CN107034143A - Prevent and treat the non-pathogenic mutation bacterial strain of Fusarium oxysporum sweet potato specialized form of stem rot of sweet potato - Google Patents
Prevent and treat the non-pathogenic mutation bacterial strain of Fusarium oxysporum sweet potato specialized form of stem rot of sweet potato Download PDFInfo
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Abstract
The invention provides a kind of non-pathogenic mutation bacterial strain of Fusarium oxysporum sweet potato specialized form for preventing and treating stem rot of sweet potato, the bacterial strain is the non-pathogenic mutation bacterial strain MG103 4 of Fusarium oxysporum sweet potato specialized form (Fusarium oxysporum f.sp.batatas), China typical culture collection center was preserved on 08 29th, 2016, deposit number is CCTCC M 2016435.
Description
Technical field
Present invention relates particularly to a kind of non-pathogenic mutation bacterial strain of Fusarium oxysporum sweet potato specialized form for preventing and treating stem rot of sweet potato.
Background technology
Stem rot of sweet potato is caused by Fusarium oxysporum sweet potato specialized form Fusarium oxysporum f.sp.batatas
Sweet potato tracheomycosis evil.This disease is mainly distributed on southeastern coast each province in China, wherein in Zhejiang, Fujian harm the most
Seriously, the sweet potato underproduction 10%~20% can typically be made, severe one is up to 50%.Using preventing and treating hand main in existing sweet potato production
Section is exactly breeding resistant variety, but is always difficult to the excellent production traits being combined with disease resistance trait in actual breeding, such as
In Fujian Liancheng " 90 days early " of large-scale plantation, " the not anti-potatos of rock potato 7-3 " cut disease, it is necessary to using the progress of the other measures such as medicament
The preventing and treating of stem rot of sweet potato.Raising with people to food and environmental safety requirements, biological control is used as a kind of green safety
Measure be increasingly valued by people, thus, explore stem rot of sweet potato biological prevention may advantageously facilitate sweet potato production
The development of industry.
Fusarium oxysporum bio-control factors species includes fungi (trichoderma Trichoderma, AMF at this stage
It is Arbuscular mycorrhizae, Nonpathogenic Fusarium oxysporum nonpathogenic Fusarium oxysporum, pale purple
Paecilomyces varioti Paecilomyces lilacinus), bacterium (Pseudomonas alba Pseudomonas, bacillus Bacillus), plant
Thing extract, actinomyces etc..Due to the non-pathogenic type reaping hook bacteria strain ecological condition similar with pathogenic strain needs, there is similar
Behavior and nutritional need are colonized, stronger Competition can be produced with pathological form bacterial strain, thus sickle-like bacteria non pathogenic strain is anti-
One of best, most stable of bio-control factors of effect.
The content of the invention
The technical problem to be solved in the present invention, is that providing a kind of Fusarium oxysporum sweet potato for preventing and treating stem rot of sweet potato specially changes
The non-pathogenic mutation bacterial strain of type.
What the present invention was realized in：A kind of non-pathogenic mutation of Fusarium oxysporum sweet potato specialized form for preventing and treating stem rot of sweet potato
Bacterial strain, the bacterial strain is non-pathogenic mutation bacterial strain MG103-4 (the Fusarium oxysporum of Fusarium oxysporum sweet potato specialized form
F.sp.batatas), China typical culture collection center was preserved on 08 29th, 2016, deposit number is CCTCC M
2016435。
The advantage of the invention is that：There is biological control effect to stem rot of sweet potato, shown in PDA culture medium to causing
The antagonistic effect of germ Vegetation space competition, this biocontrol bacterial strain is treated after sweet potato, can significantly reduce the incidence of disease of dead arm；
GFP Green Fluorescent Protein genes are added in T-DNA, are easy to the environmental behaviour of biocontrol microorganisms to monitor；The present invention is from nature
Method of the problem of biocontrol microorganisms are present there is provided solution is separated in environment, also causes disease for Fusarium oxysporum on other crops
Biocontrol microorganisms exploitation is offered reference.
Brief description of the drawings
The present invention is further illustrated in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is electrophoretograms of the pCAMBIA1300-ptrpC-hph-gfp after EcoR I and Sal I digestions in the present invention.
Fig. 2 is showing for the production spore ability of the non-pathogenic mutation bacterial strain MG103-4 of Fusarium oxysporum sweet potato specialized form in the present invention
It is intended to.
MG-Fusarium oxysporum sweet potato specialized form bacterial strain MG
The non-pathogenic mutation bacterial strain MG103-4 of MG50-5-Fusarium oxysporum sweet potato specialized form
Embodiment
Bacterial strain preservation
Non- pathogenic mutation bacterial strain MG103-4 (the Fusarium oxysporum of Fusarium oxysporum sweet potato specialized form
F.sp.batatas MG103-4), it was preserved in China typical culture collection center (CCTCC), ground on 08 29th, 2016
Location is Wuhan City, Hubei Province Wuchang District Bayi Road Luo Jia Shan, and deposit number is CCTCC M 2016435
Strains tested:Fusarium oxysporum sweet potato specialized form bacterial strain MG (Fusarium oxysporum f.sp.batatas
MG), China typical culture collection center (CCTCC) was preserved on 08 29th, 2016, address is that Wuhan City, Hubei Province is military
Prosperous area's Bayi Road Luo Jia Shan, deposit number is CCTCC M 2016432.
The non-pathogenic mutation bacterial strain MG103-4 of Fusarium oxysporum sweet potato specialized form specific preparation method is as follows：
1.1 test material
Sweet potato variety novel species is spent (dead arm responsive type) to be preserved by this research department and planted, pCT74 (Shanghai north promise biotechnologies
Co., Ltd), pCAMBIA1300 (ocean bio tech ltd of Beijing CHMC), pVOK21 is given by China Agricultural University,
PMD18-T is purchased from precious bioengineering (Dalian) Co., Ltds of Takara-；All restriction enzyme BstX I, Sal I, Xho I
Precious bioengineering (Dalian) Co., Ltds of Takara- are purchased from EcoR I, hygromycin B is purchased from Roche companies, and Ticarcillin/Clavulanate Acid is purchased from
The Shanghai past bio tech ltd, acetosyringone, MES, composite fibre miillpore filter be purchased from raw work bioengineering (on
Sea) limited company, remaining Agrobacterium-mediated Transformation is purchased from Chemical Reagent Co., Ltd., Sinopharm Group with medicine.
1.2 build double base fluorescence carrier pCAMBIA1300-ptrpC-hph-gfp
First with EcoR I and Sal I two kinds of restriction enzymes difference digested plasmid pCT74 and pCAMBIA1300, return
Harvest to obtain Sal I-ptoxA-gfp-nos-EcoR I and Sal I-pCAMBIA1300-EcoR I, structure after two fragments are connected
Build completion carrier pCAMBIA1300-gfp；Then two kinds of digestion with restriction enzyme plasmids of Xho I and BstX I are used
PCAMBIA1300-gfp (removes CaMV35S-hph fragments), and is that template (is used in fungi and commonly used using plasmid pKOV21
PtrpC promoters start hygromycin B resistant gene expression), PCR amplification ptrpC-hph fragments are separately added into fragment head and the tail
Xho I and BstX I restriction enzyme sites, primer is HphRC-F-Xho I and ptrpCRC-R-BstX I.
HphRC-F-Xho I：5 '-CTCGAGCTATTTCTTTGCCCTCGGAC-3 ' (such as SEQ ID NO：Shown in 1)；
ptrpCRC-R-BstX I：
5 '-CCAACATGGTGGGTCGACAGAAGATGATATTGAAGG-3 ' (such as SEQ ID NO：Shown in 2).
Reaction system：μ L, the dNTP Mixture (2.5mmol/L each) 8 of 50 μ L, 2 × GC Buffer I of cumulative volume 25
μ L, primer HphRC-F-Xho I (10 μm of ol/L) 1.25 μ L, primer ptrpCRC-R-BstX I (10 μm of ol/L) 1.25 μ L, mould
The μ L of plate pKOV21 (the final concentration of 0.1-10ng of plasmid in system) 1.5 μ L, LA taq 0.5, use ddH2The μ L of O polishings 50, wherein,
Reagent is purchased from precious bioengineering (Dalian) Co., Ltds of Takara-.
Response procedures：94 DEG C of pre-degeneration 1min of the first step；Second step 94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extensions
2min, 30 circulations of second step；3rd 72 DEG C of step extends 10min.
PCR primer is inserted after pMD18-T carriers by coupled reaction, is obtained pMD18-ptrpC-hph and is sent to platinum and still gives birth to
Thing Technology Co., Ltd. is sequenced, pMD18-ptrpC-hph sequences such as SEQ ID NO：Shown in 3, sequence alignment, sequencing result are carried out
After correct, with Xho I and BstX I digestion pMD18-ptrpC-hph plasmids and acquisition BstX I-ptrpC-hph-Xho I are reclaimed
Fragment.PCAMBIA1300-gfp skeletons are finally connected acquisition with BstX I-ptrpC-hph-Xho I fragments
PCAMBIA1300-ptrpC-hph-gfp carriers.
After the completion of pCAMBIA1300-ptrpC-hph-gfp plasmid constructions, first to pCAMBIA1300-ptrpC-
Hph-gfp plasmids carry out EcoR I and Sal I digestions and primarily determine that SalI-ptoxA-gfp-nos-EcoR I fragments have been connected into
(see Fig. 1, wherein mark 1 represents marker standard molecular weights, mark 2 is in pCAMBIA1300-ptrpC-hph skeletons
Band of the pCAMBIA1300-ptrpC-hph-gfp plasmids through digestion verification), performing PCR amplification ptrpC-hph fragment (tools are entered again
Body is shown in 1.2 test method) and GFP fragments.
The response procedures and reaction system of PCR amplification GFP fragments are as follows：
Primer P28：5′-TAGTGGACTGATTGGAATGCATGGAGGAGT-3′；
Primer P29：5′-GATAGAACCCATGGCCTATATTCATTCAAT-3′.
Reaction system：The μ L of cumulative volume 20, reagent is purchased from precious bioengineering (Dalian) Co., Ltds of Takara-)：10×
PCR Buffer I 2 μ L, dNTP Mixture (2.5mmol/L each) 1.6 μ L, primer P28 (10 μm of ol/L) 0.5 μ L, draw
Thing P29 (10 μm of ol/L) 0.5 μ L, template is pCAMBIA1300-ptrpC-hph-gfp (final concentration of 0.1- of plasmid in system
10ng) 1 μ L, rTaq0.2 μ L, use ddH2The μ L of O polishings 50.
Response procedures：94 DEG C of pre-degeneration 4min of the first step；Second step 94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extensions
1.5min, 35 circulations of second step；3rd 72 DEG C of step extends 10min.
Two PCR primers pass through 1% agarose gel electrophoresis and gel image analysis system detectio, ptrpC-hph PCR
Primer size is 1392bp, and GFP PCR primer size is 417bp, reclaims purpose fragment and is sent to Bo Shang Bioisystech Co., Ltd
Sequencing, and carry out sequence alignment, GFP PCR primer sequence such as SEQ ID NO：Shown in 4, it is determined that by ptrpC-hph fragments
It is imported into GFP in carrier pCAMBIA1300.
1.3 Agrobacterium-mediated Transformations, the detection of conversion situation and transformant single spore separation are preserved
Fusarium oxysporum Agrobacterium-mediated Transformation process and culture medium reference literature (Khang C H, Park the S Y, Rho used
H S,et al.Filamentous fungi(Magnaporthe grisea and Fusarium oxysporum)//
Agrobacterium Protocols[M].Totowa:Humana Press, 2007,403-420.) method slightly change,
Change part as follows：
By Fusarium oxysporum sweet potato specialized form bacterial strain MG PDA solid mediums (in 1L culture mediums contain 200g potatos,
15g glucose, 15g agar powders) purify after culture 7d, addition sterilized water, which scrapes conidium, to come out with 2 layers of sterile lens wiping paper
Filtering, and conidium concentration is adjusted to 1 × 10 with IM fluid nutrient mediums (being shown in Table 1)6CFU/mL, obtains Fusarium oxysporum sweet potato
Specialized form bacterial strain MG spore suspensions, it is standby.The Agrobacterium AGL-1 of pCAMBIA1300-ptrpC-hph-gfp carriers will be transferred to
(1 is shown in Table in MM fluid nutrient mediums) 28 DEG C, and 250r/min is shaken after training 2d, with the IM liquid containing 200 μm of ol/L acetosyringones
Bacterial concentration is adjusted to OD by culture medium600=0.15, then shake training 6h (OD600About 0.6), agrobacterium liquid is obtained.Will mixing fibre
Dimension miillpore filter is placed on the CM solid mediums (being shown in Table 1) containing 200 μm of ol/L acetosyringones, afterwards by 100 μ L points
Fusarium oxysporum sweet potato specialized form bacterial strain MG spore suspensions and 100 μ L agrobacterium liquids, which are mixed, is coated on composite fibre miillpore filter
On, 25 DEG C, light culture 2d.Composite fibre miillpore filter is divided into fritter with knife after co-cultivation to disperse to put screening and culturing medium
In (the μ g/ml containing Hygromycin B concen 100, the μ g/ml of Ticarcillin/Clavulanate Acid concentration 200 PDA culture medium), 28 DEG C of light cultures obtain transformant.
Various bacterium used in conversion process are both placed in water isolation type constant incubator GNP-9080 types and cultivated.Cultivated on screening and culturing medium
After 3d, the whole ware of transformant is placed in BD-BGC1 blue light bale cutting instruments, colony colour is observed under blue excitation light.For energy
Its spore progress monospore purifying of the bacterium colony picking of green fluorescence is sent, the bacterium colony that monospore is formed is then transferred to placement aseptic filter paper
On the screening and culturing medium of piece.Bacterium colony is put again after bacterium colony grows to and do not have filter paper and observes colony colour under blue light, if bacterium colony according to
Green fluorescence so is sent, then filter paper is put into sterile envelope and preserved.
Reagent and culture medium prescription table needed for the Agrobacterium-mediated Transformation of table 1
The pCAMBIA1300-ptrpC-hph-gfp carriers built enter Fusarium oxysporum by AGL-1 Agrobacterium-mediated Transformations
Sweet potato specialized form bacterial strain MG, it is glimmering that the transformant grown after hygromycin resistance screens 3d can send green under blue excitation light
Light, illustrates that the T-DNA with GFP Green Fluorescent Protein genes has successfully been integrated into Fusarium oxysporum sweet potato specialized form gene
Group.In conversion process 1 × 10 is about coated with a 5cm diameter composite fibre miillpore filter6Individual conidium, final every film is about
10 transformants are grown, after a small amount of spore single spore separation of bacterium colony picking for sending green fluorescence and filter paper preservation, are finally obtained
Obtained the Fusarium oxysporum sweet potato specialized form mutant of 711 purifying cultures.
The screening (pathogenicity Preliminary detection) of the 1.4 non-pathogenic mutation bodies of Fusarium oxysporum sweet potato specialized form
With 5 × 10 in production5CFU/mL Fusarium oxysporum sweet potato specialized form bacterial strain MG spore suspensions do Pathogenic Tests
It can distinguish the sweet potato of Different resistance levels well, therefore Fusarium oxysporum sweet potato specialized form bacterial strain MG all in text and turn
It is all to use 5 × 10 that beggar, which connects the experiment of bacterium sweet potato,5CFU/mL spore suspension concentration.Fusarium oxysporum sweet potato is prepared specially to change
After type bacterial strain MG spore suspensions and transformant spore suspension, the about 20cm stems at the top of the novel species flower stem of field clip health
Section, wound is soaked into spore suspension down bacterium solution is abandoned after 20min, and stem section is inserted into culture in clear water (in match good fortune cold light
In the phytoclimate case of source, 28 DEG C of temperature, photoperiod 16L:8D), with Fusarium oxysporum sweet potato specialized form bacterial strain MG spore suspensions
There are more than 4 grades typical disease symptoms and is defined starting statistics incidence and (being inoculated with sharp spore under suitable conditions in novel species flower after processing
Sickle-like bacteria sweet potato specialized form bacterial strain MG spore suspensions 5d can fall ill to 4 grades, dead after 7d), it is special with Fusarium oxysporum sweet potato
Change type bacterial strain MG spore suspensions and clear water are processed as control, and single bacterial strain handles 10 stem sections, 3 repetitions.
Dead arm severity Scaling standard：In units of strain, 0 (0.0) level：It is disease-free；1 (0.1) level：Overground part growth is normal,
Basal part of stem vascular bundle browning is in 3cm or so；2 (0.2) levels：Overground part is normal, and basal part of stem vascular bundle browning is 1/4 or so；3
(0.5) level：Basal part of stem blade turns yellow, and vascular bundle browning is more than 1/2；4 (0.8) levels：Withered, the stem of plant leaf majority flavescence
Vascular bundle browning extends and terminal bud；5 (1.0) levels：Complete stool is withered.
Disease index=(0.1 × n1+0.2×n2+0.5×n3+0.8×n4+1.0×n5)/N×100。
N is diseased plant numbers at different levels；N is total strain number of participating in the experiment.When disease index is 0.0~20.5, plant shows high anti-reflective at once,
It is non-pathogenic mutation body to regard as mutant.
711 mutant of Fusarium oxysporum sweet potato specialized form are carried out with pathogenicity detection experiment, 40 pathogenicities are obtained
The mutant of decline.The Detection of Stability of the non-pathogenic mutation body of mutant declined to 40 pathogenicities, including flat board switching are steady
The Detection of Stability of qualitative and non-pathogenic, non-pathogenic mutation body was at least transferred on PDA solid mediums after 6 generations, observed bacterium
Fall form and place to see whether that green fluorescence can be sent under blue light.The stable mutant of character inoculates novel species flower and caused
Sick power repeats to detect, each to detect 10 plants, 3 repetitions.Finally give the non-pathogenic mutation bacterial strain of Fusarium oxysporum sweet potato specialized form
MG103-4, and the non-pathogenic mutation bacterial strain MG103-4 pathogenicities of Fusarium oxysporum sweet potato specialized form are that disease index is 13.03, phase
Significantly reduced for wild type Fusarium oxysporum sweet potato specialized form bacterial strain MG disease index 95.03, novel species flower outward appearance is not showed
Go out disease symptom.
1.5 non-pathogenic mutation body safety detections
Will be with Fusarium oxysporum sweet potato specialized form bacterial strain MG and the non-pathogenic mutation bacterial strain of Fusarium oxysporum sweet potato specialized form
It is about 2.67 × 10 that MG103-4, which is configured to concentration,6CFU/mL spore suspension, takes 150mL and 400mL sterile soils respectively
Dress basin is mixed, sets clear water to mix native control.Health " novel species flower " stem section that field is gathered is transplanted to basin, often 10 plants, 3 times of basin
Repeat.Treated potted plant be placed in cold light source incubator is cultivated, condition setting is 28 DEG C of temperature, humidity 95%, photoperiod
16L:8D。
Because the pathogenicity Preliminary detection of above-mentioned steps 1.4 is only to mutant spore suspension of the sterilized water for medium
Novel species flower 20min is handled, and really biocontrol strains need to act on sweet potato plant in soil for a long time.Therefore test
Further connect in the way of the non-pathogenic mutation bacterial strain MG103-4 spore suspensions of Fusarium oxysporum sweet potato specialized form mix Nutrition Soil
Bacterium novel species flower is to determine its security.Soil is mixed with Fusarium oxysporum sweet potato specialized form bacterial strain MG spore suspensions and sterilized water mixes soil
For control, as a result show the novel species flower of Fusarium oxysporum sweet potato specialized form bacterial strain MG processing under same culture conditions in 7d sequela
More than 4 grades, no greenery are close to death, and the non-pathogenic mutation bacterial strain MG103-4 processing of Fusarium oxysporum sweet potato specialized form is new
Plant flower and do not show disease symptom as water process, disease index is significantly reduced, equivalent to the high anti-kind of sweet potato for sharp spore
Sickle-like bacteria sweet potato specialized form bacterial strain MG Resistant reaction level, as a result further demonstrate the non-cause of Fusarium oxysporum sweet potato specialized form
This mutant pathogenicity of sick mutant strain MG103-4 is lost, and continuous contact novel species flower will not still cause a disease.
The production spore situation detection of 1.6 transformants
The non-pathogenic mutation bacterial strain MG103-4 of transformant Fusarium oxysporum sweet potato specialized form of single spore separation after purification is existed
In 28 DEG C of 10~14d of activation culture in PDA culture medium, conidium is scraped wash-off by addition sterilized water with spreader in culture dish
Come.Scrape washing lotion and spore suspension is made with 2 layers of sterile lens wiping paper filtering, spore count is carried out with blood counting chamber, 1 ware is calculated
All spore counts in (a diameter of 6cm) transformant.Experiment is repeated 3 times, every time 3 ware.
Experimental result is as follows：
In PDA culture medium, either Fusarium oxysporum sweet potato specialized form bacterial strain MG, is also the sharp spore sickle of non-pathogenic mutation body
Felt shape, the back side Magnifying chromoscopy depth is all presented in the non-pathogenic mutation bacterial strain MG103-4 of knife bacterium sweet potato specialized form, positive mycelia quality
And wheel line is different, and with different incubation time batches difference.Non- cause a disease of Fusarium oxysporum sweet potato specialized form is dashed forward
The conidium yield for becoming bacterial strain MG103-4 is also remarkably decreased (see Fig. 2).Caused a disease in addition, Fusarium oxysporum sweet potato specialized form is non-
Mutant strain MG103-4 spore shape does not occur significantly relative to wild type Fusarium oxysporum sweet potato specialized form bacterial strain MG
Change.
1.7 biocontrol effects are detected
1.7.1 opposite culture is tested 28 DEG C of light culture 5d Fusarium oxysporum sweet potato specialized form bacterial strain MG and sharp spore reaping hook
The edge of the non-pathogenic mutation bacterial strain MG103-4 of bacterium sweet potato specialized form bacteria cake is punched with a diameter of 0.5cm card punch, by sharp spore
Sickle-like bacteria sweet potato specialized form bacterial strain MG and the non-pathogenic mutation bacterial strain MG103-4 of Fusarium oxysporum sweet potato specialized form bacteria cake are right respectively
Title is placed in PDA culture medium away from edge about diameter 1/4, and 28 DEG C of light cultures, experiment is repeated 3 times.
1.7.2 biocontrol effect pot experiment is by the non-pathogenic mutation bacterial strain MG103-4 spores of Fusarium oxysporum sweet potato specialized form
Configuration concentration is into 5 × 105CFU/mL spore suspension, 20cm stem section wounds at the top of stem are spent by the healthy novel species of field clip
Soak wherein, taken out respectively after immersion 2,4,8,16,24h down, (every basin is about in preprepared basin containing bacterium alms bowl for cuttage
400mL sterile vegetative soil and 150mL 2.67 × 106The Fusarium oxysporum sweet potato specialized form bacterial strain MG spores of CFU/mL concentration
Uniform suspension is mixed), 10 plants are often handled, is repeated 3 times, seedling cuttage is soaked in identical Fusarium oxysporum sweet potato specialized form bacterium with clear water
The soil and sterile soil of strain MG spore concentrations are control.Treated potted plant be placed in cold light source incubator is cultivated, condition is set
It is set to 28 DEG C of temperature, humidity 95%, photoperiod 16L:8D.
Opposite culture result：
The Fusarium oxysporum sweet potato specialized form bacterial strain MG and non-pathogenic mutation bacterial strain MG103-4 of Fusarium oxysporum sweet potato specialized form
It can be seen that obvious linearly bacterium colony boundary, mycelia occur in the middle of culture dish in two bacterium colonies of each ware after opposite culture 5d
Between do not merge into each other, illustrate the non-pathogenic mutation bacterial strain MG103-4 of Fusarium oxysporum sweet potato specialized form and Fusarium oxysporum sweet potato
There is nutrient competition and competition for space in specialized form bacterial strain MG, mycelia expansion rate is suitable.But, relative to Fusarium oxysporum sweet potato
For the non-pathogenic mutation bacterial strain MG103-4 of specialized form, Fusarium oxysporum sweet potato specialized form bacterial strain MG bacterium colonies are thicker denser, growth
Important and influential persons is more preferable.
Prevention effect of the different pretreatments duration of table 2 to stem rot of sweet potato
Note：MG refers to Fusarium oxysporum sweet potato specialized form bacterial strain, and MG55-7 refers to the non-cause of Fusarium oxysporum sweet potato specialized form
Sick mutant strain MG55-7；The different capitalizations that disease index is arranged and prevention effect is arranged represent difference extremely significantly (P ＜ 0.01)
Biocontrol effect results from pot experiment test：
The non-non- pathogenic mutation bacterial strain MG103-4 of pathogenic mutation body Fusarium oxysporum sweet potato specialized form is in processing novel species flower 2h
Afterwards, there is prevention effect to dead arm, with the extension of processing time, the non-pathogenic mutation bacterial strain of Fusarium oxysporum sweet potato specialized form
MG103-4 biocontrol effect is also gradually obvious, and after processing 16h, biocontrol effect tends towards stability, and after processing 24h, biocontrol effect reaches
To 86.98% (being shown in Table 2).
T-DNA insertional mutagenesis libraries of the invention by building Fusarium oxysporum sweet potato specialized form, filter out non-cause a disease and dash forward
Variant is finally obtained that the biocontrol bacterial strain Fusarium oxysporum sweet potato specialized form for stem rot of sweet potato is non-to cause a disease as biological and ecological methods to prevent plant disease, pests, and erosion resource
Mutant strain MG103-4.Due to being that directly Fusarium oxysporum sweet potato specialized form is transformed, therefore the biocontrol microorganisms obtained are theoretical
Preferably, Vegetation space competition is also the fiercest using rear environmental suitability preferably for upper and pathogen niche overlap.Secondly,
Because T-DNA insertion positions can be positioned by TAIL-PCR, therefore the heredity of non-pathogenic mutation body can be expressly understood that
Background.Again, the present invention also adds GFP Green Fluorescent Protein genes in T-DNA, is easy to the environmental behaviour of biocontrol microorganisms
Monitoring.The present invention is separates the method that the problem of biocontrol microorganisms are present provides solution from natural environment, also on other crops
Fusarium oxysporum causes the biocontrol microorganisms exploitation of disease to be offered reference.
Claims (1)
1. a kind of non-pathogenic mutation bacterial strain of Fusarium oxysporum sweet potato specialized form for preventing and treating stem rot of sweet potato, it is characterised in that：It is described
Bacterial strain is non-pathogenic mutation bacterial strain MG103-4 (the Fusarium oxysporum of Fusarium oxysporum sweet potato specialized form
F.sp.batatas), China typical culture collection center was preserved on 08 29th, 2016, deposit number is CCTCC M
2016435。
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| CN201610856906.2A CN107034143A (en) | 2016-09-28 | 2016-09-28 | Prevent and treat the non-pathogenic mutation bacterial strain of Fusarium oxysporum sweet potato specialized form of stem rot of sweet potato |
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| CN201610856906.2A CN107034143A (en) | 2016-09-28 | 2016-09-28 | Prevent and treat the non-pathogenic mutation bacterial strain of Fusarium oxysporum sweet potato specialized form of stem rot of sweet potato |
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| CN201610856906.2A Pending CN107034143A (en) | 2016-09-28 | 2016-09-28 | Prevent and treat the non-pathogenic mutation bacterial strain of Fusarium oxysporum sweet potato specialized form of stem rot of sweet potato |
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| JP2007082499A (en) * | 2005-09-26 | 2007-04-05 | Ibaraki Prefecture | New strain of fusarium oxysporum |
| CN102492715A (en) * | 2011-12-12 | 2012-06-13 | 河南省农业科学院 | Genetic transformation method for agrobacterium tumefacien-mediated fusarium oxysporum sesame specialized strain |
| CN103503924A (en) * | 2013-08-29 | 2014-01-15 | 中国农业科学院蔬菜花卉研究所 | Preparation method and applications of fusarium oxysporum inoculant |
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| JP2007082499A (en) * | 2005-09-26 | 2007-04-05 | Ibaraki Prefecture | New strain of fusarium oxysporum |
| CN102492715A (en) * | 2011-12-12 | 2012-06-13 | 河南省农业科学院 | Genetic transformation method for agrobacterium tumefacien-mediated fusarium oxysporum sesame specialized strain |
| CN103503924A (en) * | 2013-08-29 | 2014-01-15 | 中国农业科学院蔬菜花卉研究所 | Preparation method and applications of fusarium oxysporum inoculant |
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Application publication date: 20170811 |