CN106967654A - A kind of Methylotrophic bacillus and biological compound seed coating material and application - Google Patents
A kind of Methylotrophic bacillus and biological compound seed coating material and application Download PDFInfo
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- CN106967654A CN106967654A CN201710370877.3A CN201710370877A CN106967654A CN 106967654 A CN106967654 A CN 106967654A CN 201710370877 A CN201710370877 A CN 201710370877A CN 106967654 A CN106967654 A CN 106967654A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- 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
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
- A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
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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
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having one or more single bonds to nitrogen atoms
- A01N47/10—Carbamic acid derivatives, i.e. containing the group —O—CO—N<; Thio analogues thereof
- A01N47/22—O-Aryl or S-Aryl esters thereof
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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
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having one or more single bonds to nitrogen atoms
- A01N47/10—Carbamic acid derivatives, i.e. containing the group —O—CO—N<; Thio analogues thereof
- A01N47/26—Oxidation products of dithiocarbamic acid derivatives, e.g. thiuram sulfides
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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
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
Abstract
The present invention discloses a kind of Methylotrophic bacillus and biological compound seed coating material and application.Methylotrophic bacillus (Bacillus methylotrophicus) the LH L3, its preserving number is CGMCC No.12565.Biological seed coating agent of the present invention mainly contains fludioxonil, thiram, carbofuran, various auxiliary agents, and adds the bacterial strain and be prepared.Bacterial strain and its compound seed coating material of the present invention can be obviously promoted seed sprouting, plant strain growth, and can effectively prevent and treat verticillium wilt, cotton leafroller.Biology compound seed coating material combination chemical pesticide of the invention and the respective prevention and control feature of biological agent, are a kind of environmentally friendly Biological compound preparations.
Description
Technical field
The invention belongs to field of pesticide preparation, and in particular to a kind of somatotrophic microorganism composite suspension kind clothing of diseases prevention insect prevention
Agent, is mainly used in seed pelleting processing.
Background technology
In growing process, pest and disease damage is to influence one of key factor of its production, to soil-borne disease and seedling stage disease
Harmful preventing and treating is the problem during crop production.In view of the above-mentioned problems, prevented and treated at present using a large amount of chemical pesticides, this
Residues of pesticides and food-safety problem are not only caused, and causes soil-borne disease and some other insects also to generate anti-medicine
Property, cause prevention effect to reduce.
In recent years, this crop seed processing formulation of coating agent for seed receives extensive concern.Seed coat agent be using seed as
Carrier, is sticked on seed by film forming agent, and uniform medicine film is solidified into quickly, after planting to one protection screen of Seed Development
Barrier, with Seed sprouting, germinates, emerges, grows up, and active ingredient is gradually absorbed by plant root passes to each position of seedling plants,
And then play a part of disease-prevention and insecticide.Known seed coat agent is by bactericide, insecticide, compound fertilizer, trace element, plant life
The fertile compound seed pelleting product of medicine that long conditioning agent, sustained release agent and film forming agent etc. are processed into.Current major part seed coat agent
In the utilization for resting on pure chemistry agricultural chemicals, biological seed coating agent or biological compound seed coating material are less.It is this kind of for cotton verticillium wilt
Plant and pass and soil-borne disease, it is the important means for controlling disease to occur that seed progress, which is handled,.It is compound with biological seed coating agent and biology
Seed coat agent is the new seed coat agent of representative, and its development prospect is quite wide.
Because the gemma produced by bacillus is resistant to poor environment, it is easier to realize large-scale culture and life
Production, particularly in seed coat agent in this extreme environment, compared to other microorganisms, bacillus is more easy to survival, so as to be coated
Seed after planting, remains to produce a variety of antibacterial materials to suppress phytopathogen, therefore, is accounted in field of biological pesticide bacillus
There is consequence.Methylotrophic bacillus is the novel species of report in 2010, antifungal lipopeptid can be produced, to cucumber anthracnose
Germ, cucumber fusarium axysporum, Rhizoctonia solani Kuhn, Strain of Pseudomonas Solanacearum, botrytis cinerea, tobacco black shank bacterium, banana blight
The various plants pathogen such as bacterium and rice blast fungus is respectively provided with stronger inhibitory activity, while there is the work(for promoting plant growth
Energy.The antibacterial peptide that such bacterial strain is produced is alternatively arranged as the bio-preservative of Chilled Meats, there is wide application in field of biological pharmacy
Prospect.
The content of the invention
It is an object of the present invention to provide one kind can effectively reduce cotton verticillium wilt, cotton leafroller occur, have concurrently raising cotton,
The budding such as wheat, corn, romaine lettuce, wild cabbage rate, the Methylotrophic bacillus for promoting its growth and its biology are multiple
Close seed coat agent.
To achieve these goals, the technical scheme that the present invention takes is as follows：
1. one plant of Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3, its preserving number is：
CGMCC No.12565。
2. a kind of biological compound seed coating material, is made up of the following raw material in terms of mass fraction：Living bacteria count 7.0 ×
109Cfu/mL Methylotrophics bacillus (Bacillus methylotrophicus) LH-L3CGMCC No.12565 bacterium
Liquid 1%, fludioxonil 2.5%, thiram 10%, carbofuran 10%, Ricinate 4.2%, thickener 1.45%, preservative
0.2%, antifreezing agent 5%, film forming agent 1.25%, warning coloration 2%, surplus is sterilized water, and pH value is 7.0~7.2.
3. a kind of biological compound seed coating material according to technical scheme 2, the Ricinate is agriculture breast 602 and naphthalene sulphur
Sour sodium formaldehyde condensation product, agriculture breast 602：Mass ratio=1 of naphthalenesulfonic acid-formaldehyde condensate:2；The thickener is xanthans and paste
Change starch, xanthans：Mass ratio=1 of gelatinized starch:6；The preservative is sodium benzoate；The antifreezing agent is glycerine；Institute
Film forming agent is stated for polyvinyl alcohol 1750；The warning coloration is rhodamine toner.
4. a kind of preparation method of biological compound seed coating material according to technical scheme 2 or 3, comprises the following steps：
(1) by Methylotrophic bacillus (Bacillus methylotrophicus) LH- described in technical scheme 1
After L3CGMCC No.12565 activation, LB nutrient solutions are inoculated into, in 37 DEG C of cultivation temperature, rotating speed 180r/min shaking table cultures 4d productions
After gemma, part LB nutrient solutions are centrifuged off, make bacterium solution bacillus containing Methylotrophic (Bacillus
Methylotrophicus) LH-L3CGMCC No.12565 concentration reaches 7.0 × 109Cfu/mL produces living bacteria count 7.0
×109Cfu/mL Methylotrophics bacillus (Bacillus methylotrophicus) LH-L3CGMCC No.12565's
Bacterium solution；
(2) content of each raw material weighs each raw material in a kind of biological compound seed coating material as described in technical scheme 2 or 3, first
By thickener and film forming agent in container, plus sterilized water, the container is placed in heating in boiling water and stirs into pasty state, 48 DEG C are cooled to
~52 DEG C, add except living bacteria count 7.0 × 109Cfu/mL Methylotrophic bacillus (Bacillus
Methylotrophicus remaining raw material) beyond LH-L3CGMCC No.12565 bacterium solution simultaneously mixes and stirs evenly to obtain mixed material,
Then mixed material is poured into sand mill, the granularity for being ground to 95% raw material mixed above in sand mill is 5 μm, and mixing is former
The viscosity of material is more than 600mpa.s, that is, chemical agent suspending agent is made, and metered step is then pressed in chemical agent suspending agent
Suddenly living bacteria count 7.0 × 10 made from (1)9Cfu/mL Methylotrophic bacillus (Bacillus
Methylotrophicus) LH-L3CGMCC No.12565 bacterium solution, it is the biological compound seed coating material to mix.
In made above described in a kind of biological compound seed coating material of each raw material dosage as described in technical scheme 2 or 3
The consumption of each raw material, wherein in the chemical agent suspending agent living bacteria count 7.0 as made from metered step (1) ×
109Cfu/mL Methylotrophics bacillus (Bacillus methylotrophicus) LH-L3CGMCC No.12565 bacterium
The quality of liquid accounts for the 1% of the biological compound seed coating material quality, that is, the quality of the bacterium solution added accounts for the biological aggregate species
The 1% of clothing agent quality.
5. one plant of Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3 described in technical scheme 1
Or application of a kind of biological compound seed coating material on verticillium wilt is prevented and treated described in technical scheme 2 or 3.
6. one plant of Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3 described in technical scheme 1
Or a kind of application of the biological compound seed coating material described in technical scheme 2 or 3 on seeding ratio is improved.
7. the application according to technical scheme 6, described seed is cotton, corn, wheat, romaine lettuce and/or wild cabbage
Seed.
8. one plant of Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3 described in technical scheme 1
Or a kind of application of the biological compound seed coating material described in technical scheme 2 or 3 on plant growth is promoted.
9. the application according to technical scheme 8, described crop is cotton, corn, wheat, romaine lettuce and/or wild cabbage.
10. one plant of Methylotrophic bacillus (Bacillus methylotrophicus) LH- described in technical scheme 1
L3 or application of a kind of biological compound seed coating material on preventing and treating cotton leafroller described in technical scheme 2 or 3.
The present invention compared with prior art, has the advantages that：
1st, it is applied widely.It can be used in multiple kinds of crops such as cotton, corn, wheat, romaine lettuce, rape, wild cabbages, emerge
Rate improves notable, and plant is strongr, significantly promotes the growth of crop.
Following seed is coated using compound seed coating material of the invention biological, its cotton, corn, wheat, romaine lettuce, wild cabbage are emerged
Rate respectively reaches 93.33%, 97.86%, 91.67%, 87.50%, 86.32%, be respectively increased 3.7% than control (CK),
3.51%th, 3.78%, 4.77%, 4.63%, reach significant difference.
After biological compound seed coating material capsuled seed of the invention, cotton, corn, wheat, romaine lettuce, the strain of wild cabbage are promoted
Height, root length, overground part fresh weight, underground part fresh weight or root weight, index of correlation reach significant difference compared with the control.
2nd, seed coat agent of the present invention can coordinate plant growth, pest and disease damage can be prevented and treated again, is a kind of multi-functional compound seed coating material.
85.24% is reached to cotton verticillium wilt prevention effect, incidence of disease reduction by 71.63% reaches significant difference compared with the control.
3rd, the chemical sterilization active compound that the present invention is used, is the medicament of low toxicity and low residue.Due to being Multiple components composition
Composite seed coating agent, effect is better than the preparation of single formula, and has saved cost and time.
4th, bacterial strain Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3 CGMCC of the present invention
No.12565 is easy to culture, and fermentation preparation process technique is simple.
5th, due to there is bacterial strain Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3 of the present invention
CGMCC No.12565 participation, present invention biology compound seed coating material has effectively facilitated the growth of crop, have activated exempting from for crop
Epidemic disease system, improves its disease resistance ability.
Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3 of the present invention is in 2016
On May 30, in is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (referred to as：CGMCC), preserving number
For CGMCC No.12565, Classification And Nomenclature is：Methylotrophic bacillus methylotrophicus, and in
It is detected as survival on May 30th, 2016.The common micro-organisms center address：City of BeiJing, China Chaoyang District North Star West Road 1 institute 3
Number, postcode:100101.
The various raw materials of biology compound seed coating material of the invention.Except Methylotrophic bacillus (Bacillus of the present invention
Methylotrophicus) to be deposited in above-mentioned China Committee for Culture Collection of Microorganisms general by LH-L3CGMCC No.12565
Outside logical microorganism center, remaining raw material is commercially available.
Embodiment
Further illustrated with reference to embodiment.Following examples are not limited the invention, people in the art
Member's modified technique parameter on this basis, changes specific auxiliary material type etc. and replaces and change, still belong to the protection of the present invention
Scope.Following examples are conventional method without specified otherwise.
LH-L3 is (hereinafter referred to as the Methylotrophic bacillus of embodiment 1 (Bacillus methylotrophicus)：Bacterium
Strain LH-L3) acquisition, identification and preservation
1st, bacillus LH-L3 acquisition
1.1 material
Cotton plant is picked up from the local joint project research of Yunnan Province Anning City Qinglong Zhen microorganism strains filtration and application country
The warmhouse booth of the heart.
It is prepared by 1.2 culture mediums
LB culture mediums pour into sterilizing culture dish after high pressure steam sterilization, cooling, and LB flat boards are made.
It 1.3 is separately cultured
By cotton plant root, leaf, stem each 5g, the flower 2g of collection, with 75% alcohol surface sterilization 10min, aqua sterilisa is rinsed 3 times
Afterwards, it is fully ground in sterilizing mortar, then adds aqua sterilisa to dilute 10 times, all liq and residue are transferred to sterile triangular flask
In, it is placed in 80 DEG C of thermostat water baths and heats 10min, takes 100 μ L to be coated on LB flat boards separation cotton plant endophytic Bacillus.By cotton
Flower verticillium wilt pathogen bacteria suspension is spread evenly across PDA plate, and verticillium wilt pathogen is purchased from Institute of Plant Protection of agricultural and forest science institute, Hebei, will be separated to
Bacillus be inoculated into the flat board that carries disease germs successively, the stronger bacterial strain of screening fungistatic effect.Purified with LB culture mediums, a bacterium of gained
Strain is named as LH-L3.
2nd, Methylotrophic bacillus LH-L3 identification
2.1st, Morphological Identification
Take bacterial strain LH-L3 in the flat lining out cultures of LB, cultivated 36 hours in 37 DEG C, carry out morphologic observation identification.In LB
Occur opaque white color circular colonies on flat board, most of the smooth of the edge is neat, dry tack free has fold, centre has volcano shape to dash forward
Rise.In carrying out morphologic observation identification under microscope, thalline is rod-short, produces oval gemma.With《Common bacteria system identification
Handbook》Bacillus morphological feature described in (east show pearl etc. is write, Science Press, 2001) is basically identical, tentatively
Judge that bacterial strain LH-L3 belongs to bacillus (Bacillus spp.).
2.2nd, Physiology and biochemistry is identified
The bacterial strain can utilize malonic acid, citrate, hydrolysis starch, it is impossible to which decomposition of protein peptone produces indoles, it is impossible to hydrolyze
Arginine, can produce hydrogen sulfide and Phenylalanine dehydrogenase, and clark and Lubsreaction and V-P determine negative, Gram's staining, contact
Enzyme, glucose oxidative fermentation, sugar, alcoholic fermentation reaction etc. are positive.
2.3rd, 16S rDNA sequence analyses
According to sequencing result, the 16S rDNA sequences of bacterial strain and the Methylotrophic gemma bar that strain number is CBMB205
Bacterium Bacillus methylotrophicus (accession number is EU 194897) similarity highest, up to 99%.With each gemma bar
The chadogram of the 16S rDNA sequence constructs of bacterium type strain shows that the bacterial strain divides with Bacillus methylotrophicus
Cloth is in same branch.Therefore, bacterial strain LH-L3 is accredited as Methylotrophic bacillus (Bacillus
methylotrophicus)。
3rd, Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3 preservation
Identify that the bacterial strain for showing separated acquisition is Methylotrophic bacillus (Bacillus through above-mentioned
Methylotrophicus), its code name is LH-L3, described Methylotrophic bacillus (Bacillus
Methylotrophicus) to be deposited in China Committee for Culture Collection of Microorganisms on May 30th, 2016 commonly micro- by LH-L3
Bio-Centers are (referred to as：CGMCC), preserving number is CGMCC No.12565, and Classification And Nomenclature is：Methylotrophic bacillus
Bacillus methylotrophicus, and it is detected as on May 30th, 2016 survival.The common micro-organisms center address：In
State's Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode:100101.
Therefore, bacterial strain of the present invention is Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3,
Its preserving number is：CGMCC No.12565, hereinafter referred to as：Bacterial strain or Methylotrophic bacillus (Bacillus of the present invention
methylotrophicus)LH-L3CGMCC No.12565。
The bacterial strain of the present invention of embodiment 2 is determined to the growth promotion of cotton with verticillium wilt ability is prevented and treated
1st, growth promotion ability is determined
Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3CGMCC No.12565 are lived
After change, LB nutrient solutions are inoculated into, after 37 DEG C of cultivation temperature, rotating speed 180r/min shaking table cultures 4d production gemma, dilution coated plate is surveyed
It is 7.2 × 10 to determine bacterial concentration8cfu/mL.Plus running water dilutes 10 times successively, it is 7.2 × 10 that concentration, which is made,7cfu/mL、7.2×
106cfu/mL、7.2×105Cfu/mL bacterium solution.
Experiment is located at Yunnan Prov Agriculture University greenhouse in the school, and experiment sets 3 processing, processing 1 altogether：Methylotrophic bacillus
(Bacillus methylotrophicus) LH-L3CGMCC No.12565 concentration is 7.2 × 105Cfu/mL bacterium solution；Place
Reason 2：Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3CGMCC No.12565 concentration is
7.2×106Cfu/mL bacterium solution；Processing 3：Methylotrophic bacillus (Bacillus methylotrophicus) LH-
L3CGMCC No.12565 concentration is 7.2 × 107Cfu/mL bacterium solution；Processing 4：LB fluid nutrient mediums are compareed.Respectively handle
Operation：Cotton seed is through 75LL- 1After alcohol disinfecting, rinsed with sterile water 2~3 times, the flower of 14cm × 20cm equipped with soil is seeded in
In basin, soil then is poured with the bacterium solution 250mL used in processing, 3 repetitions are often handled, per 10 cotton seeds of basin.Hereafter every 7d
The bacterium solution used in single treatment is poured, the bacterium solution 3 times used in processing is poured altogether, and compared with LB fluid nutrient mediums.After planting
38 days, whole basin plant is poured out, gently clean soil with water, take back laboratory measurement and count its economical character.
Influence of the bacterial strain different administration concentration of the present invention of table 1 to cotton growth
As can be known from Table 1：Compared with LB fluid nutrient mediums are compareed, with regard to emergence rate, plant height, root length, overground part fresh weight, underground
For portion's fresh weight, it is 7.2 × 10 that bacterial strain bacterium solution of the present invention, which pours concentration,5During cfu/mL, be respectively increased 35.70%, 1.99%,
19.99%th, 7.93%, 71.43%.It is 7.2 × 10 that bacterial strain bacterium solution of the present invention, which pours concentration,6During cfu/mL, it is respectively increased
42.85%th, 11.49%, 23.83%, 22.82%, 97.62%, emergence rate is significantly improved, and plant is more healthy and strong, root system is relatively sent out
Reach.Significant difference (P<0.05)；It is 7.2 × 10 that bacterial strain bacterium solution of the present invention, which pours concentration,7During cfu/mL, be respectively increased 57.12%,
0.56%th, 29.60%, 1.74%, 95.24%.Therefore, it is 7.2 × 10 to pour bacterium solution optium concentration6cfu/mL。
2nd, to the indoor control effect of cotton verticillium wilt
Experiment is located at Yunnan Prov Agriculture University greenhouse in the school, and experiment sets 2 processing, processing 1 altogether：Methylotrophic bacillus
(Bacillus methylotrophicus) LH-L3CGMCC No.12565 concentration is 7.2 × 106Cfu/mL bacterium solution；Place
Reason 2：LB fluid nutrient mediums are compareed.Experiment soil is about 7.2 × 10 artificially to make the verticillium wilt spore amount mixed thoroughly5Cfu/g band
Bacterium soil, verticillium wilt pathogen is purchased from Institute of Plant Protection of agricultural and forest science institute, Hebei.Pour bacterium and type of seeding is consistent with previous step, treat LB to approved for distribution
Incidence is counted when sick 10th day.Disease index and prevention effect are calculated by 5 grades of criteria for classifications, it is specific as follows：It is 0 grade, disease-free
Plant；1 grade, the plant of 25% blade morbidity；2 grades, the plant of 25%~50% blade morbidity；3 grades, 50%~75% blade hair
The plant of disease；4 grades, 75% plant fallen ill with blade.
Greenhouse prevention effect of the bacterial strain of the present invention of table 2 to cotton verticillium wilt
Verticillium wilt result of the test is prevented and treated to show：Control cotton plant, which is emerged the 50th day, to be started to show verticillium wilt symptom, but the present invention
After bacterial strain processing, there is small part blade symptom occur in the 58th talent of emerging, disease time is substantially postponed.In control cotton plant morbidity
After 10d, adjoining tree major part incidence of leaf has dropped, but the cotton plant incidence of disease of bacterial strain of the present invention processing is still relatively low, plant
Robust growth, the incidence of disease and disease index reduce by 71.63% and 85.52% respectively compared with the control, and prevention effect reaches
85.24%, significant difference.
The formula of the biological compound seed coating material of the present invention of embodiment 3 and its preparation
1st, the formula of biological compound seed coating material is shown in such as table 3 below-table 6
No. 1 formula of biological compound seed coating material of table 3
No. 2 formulas of biological compound seed coating material of table 4
No. 3 formulas of biological compound seed coating material of table 5
No. 4 formulas of biological compound seed coating material of table 6
Each biological compound seed coating material of table 3, table 4, table 5, table 6 be briefly referred to as No. 1 formula, No. 2 formula, No. 3 formula, No. 4
Formula.
2nd, the preparation method of each biological compound seed coating material of formula more than：
(1) by Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3CGMCC No.12565
After activation, LB nutrient solutions are inoculated into, after 37 DEG C of cultivation temperature, rotating speed 180r/min shaking table cultures 4d production gemma, are centrifuged off
Part LB nutrient solutions, make bacterium solution bacillus containing Methylotrophic (Bacillus methylotrophicus) LH-L3CGMCC
No.12565 concentration reaches 7.0 × 109Cfu/mL produces living bacteria count 7.0 × 109Cfu/mL Methylotrophic bacillus
(Bacillus methylotrophicus) LH-L3CGMCC No.12565 bacterium solution.
(2) weigh each raw material by the content of each raw material in biological compound seed coating material formula in embodiment 3, first by thickener and
Film forming agent is in container, plus sterilized water, and the container is placed in into heating water bath in boiling water stirs into pasty state, is cooled to 48 DEG C~52
DEG C, add except living bacteria count 7.0 × 109Cfu/mL Methylotrophic bacillus (Bacillus
Methylotrophicus) remaining raw material mixing beyond LH-L3CGMCC No.12565 bacterium solution stirs evenly to obtain mixed material, and
Stay a part of sterilized water to be used for eluant container, then pour into mixed material in sand mill, with stayed a part of aseptic water washing
Raw material on chamber wall is simultaneously poured into sand mill, and the granularity for being then ground to 95% raw material mixed above in sand mill is 5 μm, and
The viscosity of mixed material is more than 600mpa.s, that is, chemical agent suspending agent is made, then by biology in chemical agent suspending agent
Living bacteria count 7.0 × 10 made from metered step (1) in compound seed coating material formula9Cfu/mL Methylotrophic gemma
Bacillus (Bacillus methylotrophicus) LH-L3CGMCC No.12565 bacterium solution, it is biological aggregate species clothing to mix
Agent.
3rd, bactericidal action of the 4 biological compound seed coating materials of above-mentioned No. 1-4 formula to infected seed is tested
Test method：This 4 biological compound seed coating materials are pressed into biological aggregate species to the cotton seeds with verticillium wilt pathogen respectively
Clothing agent：The mass ratio of cotton seeds with verticillium wilt pathogen is 1:50 pesticide-seeds ratio is coated, and is used respectively at after processing 24h, 48h
Lower surface spore is washed in sterilized water vibration, the survival condition of verticillium wilt pathogen is detected using dilution coated plate method, while setting the band not being coated
The cotton seeds of verticillium wilt pathogen do blank control.
The content of the cotton seeds surface verticillium wilt pathogen of table 7
Result of the test：As seen from Table 7, No. 1-4 formula is compared with blank control, respectively to cotton seeds surface verticillium wilt pathogen
Bactericidal effect respectively up to 99.22%, 99.52%, 99.93%, 100%, reach pole significant difference, wherein No. 4 formula pair
The bactericidal effect of cotton seeds surface verticillium wilt pathogen preferably, reaches 100%, surface germ can be killed completely.It is therefore preferable that No. 4
The biological compound seed coating material formula of the present invention for being formulated finally to determine.
The biological compound seed coating material of the present invention of embodiment 4 is improving seeding ratio, is promoting plant growth, preventing and treating cotton yellow to wither
Application in disease and cotton leafroller
Biology compound seed coating material of the invention is No. 4 formulas, hereinafter referred to as：This product.
First, overall operation
1. test site：Plant protection institute of Yunnan Prov Agriculture University greenhouse is carried out.
2. test kind
Cotton variety is Ji cotton 11；Corn variety is cloud standing grain list 2；Wheat breed is elder brother wheat No. 5；Romaine lettuce kind is green life
Big fast-growing；Head cabbage varieties are sweet 21 in being.
3. seed pelleting processing
This product is according to this product：Seed quality ratio=1:50 ratio is coated, and is dried after mixing after film-forming
Use.
4. test method
Capsuled seed is seeded in 14cm × 20cm flowerpot, per 10, basin, 3 repetitions are often handled, normally water guarantor
Hold humidity.
2nd, instantiation
(1) plant growth is promoted
1. corn coating
Test period：On May 11,16 days -2016 April in 2016.
Experiment sets 3 processing altogether：
Processing 1：By this product：Mass ratio=1 of seed:50 pesticide-seeds ratios are coated.
Processing 2：By 2.5% fludioxonil suspension seed-coating agent：Mass ratio=1 of seed:250 pesticide-seeds ratios are coated.
Processing 3：Seed (CK) without any processing.
After emergence of corn, the result of the test (being shown in Table 8) of the 20th day.
Influence of this product of table 8 to corn growth character
This product (processing 1) averagely improves 3.51% than the emergence rate of control (processing 3), and root length averagely increases by 9.66%,
Plant height averagely improves 6.01%, and every plant of overground part fresh weight averagely increases by 8.30%, and every plant of underground part fresh weight averagely increases
38.25%, reach significant difference.This product (processing 1) is averagely improved than the emergence rate of 2.5% fludioxonil seed coat agent (processing 2)
2.51%, root length averagely increases by 4.35%, and plant height averagely increases by 3.24%, and every plant of overground part fresh weight averagely increases by 3.07%,
Every plant of underground part fresh weight averagely increases by 8.70%, reaches significant difference.
Show：This product is significantly higher than the processing of 2.5% fludioxonil seed coat agent and compareed on corn growth is promoted.
2. the application on wheat, romaine lettuce, wild cabbage
Test period：On April 22,14 days -2016 March in 2016.
Experiment sets 2 processing altogether：
Processing 1：By this product：Mass ratio=1 of seed:50 pesticide-seeds ratios are coated.
Processing 2：Seed (CK) without any processing.
After emerging, the result of the test (being shown in Table 9,10) of the 20th day：
Influence of this product of table 9 to wheat growth character
After wheat is emerged, the result of the test of the 20th day shows：This product improves 3.78% than the emergence rate of control, and plant height is carried
It is high by 4.14%, every plant of overground part fresh weight increase by 6.90%, every plant of underground part fresh weight averagely increases by 18.75%, reaches significantly
Difference.This product is particularly evident to the growth-promoting effect of root.
This product of table 10 is to romaine lettuce, the influence of wild cabbage growth traits
After romaine lettuce is emerged, the result of the test of the 20th day shows：This product improves 4.77% than the emergence rate of control, and root length increases
Plus 11.05%, plant height improves 6.08%, and every plant of overground part fresh weight increases by 23.99%, and every plant of underground part fresh weight averagely increases
36.67%, reach significant difference.
After wild cabbage is emerged, the result of the test of the 20th day shows：This product improves 4.63% than the emergence rate of control, and root length increases
Plus 9.27%, plant height improves 25.05%, and every plant of overground part fresh weight increases by 3.87%, and every plant of underground part fresh weight averagely increases
8.93, reach significant difference.
(2) growth promotion and the effect of preventing and treating verticillium wilt of this product to cotton
Test period：On May 5,11 days -2016 March in 2016.
Experiment soil：It is about 10 artificially to make the verticillium wilt spore amount mixed thoroughly5The cfu/g soil that carries disease germs.
Experiment sets 2 processing altogether：
Processing 1：By this product：Mass ratio=1 of seed:50 pesticide-seeds ratios are coated.
Processing 2：Seed (CK) without any processing.
Test method：Cotton seed will be coated to be seeded in 14cm × 20cm flowerpot, per 10, basin, 3 repetitions are often handled.It is right
Incidence is counted when approved for distribution sick 10th day, 5 as described in embodiment 2 grade criteria for classification calculates disease index and prevention effect.
After cotton emerges, the result of the test (being shown in Table 11) of the 48th day：
The coating cotton seed growth of this product of table 11 prevents and treats situation with verticillium wilt
As can be known from Table 11：Compared with the control, it is coated with this product after cotton seed, emergence rate improves 3.7%, plant height increase
9.09%, the verticillium wilt incidence of disease is substantially reduced, and prevention effect reaches 82.10%, and incidence of disease reduction by 66.70% reaches extremely notable
Difference.
(3) prevention effect of this product to cotton leafroller
Test period：On June 3,17 days -2016 May in 2016.
Experiment sets 2 processing altogether：
Processing 1：By this product：Mass ratio=1 of seed:50 pesticide-seeds ratios are coated.
Processing 2：Seed (CK) without any processing.
Test method：Cotton seed will be coated to be seeded in 14cm × 20cm flowerpot, per 10, basin, often processing repeats 5 basins.Examination
Test it is potted plant be placed in greenhouse window opening, after whether there is insect pest on observation blade daily after seed sprouting, and investigate insect pest and occur feelings
Condition, records worm channel quantity in number of pest on every plant of seedling, and every blade.Percentage of injury and prevention effect are calculated according to formula,
Formula is as follows：
After cotton emerges, the result of the test (being shown in Table 12) of the 6th day：
This product of table 12 is coated cotton seed and prevents and treats situation to cotton leafroller
As can be known from Table 12：The seedling insect pest of capsuled seed a situation arises substantially reduction, attacked trees rate is 13.64%, than
Worm channel number is up to worm channel on 39.65, the every blade of Cotton seeds in blank control reduction by 83.07%, and the every blade of blank control
Number is only 5.52, and prevention effect reaches 92.33%, reaches pole significant difference.
Claims (10)
1. one plant of Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3, its preserving number is：CGMCC
No.12565。
2. a kind of biological compound seed coating material, is made up of the following raw material in terms of mass fraction：Living bacteria count 7.0 × 109cfu/mL
Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3 CGMCC No.12565 bacterium solution 1%, is coughed up
Bacterium nitrile 2.5%, thiram 10%, carbofuran 10%, Ricinate 4.2%, thickener 1.45%, preservative 0.2% is antifreeze
Agent 5%, film forming agent 1.25%, warning coloration 2%, surplus is sterilized water, and pH value is 7.0~7.2.
3. a kind of biological compound seed coating material according to claim 2, the Ricinate is agriculture breast 602 and sodium naphthalene sulfonate
Formaldehyde condensation products, agriculture breast 602：Mass ratio=1 of naphthalenesulfonic acid-formaldehyde condensate:2；The thickener is that xanthans and gelatinization are formed sediment
Powder, xanthans：Mass ratio=1 of gelatinized starch:6；The preservative is sodium benzoate；The antifreezing agent is glycerine；It is described into
Film is polyvinyl alcohol 1750；The warning coloration is rhodamine toner.
4. a kind of preparation method of biological compound seed coating material according to Claims 2 or 3, comprises the following steps：
(1) by Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3 described in claim 1
After CGMCC No.12565 activation, LB nutrient solutions are inoculated into, in 37 DEG C of cultivation temperature, rotating speed 180r/min shaking table cultures 4d production buds
After spore, part LB nutrient solutions are centrifuged off, make bacterium solution bacillus containing Methylotrophic (Bacillus
Methylotrophicus) LH-L3 CGMCC No.12565 concentration reaches 7.0 × 109Cfu/mL produces living bacteria count
7.0×109Cfu/mL Methylotrophics bacillus (Bacillus methylotrophicus) LH-L3 CGMCC
No.12565 bacterium solution；
(2) content of each raw material weighs each raw material in a kind of biological compound seed coating material as described in Claims 2 or 3, will first increase
Thick dose and film forming agent are in container, plus sterilized water, and the container is placed in into heating in boiling water stirs into pasty state, is cooled to 48 DEG C~52
DEG C, add except living bacteria count 7.0 × 109Cfu/mL Methylotrophic bacillus (Bacillus
Methylotrophicus remaining raw material and mixing) beyond LH-L3 CGMCC No.12565 bacterium solution, which is stirred evenly, must mix original
Material, then pours into mixed material in sand mill, and the granularity for being ground to 95% raw material mixed above in sand mill is 5 μm, and mixed
The viscosity for closing raw material is more than 600mpa.s, that is, chemical agent suspending agent is made, and then adds in chemical agent suspending agent by metering
Enter living bacteria count 7.0 × 10 made from step (1)9Cfu/mL Methylotrophic bacillus (Bacillus
Methylotrophicus) LH-L3 CGMCC No.12565 bacterium solution, the quality of the bacterium solution of addition accounts for described biological multiple
The 1% of seed coat agent quality is closed, it is the biological compound seed coating material to mix.
5. one plant of Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3 described in claim 1, or
Application of a kind of biological compound seed coating material on verticillium wilt is prevented and treated described in Claims 2 or 3.
6. one plant of Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3 described in claim 1, or
Application of a kind of biological compound seed coating material on seeding ratio is improved described in Claims 2 or 3.
7. application according to claim 6, described seed is the seed of cotton, corn, wheat, romaine lettuce and/or wild cabbage.
8. one plant of Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3 described in claim 1, or
Application of a kind of biological compound seed coating material on plant growth is promoted described in Claims 2 or 3.
9. application according to claim 8, described crop is cotton, corn, wheat, romaine lettuce and/or wild cabbage.
10. one plant of Methylotrophic bacillus (Bacillus methylotrophicus) LH-L3 described in claim 1 or
Application of a kind of biological compound seed coating material on preventing and treating cotton leafroller described in Claims 2 or 3.
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Cited By (2)
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| CN111631231A (en) * | 2020-07-02 | 2020-09-08 | 贵州大学 | Biological medicine-fertilizer composite pelletized seed coating agent pelletized seed and preparation method thereof |
| CN112369434A (en) * | 2020-11-14 | 2021-02-19 | 江苏海洋大学 | Bacillus methylotrophicus BMF04 aqueous suspension agent, preparation method and application |
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Application publication date: 20170721 |