β-1,3-dextran 5.0g, aniline blue 120mg, agar powder 12g, is settled to 1L with water, pH value is 6.8) flat board in, cultivate 48, after 72h for 30 DEG C, observe in flat board and whether occur clearing up circle, clear up circle if having and produce, show the generation having dextranase, each process three repetition (Cattelan et al., 1999).
3.2.4 detect addicted to iron element
Detect addicted to iron element CAS substratum, the bacterium to be measured of activation being inoculated in CAS detects on culture medium flat plate, after 30 DEG C of cultivation 72h, observe in flat board and whether produce safran haloing, if there is safran, show there is the generation addicted to iron element, each process three repetition (Schwyn, Neilands, 1987; Machuca et al., 2003).
Result shows that the antimicrobial spectrum being numbered the bacterial strain of LHTYBA filtered out is as shown in table 1, shows that bacterial strain LHTYBA Rhizoctonia solani and Fusarium oxysporum all have restraining effect, this bacterial strain is carried out the greenhouse biocontrol effect experiment of step 4 as biocontrol microorganisms.
The antimicrobial spectrum of table 1 biocontrol microorganisms
Note: the experimental data in table is three mean values repeated; "+" indicates that corresponding material produces, and "-" indicates and to produce without corresponding material.
4, the greenhouse biocontrol effect experiment of Biocontrol Strain
Bacterial strain LHTYBA, with bacterial strain LHTXYB for contrast, together carries out following experiment.
The preparation of biocontrol microorganisms bacterium liquid: be individually seeded to by bacterial strain LHTYBA and LHTXYB in 30 DEG C in LB liquid nutrient medium, 160rpm cultivates 60 hours, treat that gemma growth is neat, 3000rpm collected by centrifugation thalline, adds sterilized water and makes bacterium liquid.With blood counting chamber counting, calculate concentration.4 DEG C temporary for subsequent use.
The preparation of dry thread Pyrenomycetes wheat culture: add 100g wheat in 500ml triangular flask, then pour 200mL deionized water into, twice sterilizing after mixing, 121 DEG C, 20min.Get the root rot of beets bacterium-dry thread Pyrenomycetes pure culture biscuits involvng inoculation of 10 5mm in triangular flask, 30 DEG C of constant temperature culture two weeks, vibrate three times, obtain dry thread Pyrenomycetes wheat culture every days.
Three kinds of process are established in experiment: biocontrol microorganisms LHTYBA process, biocontrol microorganisms LHTXYB process and contrast.Specific experiment method is as follows: beet sweet list 302 (incubation of beet institute of the Chinese Academy of Agricultural Sciences) seed-coat sterilization 6min, sterile water wash 4 times, distilled water immersion 24h vernalization, is divided into three components and is not handled as follows: (concentration is 1.0 × 10 to biocontrol microorganisms LHTYBA process LHTYBA bacterium liquid
8cfu/ml) soak seed 6min, (concentration is 1.0 × 10 to biocontrol microorganisms LHTXYB process LHTXYB bacterium liquid
8cfu/ml) soak seed 6min, contrast tap water soaks 6min; A kind of 20-30 seed of process sowed by every basin; Water, unified beet quantity after one week, 8, every basin; Be handled as follows respectively after one week: (concentration is 1.0 × 10 to biocontrol microorganisms LHTYBA process LHTYBA bacterium liquid
8cfu/ml) fill with root, (concentration is 1.0 × 10 to every basin 20ml, biocontrol microorganisms LHTXYB process LHTXYB bacterium liquid
8cfu/ml) fill with the every basin 20ml of root, contrast clear water fills with root, every basin 20ml.Two days later each process every basin inoculation dry thread Pyrenomycetes (dry thread Pyrenomycetes wheat culture dries, after pulverizing and sterilized soil mix by the volume ratio of 1:10, water the moisturizing of 15ml tap water after surface coverage one deck).Three repetitions are established in a process, each repetition 3 basin.Cultivate and all investigate dead seedling number afterwards in 9 days, calculate death rate according to dead seedling number/experiment seedling number.Result shows, only inoculation dry thread Pyrenomycetes does not inoculate the death rate average out to 84.7% of the contrast of biocontrol microorganisms, the death rate average out to 12.5% of the biocontrol microorganisms LHTYBA process of inoculating strain LHTYBA bacterium liquid and dry thread Pyrenomycetes, the death rate average out to 29.1% of the biocontrol microorganisms LHTXYB process of inoculating strain LHTXYB bacterium liquid and dry thread Pyrenomycetes.Illustrate bacterial strain LHTYBA and LHTXYB Rhizoctonia solani inhibited, the restraining effect of bacterial strain LHTYBA Rhizoctonia solani is 2.3 times of LHTXYB.
5, the taxonomic identification of bacterial strain LHTYBA
5.1 Biolog qualifications
Biolog microbial identification system, mainly according to the utilization power of microorganism to different carbon source, detects microorganism cells and utilizes different carbon source to carry out the different substances produced in metabolic processes, carry out taxonomic identification fast and accurately to microorganism.
BUG is dull and stereotyped: 57g BUG nutrient agar water is settled to 1L, boils dissolving, adjusted to ph to 7.3 (25 DEG C) after cooling, and 121 DEG C of sterilizings 15 minutes, are cooled to 45-50 DEG C, are down flat plate.
Full automatic microorganism assessing instrument: biolog company of the U.S..
(1) single bacterium colony activates on BUG flat board
(2) aseptic cotton carrier dips in wet rear gluing and gets thalline, does not bring substratum.
(3) cotton swab contact tube wall rotates, and makes thalline stick to nutrient solution upper inner wall.
(4) by inoculation liquid, thalline is swept away from tube wall, dispersion.
(5) turbidity is adjusted.
(6) pour cultured culture into loading slot, draw with eight road electrical pipette rifles and join in 96 orifice plates, every aperture 150 μ L, cultivates 16 ~ 24h under 30 DEG C of conditions.
(7) read the Strain comparison in result and database, determine classification position (Cheng Chi etc., 2006).
Readout instrument is different to the utilization power of carbon source according to bacterium, produces different meta-bolitess and causes distinct colors reaction in identification plate, by the comparison with other bacterium in database, classify and identify (Cheng Chi etc., 2006) bacterium.
Result shows, LHTYBA can utilize dextrin, D-Maltose, D-trehalose, D-cellobiose, N-ACETYL-D-GLUCOSAMINE, D-Glucose, Trophicardyl/inosine, Serine, glycerine, D-Fructose-6-phosphoric acid, gelatin, ASPARTIC ACID, L-Histidine Pidolidone, L-silk ammonia, gluconic acid, Pyruvic Acid Methyl ester, Pfansteihl, citric acid, a-ketoglutaric acid, acetic acid, L MALIC ACID, ethyl acetate, formic acid, can not utilize ammonium sulfate, urea, D-MANNOSE, wood sugar, sorbose, lactose and N.F,USP MANNITOL.
Bacterial strain LHTYBA is accredited as bacillus cereus (Bacillus cereus), SIM value be 0.576.
The morphological specificity of 5.2 bacterial strain LHTYBA
The bacterial strain LHTYBA being in logarithmic phase is adopted after smear staining the form of observation by light microscope thalline.Result shows that somatic cells is shaft-like, end side, becomes short or long-chain, 1.0 ~ 1.2 × 3.0 ~ 5.0 microns, produces gemma, gemma circle or cylindricality, and middle life or near middle raw, 1.0 ~ 1.5 microns, sporangiocyst is without obviously expanding.Gram-positive, without pod membrane, motion.
The physiological and biochemical property of 5.2 bacterial strain LHTYBA
Carry out identification experiment according to " BergeyShi determinative bacteriology handbook " the 8th edition, result shows that bacterial strain LHTYBA bacterium colony on peptone yeast extract paste flat board is canescence, and opaque, surface is more coarse, and like ground-glass appearance or wax melting shape, bacterium colony is larger.Growth temperature range 20 ~ 45 DEG C, less than 10 DEG C poor growths or do not grow.In meat soup, grow muddiness have mycoderm or wall ring, the easy emulsification of jolting.The bacterium colony that plain agar generates is comparatively large, diameter 3-10mm, canescence, opaque, and surface irregularity is like ground-glass appearance or wax melting shape, and edge is often in expansion shape.Occasionally have and produce yellow-green colour pigment, in grass green haemolysis on blood agar plate.
According to the above results, bacterial strain LHTYBA is accredited as bacillus cereus (Bacillus cereus).Bacillus cereus (Bacillus cereus) LHTYBA is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 7th, 2013 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), deposit number is CGMCC No.7566.
Embodiment 2, bacillus cereus (Bacillus cereus) LHTYBA CGMCC No.7566 improve the field experiment of beet root output
By bacillus cereus (Bacillus cereus) LHTYBA CGMCC No.7566 (being called for short LHTYBA), with bacterial strain LHTXYB for contrast, together carry out following experiment.
One, the preparation of liquid bacterial agent
By bacterial strain LHTYBA and LHTXYB individually streak inoculation cultivate after 24h in extractum carnis solid medium 30 DEG C, inoculate a ring in extractum carnis liquid nutrient medium (500ml triangular flask 50ml loading amount), after 30 DEG C of 180rpm shake training 18h, be inoculated in extractum carnis liquid nutrient medium (500ml triangular flask 100ml loading amount) with 3% inoculum size, 30 DEG C of 180rpm shake training 48h, namely obtain LHTYBA fermented liquid and LHTXYB fermented liquid.This LHTYBA fermented liquid is LHTYBA liquid bacterial agent, and the content of the LHTYBA in LHTYBA liquid bacterial agent is 10
9cfu/mL.This LHTXYB fermented liquid is LHTXYB liquid bacterial agent, and the content of the LHTXYB in LHTXYB liquid bacterial agent is 10
9cfu/mL.
Two, right front Qi Nanying village field experiment is examined in Wulanchabu League of Inner Mongolia city
In May, 2012, with sweet list 302 (incubation of beet institute of the Chinese Academy of Agricultural Sciences) for supplying examination beet variety, before right front Qi Nanying village is examined in Wulanchabu League of Inner Mongolia city, stubble is that field experiment is carried out in the plot of beet: experiment adopts randomized block design, three iterons are set, each iteron arranges three communities at random, is respectively CK treatment zone, LHTYBA treatment zone, LHTXYB treatment zone.The area of each community is 30m
2.Each community adopted medicament to carry out first time afterwards in beet transplanting and filled with root the same day, the every young plant in LHTYBA treatment zone fills with root 50ml LHTYBA liquid bacterial agent 10 times of diluents, the every young plant in LHTXYB treatment zone fills with root 50ml LHTXYB liquid bacterial agent diluent, and the every young plant in CK treatment zone fills with root 50ml clear water.After one month, carry out second time and fill with root, the every young plant in LHTYBA treatment zone fills with root 50ml LHTYBA liquid bacterial agent 10 times of diluents, and the every young plant in LHTXYB treatment zone fills with root 50ml LHTXYB liquid bacterial agent diluent, and the every young plant in CK treatment zone fills with root 50ml clear water.Wherein, LHTYBA liquid bacterial agent 10 times of diluents with clear water, the LHTYBA liquid bacterial agent of step one are diluted 10 times to make the content of LHTYBA be 10
8cfu/mL; LHTXYB liquid bacterial agent 10 times of diluents with clear water, the LHTXYB liquid bacterial agent of step one are diluted 10 times to make the content of LHTXYB be 10
8cfu/mL.Other agricultural measures of each treatment zone is all identical.A situation arises, growth-promoting effect, output and sugar degree to investigate root rot of beets in the harvesting time of beet.Wherein, Sugarbeet Yield and beet sugar-containing quantity measuring method as follows: the 1. mensuration of Sugarbeet Yield
Each treatment zone of each iteron gets 5 points respectively, and the plant often clicking 2 square metres is investigated, and only weighs to block root, records the output of each point respectively, calculates per mu yield.
Method of calculation:
Actual output (at every turn repeating)=(2 × 5) square metre beet root output/10 of every square metre
Actual output × 666.67 square metre of every mu of theoretical yield (kg)=every square metre
Effect of increasing production (%)=(mean yield-CK treatment zone, treatment zone mean yield)/mean yield × 100, CK treatment zone
2. the mensuration of sugar-beet sugar content
Each treatment zone of each iteron repeats to select 5 points, gets 7 strain beet roots at often, and each piece of root samples at base portion mensuration sugar degree of squeezing the juice, and records determination data respectively, calculates sugar degree.
Method of calculation: increase the average sugar degree in sugar amount average sugar degree-CK treatment zone, (degree)=treatment zone.
Result shows the whole vegetative period at beet, and CK treatment zone, LHTYBA treatment zone, LHTXYB treatment zone all root rot of beets do not occur.Compared with CK process, LHTYBA process volume increase 48.56%, has no significant effect sugar degree, and LHTXYB process volume increase 20.87%, has no significant effect sugar degree, and the output of LHTYBA process is 1.23 times (table 2) of LHTXYB.
Table 2, the Inner Mongol examine right front Qi Nanying village October (harvesting time) Sugarbeet Yield and sugar degree investigation result
| Process | Per mu yield (kg) | Volume increase (%) | Sugar degree (degree) |
| LHTYBA | 11965.91 | 48.56 | 20.80 |
| LHTXYB | 9735.56 | 20.87 | 18.57 |
| CK | 8054.60 | — | 18.32 |
Note: the experimental data in table is three mean values repeated
Embodiment 3, bacillus cereus (Bacillus cereus) LHTYBA CGMCC No.7566 prevent and treat the field experiment that root rot of beets promotes beet root output
By bacillus cereus (Bacillus cereus) LHTYBA CGMCC No.7566 (being called for short LHTYBA), with bacterial strain LHTXYB for contrast, together carry out following experiment.
One, the preparation of LHTYBA and LHTXYB solid fungicide
By LHTYBA and LHTXYB individually after the streak culture 24h of extractum carnis solid medium, inoculate a ring in extractum carnis liquid nutrient medium (500ml triangular flask 50ml loading amount), after 30 DEG C of 180rpm shake training 18h, be inoculated in extractum carnis liquid nutrient medium (500ml triangular flask 100ml loading amount) with 3% inoculum size, 30 DEG C of 180rpm shake training 48h, namely obtain LHTYBA fermented liquid and LHTXYB fermented liquid.With turfy soil absorption LHTYBA fermented liquid, and to carry out being dried to water content at 28 DEG C be 10%, and obtain LHTYBA solid fungicide, the content of the LHTYBA in LHTYBA solid fungicide is 10
8cfu/g.With turfy soil absorption LHTXYB fermented liquid, and to carry out being dried to water content at 28 DEG C be 10%, and obtain LHTXYB solid fungicide, the content of the LHTXYB in LHTXYB solid fungicide is 10
8cfu/g.
Two, the preparation that root rot of beets promotes the solid microbe fertilizer of beet root output is prevented and treated
By the LHTYBA solid fungicide in step one and farm manure mixing, obtain LHTYBA solid organic fertilizer, the LHTYBA content in LHTYBA solid organic fertilizer is 10
11cfu/kg.By the LHTXYB solid fungicide in step one and farm manure mixing, obtain LHTXYB solid organic fertilizer, the LHTXYB content in LHTXYB solid organic fertilizer is 10
11cfu/kg.LHTYBA solid organic fertilizer is identical with farm manure used in LHTXYB solid organic fertilizer.
Three, Liang Jia village, Shangdu County, Wulanchabu League of Inner Mongolia city field experiment
In May, 2012, with sweet list 302 (incubation of beet institute of the Chinese Academy of Agricultural Sciences) for supplying examination beet variety, field experiment is carried out: experiment adopts randomized block design at the beet second crop soil in Liang Jia village, Shangdu County, Wulanchabu League of Inner Mongolia city, three iterons are set, each iteron arranges three communities at random, is respectively CK treatment zone, LHTYBA treatment zone, LHTXYB treatment zone.The area of each community is 3 mu.
Respectively at transplanting forward direction soil discal patch organic fertilizer in each community.Wherein, CK treatment zone (contrast) according to the amount row replacement farm manure of every mu of 5Kg, the LHTYBA solid organic fertilizer of this farm manure and step 2 is identical with farm manure used in LHTXYB solid organic fertilizer; LHTYBA treatment zone, according to the LHTYBA solid organic fertilizer of the amount row replacement step 2 of every mu of 5Kg, makes every mu to apply 5 × 10
11cfu LHTYBA; LHTXYB treatment zone, according to the LHTXYB solid organic fertilizer of the amount row replacement step 2 of every mu of 5Kg, makes every mu to apply 5 × 10
11cfu LHTXYB.Output, sugar degree and root rot prevention effect is investigated harvesting time at beet.Other agricultural measures of each treatment zone is all identical.A situation arises, growth-promoting effect, output and sugar degree to investigate root rot of beets in the harvesting time of beet.Investigation root rot of beets is when a situation arises, and each treatment zone of each iteron gets 5 points respectively, and the plant often clicking 10 square metres is investigated, and the method for calculation of the stage division of root rot of beets and disease index and prevention effect are the same.The measuring method of beet root output and sugar degree is with embodiment 2.
Result shows that the prevention effect of LHTYBA to root rot of beets reaches the prevention effect of 86.7%, LHTXYB to root rot of beets and reach 1.74 times that the prevention effect of 49.8%, LHTYBA to root rot of beets is LHTXYB.Compared with CK process, LHTYBA is 1.2 times of LHTXYB process to the beet root output of the volume increase 46.00%, LHTXYB of beet root to volume increase 22.38%, the LHTYBA process of beet root.
Table 3, Liang Jia village, Shangdu County, Wulanchabu League of Inner Mongolia city October (harvesting time) Sugarbeet Yield and sugar degree investigation result
Note: the experimental data in table is three mean values repeated.
The root colonization experiment of embodiment 4, bacillus cereus (Bacillus cereus) LHTYBA CGMCC No.7566
By bacillus cereus (Bacillus cereus) LHTYBA CGMCC No.7566 (being called for short LHTYBA), with bacterial strain LHTXYB for contrast, together carry out following experiment.
1, Rif and Nal dual anti-label L HTYBA and LHTXYB
1.1 37 DEG C of incubated overnight LHTYBA and LHTXYB in LB liquid nutrient medium respectively, 5000rpm, centrifugal 2min collects thalline, is coated on the LB flat board containing nalidixic acid sodium salt (Nal) (final concentration is 50 μ g/mL) uniformly, 37 DEG C of quiescent culture.When growing single bacterium colony of anti-Nal on flat board, pick out the best single bacterium colony streak inoculation of growing way on the LB flat board containing Nal, continuous switching 3 generation, observe the cultural colony of bacterial strain, if this bacterium colony growing way is stable and little with wild type strain difference, then elect the mutant strain that induction obtains anti-Nal as, the anti-Nal mutant strain called after LHTYBA-N obtained by LHTYBA, the anti-Nal mutant strain called after LHTXYB-N obtained by LHTXYB.
1.2 respectively on the LB liquid nutrient medium containing Nal microbiotic (final concentration is 50 μ g/mL) 37 DEG C spend the night shake training LHTYBA-N and LHTXYB-N, 5000rpm, centrifugal 2min collects thalline, be coated in containing nalidixic acid sodium salt and Rifampin (Nal and Rif) (Nal, 50ug/ml uniformly; Rif, 150ug/ml) the dual anti-flat board of LB on, 37 DEG C of quiescent culture.When growing single bacterium colony of anti-Nal and Rif on flat board, pick out the best single bacterium colony streak inoculation of growing way on the LB flat board containing Nal and Rif, continuous switching 3 generation, if this bacterium colony growing way is stable and little with wild type strain difference, then for induction obtains the mutant strain of anti-Nal and Rif, anti-Nal and the Rif mutant strain called after LHTYBA-NR obtained by LHTYBA-N, anti-Nal and the Rif mutant strain called after LHTXYB-N called after LHTXYB-NR obtained by LHTXYB-N.
2, the cultivation of bacterial strain LHTYBA-NR and LHTXYB-NR and the preparation of spore suspending liquid thereof
(contain: Nal, 50 μ g/mL in LB liquid nutrient medium; Rif, 150 μ g/mL) inoculate LHTYBA-NR and LHTXYB-NR respectively in 30 DEG C, 160rpm cultivates 60 hours, treats that gemma growth is neat, 3000rpm collected by centrifugation thalline, adds sterilized water and collect thalline, make spore suspending liquid, obtain bacteria containing amount and be 5 × 10
8the LHTYBA-NR spore suspending liquid of cfu/ml and LHTXYB-NR spore suspending liquid.With blood counting chamber counting, calculate concentration.4 DEG C temporary for subsequent use.
3, soil prepares
Test soil used and take from experimental plot, the village in China Agricultural University, sieve the removing of large soil block, by soil: the peat composed of rotten mosses: vermiculite: the ratio mixing of chicken manure=3:3:1:0.5, installs in basin.
4, beet seed process
The first vernalization of sweet list 302 (incubation of beet institute of the Chinese Academy of Agricultural Sciences) seed, (bacteria containing amount is 5 × 10 to use LHTYBA-NR spore suspending liquid and LHTXYB-NR spore suspending liquid after showing money or valuables one carries unintentionally respectively
8cfu/ml) soak 5min, plant in ready soil, fill with root by spore suspending liquid more two days later, every strain 2mL.
5, collecting soil sample and bacteria containing amount counting
Gather primary sample after filling with root, whole for thinning beet strain is pulled out gently, shakes off bulk soil, the fine earth on root surface is scraped, careful collection pedotheque 1 ~ 2g, get 3 repetitions at every turn.3 days afterwards second time collected specimens, later every 5 days collect once, continuous detecting 46 days.Accurately take soil sample 1g to be measured, put into the test tube that 10ml sterilized water is housed, vortex oscillation 3min, the microorganism cells in soil is fully disperseed, leave standstill 1min, be stoste, with 10
-3with 10
-4diluent be coated with dual anti-LB flat board (Nal, 12.5 μ g/mL; Rif, 50 μ g/ml), cultivate 24h for 37 DEG C.With excel, 3 repeating datas are analyzed.
When result shows with dual anti-labeled strain LHTYBA-NR bacteria suspension root irrigation beet root, the bacteria containing amount in rhizosphere soil reaches 3.26 × 10
7cfu g
-1, within 8 days, " Invest, Then Investigate " finds, the bacteria containing amount of bacterial strain LHTYBA-NR in beet rhizosphere soil declines, and only has 9.20 × 10
6cfu g
-1, 46 days afterwards surely the amount of growing maintain 7 × 10
6cfu g
-1.During with dual anti-labeled strain LHTXYB-NR bacteria suspension root irrigation beet root, the bacteria containing amount in rhizosphere soil reaches 2.71 × 10
7cfu g
-1, within 8 days, " Invest, Then Investigate " finds, the bacteria containing amount of bacterial strain LHTXYB-NR in beet rhizosphere soil acutely declines, and only has 3.10 × 10
6cfu g
-1, 46 days afterwards surely the amount of growing maintain 10
6cfu g
-1.
Claims (10)
1. bacillus cereus (Bacillus cereus) LHTYBA, it is CGMCC No.7566 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. a microbial inoculum, its activeconstituents is bacillus cereus according to claim 1 (Bacillus cereus) LHTYBA.
3. microbial inoculum according to claim 2, is characterized in that: described microbial inoculum is following 1)-3) in arbitrary microbial inoculum:
1) for preventing and treating the microbial inoculum of root rot of beets and raising Sugarbeet Yield;
2) for preventing and treating the microbial inoculum of root rot of beets;
3) for improving the microbial inoculum of Sugarbeet Yield.
4. bacillus cereus according to claim 1 (Bacillus cereus) LHTYBA is in preparation following 1)-3) in application in arbitrary microbial inoculum:
1) for preventing and treating the microbial inoculum of root rot of beets and raising Sugarbeet Yield;
2) for preventing and treating the microbial inoculum of root rot of beets;
3) for improving the microbial inoculum of Sugarbeet Yield.
5. contain the biological organic fertilizer of bacillus cereus (Bacillus cereus) LHTYBA or the microbial inoculum described in Claims 2 or 3 described in claim 1.
6. cultivating the method for bacillus cereus (Bacillus cereus) LHTYBA described in claim 1, comprising described bacillus cereus (Bacillus cereus) LHTYBA for cultivating the step of cultivating in the substratum of genus bacillus.
7. the preparation method of microbial inoculum described in Claims 2 or 3, comprises the steps: bacillus cereus according to claim 1 (Bacillus cereus) LHTYBA as activeconstituents, to obtain described microbial inoculum.
8. (Bacillus cereus) LHTYBA of bacillus cereus described in claim 1 or the application of the microbial inoculum described in Claims 2 or 3 in cultivated beet.
9. the method for cultivated beet, comprises and apply (Bacillus cereus) LHTYBA of bacillus cereus described in claim 1 or the microbial inoculum described in Claims 2 or 3 in the soil of cultivated beet.
10. method according to claim 9, is characterized in that: in described method, and the consumption of (Bacillus cereus) LHTYBA of bacillus cereus described in claim 1 or the microbial inoculum described in Claims 2 or 3 is every mu and applies 10
11cfu-10
12bacillus cereus described in cfu (Bacillus cereus) LHTYBA.
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