CN107460151A - A kind of spherical lysine bacillus and its application for preventing and treating Meloidogyne incognita - Google Patents
A kind of spherical lysine bacillus and its application for preventing and treating Meloidogyne incognita Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The spherical lysine bacillus in tomato root and its application as Meloidogyne incognita biocontrol microorganisms are isolated from the invention discloses a kind of.The bacterial strain is identified as spherical lysine bacillus (Lysinibacillussphaericus), the entitled RQ11 of bacterial strain, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is：CGMCC No.14201, preservation date are on 05 27th, 2017, and it has good killing activity to Meloidogyne incognita, while can also remarkably promote tomato growth.The spherical lysine bacillus RQ11 bacterial strains that the present invention obtains are the endogenetic bacterias colonized in a kind of tomato root, are not easily susceptible to the influence of external environment, overcoming the environment that biocontrol microorganisms change in by soil is influenceed.Because RQ11 has higher application value in terms of Meloidogyne incognita harm is prevented and treated, especially the bacterium is tomato endogenetic bacteria, and persistently root-knot nematode can be worked, overcome general biocontrol microorganisms can not be colonized in root and caused by preventive effect it is low or unstable the defects of.
Description
Technical field
The invention belongs to field of microbial biotechnology, and in particular to a kind of spherical lysine bacillus and its preventing and treating south
The application of square root-knot nematode.
Background technology
Plant nematode is one of important pathogen thing of plant infectious diseases, is generally occurred in the whole world, is caused harm tight
Weight.Root-knot nematode is a kind of plant nematode for seriously endangering agricultural production, and main parasitic is in vegetables, cereal crops, economy
On more than the 2000 kinds of host such as crop, fruit tree, ornamental plant and weeds, the hazard of plant in the torrid zone, subtropical zone and temperate zone is especially serious,
The underproduction 10% or so as disease generation is latter, serious up to more than 75%.Especially Meloidogyne incognita onset area is wide,
Nearly all cultivated plant can be infected.Root-knot nematode is mainly caused harm host root, and lateral root and fibrous root are aggrieved the most serious, overground part
Divide and illness also occurs sometimes.Harm one of the root-knot nematode to host root is to destroy the direct machinery damage of root epidermal cell
Wound, second, with being salivated after kiss pricking wound host, the normal function of root cell is destroyed, ultimately results in root
Deformation.Variform warty root knot not of uniform size can be formed after root system is aggrieved, is started as white, soft texture, later intensification
Into filbert, there is crackle on surface.It absorbs after root system morbidity and the ability of transport nutrient and moisture can decline, and causes plant sick and weak
Yield reduction.The performance of grave illness strain aerial part becomes apparent, and plant severe malnutrition, thin and weak wilting is slow-growing, blade hair
Xanthochromia is small, and deformed fruit diminishes or flowers and fruits come off, and serious meeting complete stool is withered.
Due to lacking the reason such as resistant variety, multiple crop index height, the preventing and treating of plant nematode is still based on chemical agent.
Nematocide has that toxicity is big, pollutes environment, threatens the drawbacks such as human and livestock health, many effective nematode killing agents such as legendary gods of farming are red, furan
Mutter it is red wait disabled, with advocating for green agriculture and the strategy of sustainable development, people more urgently need safety and environmental protection
Prophylactico-therapeutic measures.Biological control has the advantages that Environmental compatibility, green, has wide application background.AVM
Be it is isolated from Avid kyowamycin (Streptomyces avermitilis) zymotic fluid kill nematode material, be considered as
It is the best biological agent of current effect, but some nematodes generate resistance to AVM.In a word, still lack at present
The root-knot nematode biological prevention and control agent of stability and high efficiency.
The content of the invention
Goal of the invention：The present invention is intended to provide a kind of spherical lysine Bacillus strain RQ11 of tomato endogenetic bacteria and its
Application as tomato root-knot eelworm biocontrol microorganisms.By verification experimental verification, using spherical lysine bacillus provided by the invention
Bacterial strain RQ11 has good result to preventing and treating tomato root-knot eelworm.This promotes the vegetable in China for further reducing pesticide dosage
Dish industry develops significant towards green non-pollution direction.
Technical scheme：The invention provides a kind of spherical lysine bacillus, spherical lysine gemma bar is identified as
Bacterium (Lysinibacillussphaericus), the entitled RQ11 of bacterial strain, is deposited in China Committee for Culture Collection of Microorganisms
Common micro-organisms center, preservation address：City of BeiJing, China Chaoyang District North Star West Road 1 institute 3, Chinese Academy of Sciences's microbe research
Institute, deposit number are：CGMCC No.14201, preservation date are on 05 27th, 2017.
The spherical lysine bacillus is from obtained by separation screening inside tomato root.
Spherical lysine bacillus RQ11 bacterial strains provided by the invention are a kind of sporiferous bacteriums, and the bacterial strain can be adopted
Any technical method for preserving bacillus provided with this area open source literature is preserved.
What the present invention obtained has the spherical lysine bacillus RQ11 bacterial strains of bacterial strain of prevention effect to Meloidogyne incognita.
Studied to realize that the effect of the preservation of the bacterial strain, transport and Field information is further used for formulation.
Therefore, present invention also offers the microbial inoculum for including the spherical lysine bacillus.
The preparation method of the microbial inoculum comprises the following steps：
(1) use conventional method culture to logarithmic phase for nutrient solution spherical lysine Bacillus strain RQ11；
(2) nutrient solution is prepared into microbial inoculum with the freeze-drying of routine, specific preparation method is nutrient solution degreasing
Milk powder is adsorbed, and volume ratio is 5~10 portions of skimmed milk powers, 1~2 part of NaCl, 1~3 part of 50~80 parts of peptone eutectoid content
Nutrient solution, freeze-drying, produces the microbial inoculum.
Thalline content is 1.00 × 10 in the microbial inoculum8~1.00 × 109CFU/g, the microbial inoculum preserve in the dry state.
, will be described spherical present invention also offers the application of the spherical lysine bacillus preventing and treating Meloidogyne incognita
Nutrient solution, then centrifuged fermented supernatant fluid, or bacterium prepared by the thalline are obtained after the fermentation of lysine Bacillus strain
Suspension, it can be used in preventing and treating Meloidogyne incognita.
Present invention also offers the application of microbial inoculum preventing and treating Meloidogyne incognita, its application process is：Using preceding using 30
The microbial inoculum is dried in~50 DEG C of warm water activation, and 1~2 portion of microbial inoculum of volume ratio adds 50~100 parts of warm water, and stir placement 5~8
Hour, using root perforated method is hindered, (hole that a 3~6cm of depth is dug in 2~3cm away from the root of tomato seedling applies concussion uniformly
Bacteria suspension), usage amount is per young plant 10~30mL.
The present invention finally additionally provides the spherical lysine bacillus and promotes tomato life using its manufactured microbial inoculum
Long application.
Endophytic bacterium is present in host, and living environment is stable, is not easy, by external environmental interference, to be present in root
Endogenetic bacteria and root-knot nematode there is identical ecological niche, unique ecological dominance has become biological control resource
Study hotspot.Therefore, the present invention obtains the endogenetic bacteria colonized in a kind of tomato root, and it can effectively prevent and treat Root Knot line
Worm, and the growth of tomato can also be remarkably promoted.
Technique effect：Relative to prior art, the spherical lysine bacillus RQ11 bacterial strains that the present invention obtains are a kind of
The endogenetic bacteria colonized in tomato root, the influence of external environment is not easily susceptible to, overcomes the environment that biocontrol microorganisms change in by soil
Influence.Because RQ11 has higher application value in terms of Meloidogyne incognita harm is prevented and treated, especially the bacterium is raw in tomato
Bacterium, persistently root-knot nematode can be worked, overcome general biocontrol microorganisms can not be colonized in root and caused by preventive effect it is low or
The defects of unstable.
Specific implementation method
The explanation that the gives an actual example present invention, but the invention is not limited in following examples.
The endophytic bacterium being related in the present invention is isolated from the various plants body in Nanjing Suburb farmland, is stored in south
Microbiological Lab of model university life science institute of capital；Tomato is purchased from market, and kind is that the cooperation 903 of Shanghai tomato research institute is big
Fructus Lycopersici esculenti (Lycopersiconesculentum)；Meloidogyne incognita (Meloidogyne incognita) is by Nanjing agriculture
University Li Hongmei professor give, and tomato expands numerous adopt.
What the raw and auxiliary material used selected in the present invention, reagent, instrument etc. were all well known in the art, other it is well known that
Some experimental facilities may be suitable for the implementation of in the following manner of the present invention.
The separation and screening of spherical lysine bacillus RQ11 bacterial strains：
By gathering tomato root, the tomato under ground portion diced facets sterilization of normal growth, sterilization utilizes routine
As long as method, the killing bacteria on surface, the root tissue block after sterilization is placed in the tissue homogenizer of high-temperature sterilization and ground
Mill, pour into dilute in conventional bacteria culture media after grinding and cultivated in 28 DEG C of insulating boxs of juxtaposition, picking is all to be separated to
Bacterium bacterial strain, all bacterial strains of separation are trained altogether with Meloidogyne incognita second instar larvae respectively by bacteria suspension and fermented supernatant fluid
Support and carry out bacterial strain of the screening acquisition with eelworm-killing activity.There is every plant of bacterium of bacterial strain of Preliminary activation to observe under the microscope for these
Thalli morphology, and number, shaft-like bacteriophage strain numbering is RG1, RG2, RG3 ...；Spherical bacteriophage strain numbering be RQ1,
RQ2、RQ3…….The bacterial strain of these active numberings further by potted tomato artificial infection root-knot nematode and is inoculated with this
Bacterial strain of the method screening with higher preventive effect of the bacteria suspension of a little bacterial strains, inoculation method are root-knot nematode inoculations：Using worm's ovum soil
With field soil 1:Tomato is planted after 1 mixing；3~5cm hole is dug in microbionation using root inocalation method is hindered in the root of tomato, is applied
10~20mL bacteria suspension.Tomato seedling greenhouse cellar culture investigates the growth-promoting functions and colonization ability of each bacterial strain for 30~50 days,
Investigation method document with reference to disclosed in this area.Handle is provided simultaneously with external eelworm-killing activity, colonized in tomato after biometrics
The significant bacterial strain of root and growth-promoting effect passes through the bacteria suspension and Root Knot of every plant of bacterium of potted tomato artificial infection again
The investigation of nematode egg cultivation 40~50 days obtains preventive effect highest bacterial strain, and it is most preferably bacterium as a result to find to be named as RQ11 bacterial strains
Strain.
Embodiment 1：The in-vitro screening of Meloidogyne incognita biocontrol bacteria -- gelatin hydrolysate activity primary dcreening operation
The 59 plants endogenetic bacteriums obtained will be separated in the flat lining out activation of NA, 28 DEG C of culture 48h.Picking single bacterium colony
Be inoculated in 5mL NB culture mediums (beef extract 0.3% (w/v), peptone 1% (w/v), NaCl 0.5% (w/v), pH 7.0~
7.2,121 DEG C of sterilizing 20min), seed liquor is made in 180rpm, 28 DEG C of culture 12h.Draw 5mL seed liquors and be inoculated in 50mL NB trainings
Support in base, 180rpm, 28 DEG C of culture 24h.Inoculum 5000rpm centrifugations 20min removes supernatant, and bacterium is rinsed with sterile distilled water
Body twice and is resuspended.With method of dilution butteron on plate by the concentration dilution of bacteria suspension to 1.34 × 108CFU/mL.After bacteria suspension gradient dilution
It is coated on gelatin culture medium (gelatin 0.5% (w/v), K2HPO40.05% (w/v), MgSO4·7H2O 0.02% (w/v), fine jade
Fat 1.5% (w/v)) on flat board, 28 DEG C of culture 48h.It is to hydrolyze the bacterial strain enclosed to select periphery of bacterial colonies to produce transparent circle, measurement hydrolysis
Loop diameter and colony diameter and the ratio (HC values) for calculating hydrolytic circle and colony diameter, each processing repeat three times.Experiment
As a result：There are 19 plants of surrounding bacterial colonies to generate transparent circle in 59 plants of endogenetic bacterias, wherein spherical lysine bacillus RQ11
Bacterial strain HC values are 2.07.
Embodiment 2：The in-vitro screening of Meloidogyne incognita biocontrol bacteria -- eelworm-killing activity screens
60h, collected after centrifugation supernatant are cultivated on 19 plants of bacterium NB culture mediums with gelatin hydrolysate that embodiment 1 obtains
And it is degerming again twice with 0.22 μm of filtering with microporous membrane, supernatant is diluted 5 times with sterile distilled water.Draw 200 μ L of supernatant
Liquid adds 10 μ L nematodes suspensions (about containing 50 Meloidogyne incognita second instar larvaes), 25 DEG C of trainings in 24 orifice plates in each hole
Support 12h.Adding 10 μ L 1%NaCl to every hole stimulates nematode, and the survival condition of nematode, living nematode worm are observed under microscope (40X)
The soft bending of body, polypide is stiff after dead wire worm stimulates.Count hot line borer population and dead wire borer population and calculate fermented supernatant fluid to two ages
The correction fatal rate of larva.Each processing is repeated 3 times, and the NB nutrient solutions of 5 times of dilution are as control.Bacteria suspension kills nematode：Draw
The bacteria suspension of 200 19 plants of bacterial strains of μ L adds 10 μ L nematode suspensions in 24 orifice plates in each hole, co-culture 24h.NaCl is stimulated
Nematode, micro- Microscopic observation count, and calculate correction fatal rate of the bacteria suspension to second instar larvae.Each processing is repeated 3 times, sterile steaming
Distilled water is as control.Root-knot nematode corrects fatal rate formula：Fatal rate=(verge of death borer population-line insect number) × 100%.Correction
Fatal rate=(experiment fatal rate-control fatal rate)/(1- compares fatal rate) × 100%.Result of the test：19 plants of endophytes have
There is eelworm-killing activity, wherein the correction fatal rate of spherical lysine bacillus RQ11 strain fermentation supernatants is up to
66.43%, the correction fatal rate of bacteria suspension is up to 52.76%.
Embodiment 3：The potted plant control effect testing of Meloidogyne incognita biocontrol bacteria
Select the consistent tomato seeds of full seed to be sown into the seedling basin equipped with sterilizing organic matter, 28 DEG C/20 DEG C, 14h light
According to/10h dark (daytime/evening), humidity 70% greenhouse in cultivate.It is transplanted to after growing 2 true leaves equipped with 1000g Nutrition Soils
Flowerpot in, Nutrition Soil is by the healthy loam that sterilizes and organic matter 1：1(v:V) mix, diameter 15cm, high 14cm on flowerpot, often
3 plants of tomato seedlings of pot transplanting.Slow seedling hinders piece-root grafting kind 10mL bacteria suspensions for every plant after one week, i.e., seedling side away from root 1cm with knife draw 3
~5cm zanjons are completely cured, to lateral irrigation note bacteria suspension of being completely cured.Same method access 10mL sterile distilled waters are negative control (CK),
The AVM that 10mL dilutes 100 times is positive control.In tomato both sides away from one 2~3cm of each stamp at rhizome 2cm after one week
Duck eye, with pipettor into each hole respectively plus 1mL worm's ovums suspension (about 500), it is each to handle 5 basins, in triplicate.Conventional pipe
Root knot number, the pieces of an egg number of Tomato Root System under different disposal are investigated after reason 50d, calculates root knot inhibiting rate and pieces of an egg inhibiting rate, according to
Root-knot Rating Chart investigate root knot index.Root knot inhibiting rate, pieces of an egg inhibiting rate formula：Root knot inhibiting rate=(cloudy
Property control root knot number-treatment group root knot number)/negative control root knot number.Pieces of an egg inhibiting rate=(negative control pieces of an egg number-treatment group
Pieces of an egg number)/negative control pieces of an egg number.Result of the test：There are 4 plants can significantly reduce tomato in 19 plants of endophytes that embodiment 1 is screened
Root knot index, its root knot inhibiting rate, pieces of an egg inhibiting rate and AVM indifference, spherical lysine bacillus RQ11 bacterial strains
Root knot inhibiting rate and pieces of an egg inhibiting rate are respectively 75.84% and 71.01%, and root knot index is 2.43.
Embodiment 4：Growth-promoting of the spherical lysine bacillus RQ11 bacterial strains to tomato
Tomato seeds 0.5%NaClO surface sterilization 2min, sterile distilled water rinse twice, are separately immersed in 4 plants of biological and ecological methods to prevent plant disease, pests, and erosions
8h in the bacteria suspension of bacterium.Seed is seeded in flowerpot after drying in the shade, and 3 seeds are broadcast per basin, each to handle 5 basins.Hanged after emergence with bacterium
Liquid irrigating root is inoculated with again, and sterile distilled water is control (CK), and each processing repeats three times.Investigated in greenhouse after cellar culture 60d
Aerial part fresh weight, plant height, root fresh weight, root long, 60 DEG C are dried to weighing aerial part dry weight and root dry weight after constant weight.Experiment knot
Fruit：Spherical lysine bacillus RQ11 bacterial strains can remarkably promote the aerial part fresh weight and stem length of tomato, compared with the control
Upper part fresh weight and stem length add 68.91%, 22.70% respectively.
Embodiment 5：Colonization dynamics of the spherical lysine bacillus RQ11 bacterial strains in tomato root
Carrying out resistance marker with sulfuric acid mycin to spherical lysine bacillus RQ11 bacterial strains can be containing 300 μ g/mL
Normal growth in the NA culture mediums of streptomycin sulphate.Tomato hinders piece-root grafting kind 10mL 1.34 × 10 after transplanting one week8CFU/mL bacterium are hanged
Liquid, per 5d, once, each processing every time randomly selects 3 plants of tomato seedlings, and every plant of root weighs fresh weight 0.2g, uses for investigation after inoculation
0.5%NaClO and each surface sterilization 5min of 70% ethanol, sterile distilled water rinse 5 times.1mL is added into the homogenizer of sterilizing
Sterile distilled water and tomato root are fully ground, and are drawn the dilution that 1mL homogenates carry out 0,10,100,1000 times, are respectively taken 200 μ L dilute
Homogenate coating NA flat boards after releasing, each dilution gradient apply 3 flat boards, count clump count after 28 DEG C of culture 48h, calculate accordingly
Quantity of 4 plants of bacterium in tomato root.Collect the rhizosphere soil of every plant of tomato natural air drying 24h at room temperature, the soil mistake after air-drying
1mm sieve.Every part of soil weighs 10g and mixed with the concussion of 90mL sterile distilled waters.30min is stood, 1mL supernatants is drawn and carries out
10th, 100,1000,10000 times of dilution, the supernatant after 200 μ L dilutions is respectively taken to be coated with NA flat boards, every portion of supernatant is each dilute
Release gradient and apply 3 flat boards, count clump count after 28 DEG C of culture 48h, calculate number of the RQ11 bacterial strains in tomato rhizosphere soil accordingly
Amount.As a result spherical lysine bacillus RQ11 bacterial strains colonizing in tomato root maintains 4.29 × 104CFU/mL, Rhizosphere Soil
4.66 × 10 are stably maintained in earth6CFU/mL,.
Embodiment 6：The sick nursery control effect testing of spherical lysine bacillus RQ11 microbial inoculums
The spherical lysine bacillus RQ11 strain cultured solutions of 50mL are filled with 8g skimmed milk powers, 1g NaCl, 1g peptones
Divide and mix, being put into 2h in -80 DEG C of refrigerators makes liquid fully charge, places into and 48h is handled in freeze drier.Crushing block after taking-up
The microbial inoculum of shape and the viable bacteria amount surveyed with gradient dilution rubbing method in microbial inoculum.Tomato seedling with two panels true leaf is transplanted in sick nursery
(3m × 4m), every plant of tomato seedling of experimental group hinders piece-root grafting kind 10mL 1g/mL Q1 microbial inoculums after one week, every plant of negative control group
Tomato seedling hinders piece-root grafting and enters 10mL 1g/mL empty carriers (CK), and every plant of tomato seedling of positive controls hinders piece-root grafting and enters 10mL dilutions 100
AVM again.1000 Meloidogyne incognita worm's ovums are accessed in every plant of tomato root after one week.Each 20 plants of tomato seedlings of processing.
With the worm's ovum in NaClO extraction methods separation root and nematode, worm's ovum and the nematode in rhizosphere soil are separated with sucrose centrifugal process, according to
Root-knot Rating Chart investigate root knot index.Result of the test：Spherical lysine bacillus RQ11 microbial inoculums can make kind
Nematode number in eggplant root significantly reduces 68.54%.
Embodiment 7：The identification of spherical lysine bacillus RQ11 bacterial strains
Morphologic identification reference《The outstanding Bacteria Identification handbook of uncle》With《Common bacteria system identification handbook》, mainly including bacterium
Fall morphologic observation, Gram's staining and spore staining.Molecular Identification selection 16S rRNA gene order, with Ezup pillar genes
The genomic DNA of group DNA extraction agents box (Shanghai life work) extraction bacterium, the amplification of 16S rRNA gene orders are led to using bacterium
With primer 2 7F and the 1492R ,-AGAGTTTGATCCTGGCTCAG-3 ' of forward primer 5 ' (SEQ ID No：1), reverse primer is
5’-GGTTACCTTGTTACGACTT-3’(SEQ ID No：2).Reaction system (25 μ L)：20 × Taq enzyme mix (Takara)
12.5 μ L, the μ L of forward primer 1,1 μ L, NDA template of reverse primer 1 μ L, the μ L of distilled water 9.5.Reaction condition：94 DEG C of pre-degenerations
4min；94 DEG C of denaturation 0.5min, 52 DEG C of annealing 0.5min, 72 DEG C of extension 1min, 30 circulate；72 DEG C of heat treatment 1min.Obtain
16S rRNA genetic fragments transfer to Shanghai Hua Da company be sequenced, sequence results submit ncbi database carry out BLAST comparisons.Examination
Test result：RQ11 bacterial strains bacterium colony is slightly yellow on NA flat boards, and a diameter of 3.0~3.5mm, surface is smooth, under biomicroscope
Thalline is into the linear alignment, and single thalline is into shaft-like, end circle, and thalline is about 2.0 μm, wide about 1.0 μm, Gram-negative, tentatively
It is lysine bacillus (Lysinibacillus) member to determine the bacterial strain.By the homologous comparisons of BLAST, RQ11 bacterial strains
16S rRNA gene orders and Lysinibacillussphaericus homology are 100%, therefore RQ11 bacterial strains are determined
For spherical lysine bacillus (Lysinibacillussphaericus).
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Claims (8)
1. a kind of spherical lysine bacillus, it is characterised in that be identified as spherical lysine bacillus
(Lysinibacillussphaericus), the entitled RQ11 of bacterial strain, it is general to be deposited in China Committee for Culture Collection of Microorganisms
Logical microorganism center, deposit number are：CGMCC No.14201, preservation date are on 05 27th, 2017.
2. spherical lysine bacillus according to claim 1, it is characterised in that the spherical lysine bacillus
It is from obtained by separation screening inside tomato root.
3. include the microbial inoculum of the spherical lysine bacillus described in claim 1 or 2.
4. the application of the spherical lysine bacillus preventing and treating Meloidogyne incognita described in claim 1 or 2.
5. application according to claim 4, it is characterised in that will be obtained after the spherical lysine Bacillus strain fermentation
To nutrient solution, then centrifuged fermented supernatant fluid, or bacteria suspension prepared by the thalline, can be used in preventing and treating Root Knot line
Worm.
6. the application of the microbial inoculum preventing and treating Meloidogyne incognita described in claim 3.
7. the spherical lysine bacillus described in claim 1 or 2 promotes the application of tomato growth.
8. the microbial inoculum described in claim 3 promotes the application of tomato growth.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108676741A (en) * | 2018-04-26 | 2018-10-19 | 南京农业大学 | A kind of functionality complex microorganism seedling medium and the preparation method and application thereof |
| CN108676762A (en) * | 2018-07-11 | 2018-10-19 | 中国农业大学 | One plant of lysine bacillus for preventing Plant nematode and its application |
| CN110055188A (en) * | 2019-03-07 | 2019-07-26 | 南京师范大学 | One plant of Paenibacillus polymyxa XW4 for producing bacteriostatic peptide and its separation screening and application |
| CN111763646A (en) * | 2020-07-24 | 2020-10-13 | 湖南省植物保护研究所 | Long lysine bacillus, microbial agent, biocontrol agent, and preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61205202A (en) * | 1985-03-08 | 1986-09-11 | Tsubaki Toshio | Nematocide and nematocidal method |
| JPH07138118A (en) * | 1993-11-19 | 1995-05-30 | Kazuo Tsubaki | Plant pest controlling agent |
| MXPA02010086A (en) * | 2000-04-11 | 2004-08-19 | Valent Biosciences Corp | METHOD FOR PRODUCING NEMATOCIDAL COMPOSITION BY HEAT TREATING A pH ADJUSTED FERMENTATION BROTH. |
| CN103039534A (en) * | 2012-12-28 | 2013-04-17 | 李峰 | Microbial preparation for inhibiting root-knot nematode and preparation method and application thereof |
| CN104031860A (en) * | 2014-05-27 | 2014-09-10 | 南京工业大学 | Bacillus sphaericus with high yield and high-temperature xylanase resistance and application thereof |
| CN105176886A (en) * | 2015-10-21 | 2015-12-23 | 湖南师范大学 | Lysinibacillus sphaericus and application of crystal protein and active product of Lysinibacillus sphaericus |
-
2017
- 2017-09-29 CN CN201710906499.6A patent/CN107460151B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61205202A (en) * | 1985-03-08 | 1986-09-11 | Tsubaki Toshio | Nematocide and nematocidal method |
| JPH07138118A (en) * | 1993-11-19 | 1995-05-30 | Kazuo Tsubaki | Plant pest controlling agent |
| MXPA02010086A (en) * | 2000-04-11 | 2004-08-19 | Valent Biosciences Corp | METHOD FOR PRODUCING NEMATOCIDAL COMPOSITION BY HEAT TREATING A pH ADJUSTED FERMENTATION BROTH. |
| CN103039534A (en) * | 2012-12-28 | 2013-04-17 | 李峰 | Microbial preparation for inhibiting root-knot nematode and preparation method and application thereof |
| CN104031860A (en) * | 2014-05-27 | 2014-09-10 | 南京工业大学 | Bacillus sphaericus with high yield and high-temperature xylanase resistance and application thereof |
| CN105176886A (en) * | 2015-10-21 | 2015-12-23 | 湖南师范大学 | Lysinibacillus sphaericus and application of crystal protein and active product of Lysinibacillus sphaericus |
Non-Patent Citations (6)
| Title |
|---|
| KLOEPPER, JW等: "《Induced systemic resistance and promotion of plant growth by Bacillus spp.》", 《PHYTOPATHOLOGY》 * |
| LING-LING YANG等: "《Lysinibacillus mangiferahumi sp. nov., a new bacterium producing nematicidal volatiles》", 《ANTONIE VAN LEEUWENHOEK》 * |
| R. A. SIKORA等: "《Modes of action associated with microbially induced in planta suppression of plant-parasitic nematodes》", 《AUSTRALASIAN PLANT PATHOLOGY》 * |
| XIANG, NI等: "《Biological Control of Meloidogyne incognita by Spore-forming Plant Growth-promoting Rhizobacteria on Cotton》", 《PLANT DISEASE 》 * |
| ZHENG, ZIQIANG等: "《Nematicidal spore-forming Bacilli share similar virulence factors and mechanisms》", 《SCIENTIFIC REPORTS 》 * |
| 刘杏忠等主编: "《植物寄生线虫生物防治》", 31 October 2004, 中国科学技术出版社 * |
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| CN108676741A (en) * | 2018-04-26 | 2018-10-19 | 南京农业大学 | A kind of functionality complex microorganism seedling medium and the preparation method and application thereof |
| CN108676741B (en) * | 2018-04-26 | 2022-04-22 | 南京农业大学 | Functional composite microorganism seedling culture substrate and preparation method and application thereof |
| CN108676762A (en) * | 2018-07-11 | 2018-10-19 | 中国农业大学 | One plant of lysine bacillus for preventing Plant nematode and its application |
| CN110055188A (en) * | 2019-03-07 | 2019-07-26 | 南京师范大学 | One plant of Paenibacillus polymyxa XW4 for producing bacteriostatic peptide and its separation screening and application |
| CN111763646A (en) * | 2020-07-24 | 2020-10-13 | 湖南省植物保护研究所 | Long lysine bacillus, microbial agent, biocontrol agent, and preparation method and application thereof |
| CN111763646B (en) * | 2020-07-24 | 2021-11-30 | 湖南省植物保护研究所 | Long lysine bacillus, microbial agent, biocontrol agent, and preparation method and application thereof |
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