CN105274020B - A kind of curing ginger stalk rot bacterium antagonistic bacterium and application - Google Patents
A kind of curing ginger stalk rot bacterium antagonistic bacterium and application Download PDFInfo
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Abstract
The invention discloses a kind of curing ginger stalk rot bacterium antagonistic bacteriums, it is named as Burkholderia cepacia (Burkholderia cenocepacia) C3, China Committee for Culture Collection of Microorganisms's common micro-organisms center, address are preserved on October 17th, 2014：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, deposit number are CGMCC No.9791.Curing ginger stalk rot bacterium antagonistic bacterium C3 up to 41.18%, is significantly increased to curing ginger stalk rot preventive effect compared with the control, and incidence, disease index decrease compared with CK2 after processing, illustrates that C3 protection effects are ideal.
Description
Technical field
The present invention relates to a kind of curing ginger stalk rot bacterium antagonistic bacterium and applications.
Background technology
Curing ginger stalk rot is also known as ginger soft rot, ginger stem rot etc., is infected and is caused by pythium spp, reported for the first time in 1907
Road is found in Surat of India, now generally betides India, Australia, Japan, Nigeria, Hawaii, Fiji, Si Lilan
The areas such as card, South Korea and China Taiwan, the evil of being critically ill are serious.It is reported that part grave illness growing area is every year because of curing ginger stalk rot
The caused underproduction is up to 80%~90%, or even total crop failure.In Nepal's crop field loss 25% caused by the disease, wall losses is stored
24%.In India's grave illness field loss up to 90% or more.Curing ginger stalk rot pathogen is oomycota rotten mold genus, in fungi
Curing ginger stalk rot pathogen is under the jurisdiction of Oomycete, pythiaceae, pythium on kingdom.The pathogen is a kind of lower fungus,
In a manner of saprophytic long-term survival in the soil, higher plant is parasitized, causes harm root and basal part of stem causes to rot, show as sudden
, root-rot and stem rot.
For the research of curing ginger stalk rot cause of disease, occurrence regularity, Infection cycie rule and the factor etc. for influencing morbidity,
The control measure in production mainly has cultural control, Agro-chemicals control and biological control at present.The plurality of advantages of chemical agent
So that it is widely used in agricultural production, but gradually increasing with human health and environmental consciousness, pesticide it is long-term big
Use lack of standardization is measured, adjoint problems also gradually display.Such as environmental pollution, pesticide residue, anti-medicine more outstanding
Property generation and to non-target organism directly poison etc..And biological control compensates for lacking for chemical pesticide to a certain extent
It falls into, significant role will be played in the future of agriculture sustainable development.But biological control at present is limited only to indoor pot experiment,
Biological control is applied to field and still needs long time.However from Environmental security and human health angle, finally
Safely and effectively biological control method and approach are found out, gradually to replace existing chemical prevention.
Biocontrol agent was widely studied and has answered because of the advantages that its environment friendly, performance duration, preventive effect specific aim
With having a high potential.Both at home and abroad it has been reported that a large amount of biological prevention and control agent development and being applied successfully.Such as common Dipel, in vain
Stiff bacterium etc. has been commercialized production application, and achieves good preventive effect.
Invention content
For the above-mentioned prior art, the present invention provides a kind of curing ginger stalk rot bacterium antagonistic bacterium and applications.
The present invention is achieved by the following technical solutions：
Curing ginger stalk rot bacterium antagonistic bacterium provided by the invention, the Strain Designation are Burkholderia cepacia
(Burkholderia cenocepacia) C3 has been preserved in Chinese microorganism strain preservation management committee on October 17th, 2014
Member's meeting common micro-organisms center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, deposit number
For CGMCC No.9791.
The main biological property of above-mentioned bacterial strains is：Burkholderia cepacia (Burkholderia
Cenocepacia), Gram-negative bacteria, bacterium colony yellow-white, the smooth corrugationless in surface, single bacterium colony is round, in PDA culture medium
Colony growth rate is fast.
The method that curing ginger stalk rot bacterium antagonistic bacterium C3 is used to prepare biocontrol agent is：With Optimal Medium culture ginger
Bacterial suspension 6000rpm when 600nm OD values are 1, is centrifuged 10min, thalline is in equal volume by brown foot rot germ antagonistic bacterium
Sterile water or phosphate buffer suspend again, and it is 5 × 10 to be diluted to bacterial concentration8Cfu/ml, 4 DEG C of placements are spare after packing.
Beneficial effects of the present invention,
1. protection effect measurement shows that curing ginger stalk rot bacterium antagonistic bacterium C3 reaches curing ginger stalk rot preventive effect in Room
41.18%, it is significantly increased compared with the control, incidence, disease index decrease compared with CK2 after processing, illustrate C3
Protection effect is ideal；
2. a pair curing ginger stalk rot bacterium antagonistic bacterium C3 fermentation conditions optimize, to basal medium, nutrient media components
Single factor experiment is studied, the results showed that effect is best when glycerine and yeast powder are respectively as culture medium carbon nitrogen source, culture medium
The orthogonal test of component shows glycerine 2%, yeast powder 1%, K2HPO40.2%, MgSO40.1% proportioning is optimal proportion, is led to
Cross the influence to thalli growth amount such as incubation time, inoculum concentration, Medium bottling volume and pH studies have shown that with inoculum concentration 3%,
When the bottled amounts of 150ml/250ml, incubation time 48h, pH are 8-9, the bacterial strain cell concentration is maximum；
3. pair Activities of Fermentation Broth substance is into trip temperature, the stability experiment of protease and the aspect of ultraviolet light three.Hair
Zymotic fluid changes unobvious after trypsase and pepsin to the inhibition of pathogen；After treatment of different temperature 30min
It is compared with a control, pathogen colony diameter is without significant change；Fungistatic effect after ultraviolet light Continuous irradiation 12h with to photograph
Compare no significant difference.Therefore the bacterium active material has stronger stability to heat, protease and ultraviolet light.
Description of the drawings
Fig. 1 is curing ginger stalk rot bacterium antagonistic bacterium C3 colonial morphologies；
Fig. 2 is inhibiting effect of the antagonistic bacterium C3 to curing ginger stalk rot bacterium；
Fig. 3 is that for antagonistic bacterium C3 to the mycelial effect of curing ginger stalk rot bacterium, wherein A is control, B under light microscope
For with C3 handle 2 days after the mycelial form of cause of disease；
Fig. 4 is inhibition zone sizes of the antagonistic bacterium C3 to curing ginger stalk rot bacterium and other pathogens；
Fig. 5 is bacteriostasis figures of the antagonistic bacterium C3 to curing ginger stalk rot bacterium and other pathogens；
Fig. 6 is influence of the different culture media to thalline fermentation concentration；
Fig. 7 is influence of the different carbon source to thalline fermentation concentration；
Fig. 8 is influence of the different nitrogen sources to thalline fermentation concentration；
Fig. 9 is influence of the different bottling amounts to thalline fermentation concentration；
Figure 10 is influence of the different vaccination amount to thalline fermentation concentration；
Figure 11 is influence of the different fermentations time to thalline fermentation concentration；
Figure 12 is influences of the different pH to thalline fermentation concentration；
Figure 13 is treatment of different temperature on the active influence of antibacterial material in zymotic fluid；
Figure 14 is UV treatment on the active influence of antibacterial material in zymotic fluid；
Figure 15 is Protease Treatment on the active influence of antibacterial material in zymotic fluid.
Specific implementation mode
In conjunction with embodiment, the present invention is further illustrated, it should which explanation, following the description is merely to explain
The present invention is not defined its content.
The reagent of not detailed description, method are the conventional reagent of fields, method in the present invention.
Embodiment 1：The acquisition and identification of curing ginger stalk rot bacterium antagonistic bacterium C3
Antagonistic bacterium is screened from shallot rhizosphere, the antagonistic bacterium obtained to screening using morphology and molecular biology method
It is identified.
(1) Morphological Identification：
The antagonistic bacterium filtered out is lined NA culture mediums, culture is for 24 hours by ne ar in the case that suitable for stablizing relatively
Single bacterium colony feature is observed afterwards, including whether there is or not the progress such as discoloration for the homogeneity of colonial morphology, size, color, thalline smell, culture medium
Preliminary Identification, the results are shown in Figure 1；
(2) molecular biology identification：
By 16S rDNA sequencing and RecA gene sequencing, will have been filed in sequencing result sequence and GenBank sequence into
Row compares, and bacterial strain C3 is accredited as Burkholderia cepacia (Burkholderia cenocepacia).
By identifying above, confirmation bacterial strain C3 is Burkholderia cepacia (Burkholderia cenocepacia),
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address：BeiChen West Road, Chaoyang District, BeiJing City 1
Institute of microbiology of the Chinese Academy of Sciences of institute, deposit number are CGMCC No.9791, and the preservation time is on October 17th, 2014.
Embodiment 2：Fungistatic effect
(1) inhibitions of the curing ginger stalk rot bacterium antagonistic bacterium C3 to pathogen
C3 is around ginger base rot disease as shown in Fig. 2, being C3 among culture dish to the inhibiting effect of ginger brown foot rot germ
Bacterium, therefore, C3 obviously have inhibiting effect to ginger brown foot rot germ.As shown in Figure 3, wherein A figures are control, and mycelium is shown as
Smooth surface, even thickness, in a tubular form；B figures are C3 and mycelium of the ginger brown foot rot germ opposite culture after 2 days, show as bacterium
The teratogenesis such as filament local bulkiness, hooks.
(2) inhibitions of the curing ginger stalk rot bacterium antagonistic bacterium C3 to instruction pathogen
As shown in Figure 4,5, C3 shows brown foot rot germ, tobacco ralstonia solanacearum and Rhizoctonia solani Kuhn apparent inhibition and makees
With；Geotrichum candidum is screening indicator bacteria.
Embodiment 3：Control efficacies of the curing ginger stalk rot bacterium antagonistic bacterium C3 to base rot disease
(1) preparation of biocontrol agent and cause of disease inoculum
Bacterial suspension prepares：It is 1 with Optimal Medium culture curing ginger stalk rot bacterium antagonistic bacterium C3,600nm OD values
When, bacterial suspension 6000rpm is centrifuged into 10min, thalline is suspended with isometric sterile water or phosphate buffer, is diluted to again
Bacterial concentration is 5 × 108Cfu/ml, 4 DEG C of placements are spare after packing.
Assistant carrier：According to three kinds of substance mixings of proportioning of maltodextrin 60%, yeast powder 10%, wheat bran 30%.
Cause of disease inoculum：Base rot disease opportunistic pathogen is inoculated in the wheat berry of sterilizing, 28 DEG C of culture 7d, spare, experiment
Design is specifically shown in Table 1.
1 experimental design of table and different disposal
First by soil and matrix 3:160 DEG C of sterilizing 2h, are dispensed into flowerpot after 1 mixing.A few days ago, auxiliary is carried for plantation
Body is with every basin 10g and 1/3 soil mixing of upper layer.It is according to above-mentioned different disposal, prepared curing ginger stalk rot bacterium antagonism is thin
Bacterium C3 bacteria suspensions are pressed per basin 10ml with 1000ml water mixings, and soil is poured into.Two days later, plantation is often located without disease ginger per 5, basin
Manage 3 basins.Growth two days later, accesses base rot disease inoculum, and each ginger bud is nearby inoculated with the wheat that carries disease germs.
5d after inoculation is observed and is measured after 12d and managed onset grade everywhere with same concentrations biological and ecological methods to prevent plant disease, pests, and erosion bacteria suspension pouring root, according to
Disease scale criterion calculation disease index and protection effect are control with clear water processing.
Onset grade is with reference to sprout term diseases grade scales such as Li Changsong：
0 grade：It is disease-free
1 grade：The slightly aobvious scab of basal part of stem or root slightly changes colour
2 grades：There is scab, discoloration or rotten in 1/3~1/2 basal part of stem or root
3 grades：Scab rots around entire basal part of stem or root, discoloration
4 grades：Withered death
(2) control efficacy result
The results are shown in Table 2, curing ginger stalk rot bacterium antagonistic bacterium C3 to curing ginger stalk rot preventive effect up to 41.18%, with
Control is compared to being significantly increased, and incidence, disease index decrease compared with CK2 after processing, illustrates protection effect ideal.
Control effect of 2 different disposal of table to curing ginger stalk rot
Embodiment 4：Curing ginger stalk rot bacterium antagonistic bacterium C3 cultivation and fermentation condition optimizings
(1) screening of basal fermentation medium
With KB1, KB2, NYBD, YPG, LB, Ppm culture medium be alternative basal medium, nutrient media components and match such as table
3, seed liquor is inoculated into 4% inoculum concentration in the 100mL triangular flasks equipped with 50mL culture mediums, 28 DEG C, 180rpm culture 72h obtain
Bacteria suspension, by 4 DEG C of bacteria suspension, 10000rpm refrigerated centrifuge 20min, precipitation is microorganism, with ultraviolet spectrometry light after dilution
Degree meter measures OD values under 600nm, with sterile water tune 0, measures influence of the different culture media type to thalline fermentation concentration, as a result such as
It is as seen from the figure, maximum with KB1 culture medium thalline fermentation concentration shown in Fig. 6, so being cultivated based on choosing KB1 culture mediums
Base.
3 basic media components of table and proportioning
(2) nutrient media components experiment of single factor
The screening of carbon source and nitrogen source is carried out to basal medium KB1.According to it is identical proportioning by glycerine, sucrose, glucose,
Respectively as the carbon source in basal medium, other compositions and proportioning are constant, measure different for lactose, maltose, soluble starch
Influence of the ingredient carbon source to cell concentration.It is matched beef extract, yeast powder, tryptone, (NH according to identical4)2SO4、NH4Cl、
Peptone is respectively as nitrogen source in basal medium.Glycerine is as culture medium carbon source, yeast powder conduct it can be seen from Fig. 7-8
When culture media nitrogen source, OD values are maximum, illustrate that curing ginger stalk rot bacterium antagonistic bacterium C3 cell concentrations are maximum, therefore by glycerine and ferment
The carbon source and nitrogen source that female powder optimizes as nutrient media components.
After determining basal medium, carbon source and nitrogen source and basal medium on the basis of single factor experiment to filter out
Middle inorganic salts be constant, according to original basis Medium Proportion select three levels of upper, middle and lower, to medium component match into
The horizontal L of 4 factor of row 39(34) orthogonal test, the design of orthogonal arrage automatically generates table 4 according to DPS.By 4 DEG C of bacteria suspension,
10000rpm refrigerated centrifuge 20min, precipitation are microorganism, and OD values under 600nm are measured with ultraviolet specrophotometer after dilution,
With sterile water tune 0, different component is measured with the influence for comparing thalline fermentation concentration.
4 Optimal Medium of table matches orthogonal test factor and level
By orthogonal trial, each factor level, experimental result and range analysis are shown in Table 5, and four kinds of components ferment to thalline dense
The influence of degree is followed successively by glycerine ＞ MgSO4＞ K2HPO4＞ yeast powders, i.e., the percentage composition of yeast powder sends out thalline in zymotic fluid
Ferment influences maximum, optimum combination A1B2C3D1, i.e. glycerine 2%, yeast powder 1%, K2HPO40.2%, MgSO40.1%.
5 fermentation medium Orthogonal experiment results of table and analysis
(3) fermentation condition optimization
Based on the Medium Proportion optimized, measure respectively culture medium different vaccination amount, bottling amount, fermentation time and
Influence of the medium pH to thalline fermentation concentration.
(1) influence of the bottling amount to thalline fermentation concentration
Based on proportion optimizing culture medium, adjusted in 250ml triangular flasks under 4% seed liquor inoculum concentration, pH natural conditions
It is that 50ml, 10ml, 150ml, 200ml are surveyed after 28 DEG C of 180rpm cultivate 72h with ultraviolet specrophotometer to save Medium bottling volume
Determine OD values under 600nm, with sterile water tune 0, determines bottled amount when maximum cell concentration.In Fig. 9 it can be seen that cell concentration with
Bottled amount increases and increases, and whens 150ml/200ml bottled amounts reaches peak, and subsequent concn declines, reason may be because of with
The increase of bottled amount, the interior oxygen utilized for bacterium of bottle are reduced, and metabolism reduces.So determine that bottled amount is 150ml/250ml,
150mL culture mediums are housed i.e. in 250mL triangular flasks.
(2) influence of the inoculum concentration to thalline fermentation concentration
With Optimal Medium component and proportioning, seed liquor inoculum concentration is adjusted under the bottled amounts of 150ml/250ml, pH natural conditions
It is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, as shown in Figure 10, bacterium when 3% inoculum concentration is influenced on thalline fermentation concentration
Bulk concentration is maximum, it is thus determined that inoculum concentration is 3%.
(3) influence of the fermentation time to thalline fermentation concentration
With Optimal Medium component and proportioning, under the conditions of the bottled amounts of 150ml/250ml, pH natures, 3% inoculum concentration, setting
Different incubation time 12h, for 24 hours, 36h, 48h, 60h, 72h, 96h, 108h, ultraviolet specrophotometer measure 600nm under OD values, with
Sterile water tune 0 analyzes different incubation time section cell concentrations, as a result as shown in figure 11, as can be seen from the figure cell concentration with
Fermentation time increases and increases, and cell concentration is maximum when 48h, is in then stable state, therefore determines that fermentation time is 48h.
(4) influences of the pH to thalline fermentation concentration
With Optimal Medium component and proportioning, it is arranged different pH's under the conditions of 150ml (250ml) bottlings amount, 3% inoculum concentration
Culture medium, pH are respectively 4,5,6,7,8,9,10 to shake bacterium 48h, and by 4 DEG C of bacteria suspension, 10000rpm refrigerated centrifuge 20min, precipitation is
For microorganism, OD values under 600nm are measured with ultraviolet specrophotometer after dilution and different pH are measured to thalline with sterile water tune 0
The influence for concentration of fermenting.The result is shown in Figure 12, as can be seen from the figure pH8-9 cell concentrations are maximum, show that this condition hypothallus is given birth to
Length is most fast.
Embodiment 5：Zymotic fluid physical and chemical property determining
With the culture medium optimized and fermentation condition culture bacterium, zymotic fluid is through centrifuging, obtaining bacteria-free filtrate, using mixing
Flat band method is measured the temperature of zymotic fluid, ultraviolet light and protease stability.
(1) temperature stability：
Zymotic fluid 10mL centrifuge tubes are dispensed, respectively at 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 100 DEG C of processing 30min, with 4
DEG C place filtrate be control, use and be mixed into flat band method with filtrate：Culture medium=1:5 ratio a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, each processing repeat 3
It is secondary, measure zymotic fluid bacteriostatic activity difference.
As a result such as Figure 13, colony diameter no significant difference compared with the control and compares 100 DEG C of processing 30min fungistatic effects
It is identical, illustrate that bacteriostatic active ingredients are insensitive to high temperature.
(2) UV stable：
Take appropriate zymotic fluid to be placed in sterile petri dish, after sealing under 30W ultraviolet lamps distance 30cm Continuous irradiation 12h,
It is control to place filtrate with 4 DEG C, and bacteriostatic activity is measured with reference to the above method.
As shown in figure 14, zymotic fluid after ultraviolet light short distance Continuous irradiation 12h fungistatic effect with compare no significant difference,
Illustrate that active constituent has good stability to ultraviolet irradiation in zymotic fluid.
(3) protease stability：
Appropriate zymotic fluid is taken, appropriate trypsase and pepsin solution are separately added into, makes the final concentration of 0.5g/L of enzyme,
37 DEG C of water-bath 3h make enzyme fully reflect.It is control to place filtrate with 4 DEG C, and bacteriostatic activity is measured with reference to the above method.
Such as Figure 15, zymotic fluid fungistatic effect after trypsase and pepsin 3h illustrates to ferment without significant change
Bacteriostatic active ingredients are insensitive to protease in liquid.
To sum up, zymotic fluid after treatment of different temperature 30min compared with the control pathogen colony diameter without significant change；Hair
Zymotic fluid after ultraviolet light Continuous irradiation 12h compared with the control pathogen colony diameter without significant change；Zymotic fluid is through trypsase
With after pepsin 3h compared with the control pathogen colony diameter without significant change；Therefore the antibacterial material pair that the bacterium generates
Heat, protease and ultraviolet light have stronger stability.
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not protects model to the present invention
The limitation enclosed, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not
Need to make the creative labor the various modifications or changes that can be made still within protection scope of the present invention.
Claims (5)
1. curing ginger stalk rot bacterium antagonistic bacterium, which is characterized in that be named as new Burkholderia cepacia (Burkholderia
Cenocepacia) C3 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on October 17th, 2014
Center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, deposit number CGMCC
No.9791。
2. a kind of C3 microbial inoculums, which is characterized in that its active constituent is that curing ginger stalk rot bacterium antagonism described in claim 1 is thin
Bacterium.
3. microbial inoculum as claimed in claim 2, which is characterized in that the product packaging dosage form of the microbial inoculum is liquid bacterial agent.
4. the preparation method of microbial inoculum described in Claims 2 or 3, which is characterized in that use following steps：With Optimal Medium culture
Curing ginger stalk rot bacterium antagonistic bacterium, when 600nm OD values are 1, by bacterial suspension 6000rpm centrifugation 10min, thalline is used etc.
Volume sterile water or phosphate buffer suspend again, and it is 5 × 10 to be diluted to bacterial concentration8Cfu/ml, 4 DEG C of placements are standby after packing
With.
5. microbial inoculum described in bacterial strain or claim 2 described in claim 1 is in the application for the treatment of curing ginger stalk rot.
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Non-Patent Citations (2)
| Title |
|---|
| Zoospore Homing and Infection Events: Effects of the Biocontrol Bacterium Burkholderia cepacia AMMDR1 on Two Oomycete Pathogens of Pea (Pisum sativum L.);K. HEUNGENS等;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;20001231;第66卷(第12期);5192-5200页 * |
| 生姜茎基腐病病原拮抗细菌的筛选与鉴定;韩超 等;《山东农业科学》;20121130;第44卷(第11期);84-89页 * |
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