CN108676778A - One plant of prevention soil passes bacteriophage and its application of bacterial wilt - Google Patents
One plant of prevention soil passes bacteriophage and its application of bacterial wilt Download PDFInfo
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Abstract
The present invention discloses the native bacteriophage for passing bacterial wilt of one plant of prevention and its application, this plant of Ralstonia solanacearum specific phage NN P42 were preserved in China typical culture collection center on March 6th, 2018, and deposit number is：CCTCC NO:M 2018102, Classification And Nomenclature are Podoviridae phage.Biological control is carried out using the pathogen specific bacteriophage, can efficiently crack bacterium makes its death, and does not have toxicity to environment；High specificity will not destroy other normal floras, edaphon can be kept to balance.The present invention bacteriophage can Specific lytic Stalk Rot, can be used for tomato soil pass bacterial wilt prevention.
Description
Technical field
The invention belongs to environmentally friendly biological prevention and control agents, are related to one plant of Podoviridae NN-P42 and its application, are exclusively used in
Prevention soil passes bacterial wilt problem.
Background technology
Bacterial wilt of tomato is a kind of bacillary dimension caused by Ralstonia solanacearum (Ralstonia solanacearum)
Restrain disease), there is destructive disaster to solanaceous crops.Ralstonia solanacearum host range is wide, strong environmental adaptability, and variation is big,
With complicated inbred genetic.In China, most area has bacterial wilt at present, but southern area morbidity is serious.Tradition
Prevention and control measure such as resistant variety, grafting crop rotation, chemical pesticide etc. have that research and development are difficult, effect is unstable, destroy soil ecology
And the problem of food security, there is an urgent need for the prevention and control measures of development environment friendly.
The microbiologic population of plant rhizosphere has the ability that " instinct " resists soil-borne disease, has constructed pathogen instruction plant
The first line of defence.Edaphon mainly passes through the way such as resource contention, antagonism competition, inducing plant resistance, predation and parasitism
Diameter prevention and control disease.But rhizosphere has the Biocontrol Potential of a kind of more small but abundant quantity group-bacterial virus ignored.Bacterium
Virus is also referred to as bacteriophage, is widely present in environment, quantity is 10 times of bacterium or more.Wherein lytic phage according to
Rely host to replicate and crack host bacteria, is main biological and ecological methods to prevent plant disease, pests, and erosion " reserves ".
Bacteriophage has host specificity and the efficiently ability of cracking and fast breeding, compares the list of bacterial antibiotic resistance
To evolution, bacteriophage has more the advantage of biological and ecological methods to prevent plant disease, pests, and erosion application with host bacteria coevolution.With to bacteriophage biological characteristics,
The research of the interaction relationship of bacteriophage and host strain, the grasp to bacteriophage safety and controllability, phage genome
Research deeply and by biotechnology to the transformation of bacteriophage is studied, and it is huge that bacteriophage will play its in terms of biological prevention and control
Application potential.The specific phage of one plant of cracking performance Ralstonia solanacearum of screening is needed for the above technical problem.
Invention content
Biological control for current bacterial wilt of tomato provides a kind of new thinking and means, reduces the generation of disease.Profit
Biological control is carried out with pathogen specific bacteriophage, can efficiently crack bacterium makes its death, and does not have toxicity to environment；It is special
It is anisotropic strong, other normal floras will not be destroyed, edaphon can be kept to balance.The applicant falls ill serious in bacterial wilt
Separation screening is to one plant of cracking performance Ralstonia solanacearum specific phage in the soil of border, and is ground to its biological characteristics and cracking performance
Study carefully.
Another object of the present invention is to provide a kind of methods quickly and effectively inhibiting Ralstonia solanacearum using this bacteriophage.
The purpose of the present invention can be achieved through the following technical solutions：
One plant of Ralstonia solanacearum specific phage NN-P42 is preserved in Chinese Typical Representative culture guarantor on March 6th, 2018
Tibetan center, deposit number are：CCTCC NO:M 2018102, Classification And Nomenclature are Podoviridae phage.
Ralstonia solanacearum (Ralstonia solanancearum) the specific phage NN-P42 is isolated from bacterial wilt hair
It is Caudoviradles Podoviridae, Genome Size is about 42.28kb in the serious rhizosphere soil of disease.Bacteriophage is trained
Can be preserved 2 months or more at 4 DEG C after nutrient solution filtration sterilization, be stored at -70 DEG C in glycerol tube can preserve 2 years with
On；The bacteriophage culture solution stock concentrations are 1011PFU/mL.Greater activity can be kept in 7 days at 25 DEG C；Lead at 37 DEG C
Higher activity can be kept by crossing 30% glycerine of addition or SM buffer solutions.Bacteriophage has stable split under low-resource level
Solution ability.
The Ralstonia solanacearum specific phage NN-P42 passes bacterial wilt in prevention soil or preparation prevention soil passes bacterial wilt and bites
Significant effect in the application of fungi medicament.
A kind of prevention soil passes the phage preparation of bacterial wilt, includes that above-mentioned Ralstonia solanacearum is special in the phage preparation
Property bacteriophage NN-P42.As a kind of optimal technical scheme, content >=10 of the phage preparation pnagus medius6PFU/mL, into
One step is preferred, the content 10 of the phage preparation pnagus medius6~1011PFU/mL.The phage preparation can be bacteriophage
Culture solution is also added with SM buffer solutions or 30% glycerine in the phage preparation.
Above-mentioned phage preparation can keep higher activity and for a long time in prevention soil passes the application of bacterial wilt
Significant effect.
A method of prevention crop soil passes bacterial wilt, and bacteriophage culture solution or phage preparation are directly in a manner of pouring root
It is inoculated into the plant rhizosphere of crop, additive amount is about >=106PFU/g dry ground, diseases prevention rate reach 60%.
Beneficial effects of the present invention：
The present invention bacteriophage can Specific lytic Stalk Rot, can be used for tomato soil pass bacterial wilt prevention.
Description of the drawings
Plaque on Fig. 1 bilayer agar plates.
Form under Fig. 2 bacteriophage NN-P42 Electronic Speculum.
The SDS-PAGE of Fig. 3 bacteriophage NN-P42 albumen is analyzed.
The phylogenetic tree of Fig. 4 bacteriophage NN-P42 genes.
Influence of Fig. 5 different temperatures to bacteriophage NN-P42 potency.
Potency variations of the bacteriophage NN-P42 in different solvents under Fig. 6 different temperatures.
Bacteriostasis of Fig. 7 bacteriophages under the conditions of Different Nutrition.
Fig. 8 is inoculated with bacterial wilt incidence (A) and rhizosphere pathogen quantity (B) after bacteriophage NN-P42.
Specific implementation mode
(1) separation and identification of bacteriophage
1. bacteriophage isolates and purifies
Host bacteria：Ralstonia solanacearum, what separation obtained during tomato plant is fallen ill in this laboratory from Nanjing kylin town has strong
The Ralstonia solanacearum QL-Rs1115 (Wei et al.2011) of pathogenicity, abbreviation RS.
Bacteriophage NN-P42 is to plant tomato for a long time from Nanning (108 ° of 21 ' E, 22 ° of 49 ' N), and bacterial wilt is fallen ill more
Screening obtains in the rhizosphere soil of serious green house of vegetables.The process that it is isolated and purified is as follows：
Culture medium：
NA fluid nutrient mediums：Glucose 10g, peptone 5g, yeast extract 3g, yeast extract 0.5g, deionized water
1000mL adjusts pH 7.2-7.4,115 DEG C of high pressure sterilization 30min.
NA solid mediums：The agar powder of 1.8% (g/100ml) is added in NA fluid nutrient mediums.
NA semisolid culturemediums：The agar powder of 0.8% (g/100ml) is added in NA fluid nutrient mediums.
SM buffer solutions：Take MgSO4·7H2O 2.0g, NaCl 5.8g, 1mol/L Tris-HCl (pH7.5) 50mL, 2% is bright
Glue 5mL, adds deionized water, is settled to 1000mL, in 121 DEG C of high pressure sterilization 20min, 4 DEG C of preservations, SM buffer solutions use after packing
In the dilution and preservation of bacteriophage.
Add the ratio of 10g soil samples that shaken overnight in triangular flask is added in every 90mL SM buffer solutions the soil sample of acquisition, takes out
After stand.It takes 5mL to be added in the R.solanacearum bacterium solutions of logarithmic phase after 0.22 μm of membrane filtration Soil Slurry to shake
Culture 12h is swung, will be bacteriophage stoste after 0.22 μm of membrane filtration of co-culture media.Take the R. of 0.5mL exponential phases
Solanacearum bacterium solutions are cooled to 50 DEG C or so of NA semisolid culturemedium mixings with 6mL, and it is solid to pour into the NA solidified immediately
Double-layer plate is made on body culture medium flat plate, by tablet subregion after the solidification of upper layer culture medium, puts the gradient dilution for meeting 2 μ L respectively
Bacteriophage stoste (10,102, 103, 104, 105, 106, 107, 108), 30 DEG C are inverted culture for 24 hours~48h, and whether there is or not phagocytosis for observation
Spot (Fig. 1).
After having plaque appearance, the R.solanacearum bacterium of the single maximum plaque access exponential phase of picking
In liquid, shaking up and be put into shaking table after standing 15min, under the conditions of 30 DEG C, 120r/min shaken cultivations 12h makes bacteriophage multiplication,
By 0.22 μm of membrane filtration of co-culture media after Multiplying culture, plaque is obtained by the above-mentioned double-deck agar method.Repetition 3~
It can be obtained purer bacteriophage 5 times.
It will obtain directly preserving 4 DEG C without host strain phage solution, or mixed with 30% glycerine (v/v) together with host strain
- 70 DEG C are stored in after conjunction.
The bacteriophage NN-P42, Classification And Nomenclature are Podoviridae phage, and Chinese allusion quotation is preserved on March 6th, 2018
Type culture collection, deposit number are：CCTCC NO:M 2018102.
2. the morphologic observation of bacteriophage
The phage suspensions for taking 20 μ L fresh drop on copper mesh, and standing uses filter paper in side after making its natural sedimentation 15min
Extra liquid is sucked, 2% PTA (phosphotungstic acid) is then dripped and phage particle is dyed on copper mesh, is continued
Extra dyeing liquor is siphoned away in side with filter paper after standing 10 min, continuing to stand general 5min or so makes sample drying, most
It by the form of electron microscope observation bacteriophage and photographs to record afterwards.
The form of transmission electron microscope observing to bacteriophage NN-P42 are tadpole shape, and head is icosahedral cubic symmetry, directly
Diameter is about 100nm, has a short-tail, as shown in Figure 2.It is compared by the criteria for classification of same International Commission on Virus Classification,
Bacteriophage NN-P42 belongs to Caudoviradles (Caudovirales), Podoviridae (Podoviridae).
3. the SDS-PAGE of phage protein is analyzed
The 15 μ L of bacteriophage NN-P42 particles liquid after concentration are taken, 6 × sample-loading buffer of 3 μ L is mixed even together, is boiled at 100 DEG C
Boil 5 min makes protein-denatured purpose to reach, and gel sample-adding is respectively added in the sample, albumen Marker after high-temperature process
Hole (two ducts cannot be adjacent), powers on after marking, under the voltage of 90V after about 120min, sample to be tested meeting
Electrophoresis is brought the voltage up at this time to the intersection for concentrating glue and separation gel to 150V, until sample electrophoresis is closed to separation gel lower edge
Close electrophoresis apparatus, take out gel, sanction removes concentration glue, separation gel is placed in glass dish, according to routine protein silver staining method into
Row dyeing is taken out after protein band colour developing is clear and is scanned.
As shown in figure 3, then through albumen silver, at least six protein band can be observed, illustrate that the phage particle at least contains
There are 6 structural proteins, relative molecular mass is between 25kDa~100kDa.
4. the genomic information of bacteriophage
Phage genome DNA is extracted by operating procedure using kit (lambda bacteriophage dna extracts kit, Abigen),
The purity and concentration of genomic DNA are detected using 2000 ultramicrospectrophotometers of NanoDrop (NanoDrop companies of the U.S.),
It send to Shanghai Mei Ji biotech firms and is sequenced.
Sequencing result shows that bacteriophage NN-P42 Genome Sizes are about 42.28kb, and G/C content is about 62.10%.It will bite
The genome sequence of thalline NN-P42 carries out BLAST analyses and phylogenetic tree construction with some selected standard bacteriophages.Such as
Shown in Fig. 4, the homology highest of bacteriophage NN-P42 and Pelagibacter_phage_HTVC010P that we detach, to
It can determine that bacteriophage NN-P4 belongs to Caudoviradles (Caudovirales), Podoviridae (Podoviridae).
(2) the shelf life evaluation of bacteriophage
It develops in bacteriophage products application to actual production, it is intermediate to pass through long-distance transport, so we must examine
Consider the shelf life of bacteriophage, i.e., the condition for validity that bacteriophage preserves.
1. storage temperature
By the bacteriophage NN-P42 suspension (about 10 of activation11PFU/mL) packing multitube preserves, and is placed at 4,25,37 DEG C
Static gas wave refrigerator one week, record different time (1,2,4,7 day) phage titer variation, each processing are equipped with 3 parallel repetitions.Knot
Fruit finds that 37 DEG C of high temperature is affected to the potency of bacteriophage, and bacteriophage is still and under low temperature (4 DEG C) and room temperature (25 DEG C), in 7 days
The higher order of magnitude can be kept, slightly reduces (Fig. 5).
2. preserving solvent
By the bacteriophage NN-P42 suspension (about 10 of activation11PFU/mL) dispense multitube, respectively with 30% glycerine, sterile
Four kinds of water, 0.85% physiological saline, SM buffer solutions solvents are according to 1：1 ratio mixing is placed in static gas wave refrigerator culture at 4,25,37 DEG C
One week, record different time (1,2,4,7 day) phage titer variation, each processing was equipped with 3 parallel repetitions.It is mutually synthermal
Under, influence of the different solvents to the potency of bacteriophage is different, wherein using physiological saline as the treatment effect of solvent worst, bacteriophage
Quantity decline at any time it is most fast.30% glycerine, SM buffer solutions and ddH at 4 DEG C2Difference is not notable (Fig. 6-A) between O processing；
DdH at 25 DEG C2The processing of O and SM buffer solutions is preferably (Fig. 6-B)；SM buffer solutions and 30% glycerine effect are preferable (Fig. 6-C) at 37 DEG C.
Illustrate that we can extend time and the activity of bacteriophage preservation by both solvents even if under the high temperature conditions.
(3) bacteriophage prevention and control bacterial wilt effect
1. bacteriophage is under different resource level conditions to the inhibition of Ralstonia solanacearum QL-RS1115
Prepare 5 resource levels：NA, 1/2NA, 1/10NA, 1/20NA, 1/100NA culture medium, using microwell plate system,
The system is made of 4 links：Culture medium, inoculated bacteria, shake culture and microplate reader measure OD600.The inoculum concentration of Ralstonia solanacearum
It is 107The inoculum concentration of CFU/mL, bacteriophage are 106PFU/mL.30 DEG C of shake culture 96h measure the OD of different time respectively600。
The results are shown in Figure 7, and different nutritional conditions has an impact the growth of Ralstonia solanacearum and the fungistatic effect of bacteriophage.
Under low nutrient level (1/20NA), the growth of phagocytosis physical efficiency stabilization checking Ralstonia solanacearum；Under high-plane of nutrition, in inoculation phagocytosis
35h after body or so can inhibit the growth of Ralstonia solanacearum, and with the extension of incubation time, the inhibiting effect of bacteriophage can weaken.This
May be resistance to be generated to bacteriophage due to later stage Ralstonia solanacearum, but Ralstonia solanacearum is difficult to generate resistance under low-resource level, this is right
How actual production regulates and controls plant rhizosphere resource to which the prevention and control efficiency for improving bacteriophage has certain directive significance.
2. greenhouse pot culture is tested
Greenhouse at Yixing organic solid castoff recycling collaborative innovation center investigates bacteriophage NN-P42 preventions
Tomato soil passes the effect of bacterial wilt.
For the rice soil that examination soil is not Ralstonia solanacearum.Tomato variety is Micro-Tom (dwarfted varieties).
2 processing of experiment setting, it is specific as follows：
Control, CK：Only inoculation pathogen；
Processing, NN-P42：It is inoculated with pathogen and bacteriophage NN-P42.
Micro-Tom tomato seeds are after surface sterilization：1min is impregnated with 70% alcohol, sterile water washing 1 time is used
3% NaClO solution impregnates 5min, rinsed with sterile water 6 times.The seed disinfected is put into and is lined with aseptic double-distilled water and soaks filter
In the aseptic flat board of paper, 30 DEG C of vernalization two days.The seed of germination is transplanted in 9 hole seedlings nursing plates and (soil is housed).Often handle 3 disks (9
A repetition, totally 27 tomato seedlings), 3 leaf phase tomato seedlings are inoculated with Ralstonia solanacearum, concentration 5.0 × 107CFU/g dry ground；It is inoculated with Ralstonia solanacearum 5
Phage suspensions, concentration 5.0 × 10 are inoculated with after it6PFU/g dry ground.Incidence, final test knot are counted daily after completing inoculation
Each 3 healthy tomato seedlings are adopted in processing at random when beam, acquire rhizosphere soil.
Tomato rhizosphere Ralstonia solanacearum total amount is measured using quantitative PCR.After acquiring Rhizosphere Soil, soil DNA is extracted with kit.It is green
Withered bacterium specific primer is fliC F：5 '-GAA CGC CAA CGG TGC GAA CT-3 ', fliC R：5′-GGC GGC CTT
CAG GGA GGT C-3 ' (Schonfeld et al.2003) (Jin Sirui biologies Co., Ltd synthesizes by Nanjing).Pathogen
Quantitative fluorescent PCR uses Premix Ex TaqTM(precious bioengineering Co., Ltd, Takara, Dalian) kit is pressed
As directed on book method in ABI (AppliedBIO systems)7500Real-time PCR System amplification instruments
Upper amplification.According to the copy number for drawing obtained standard curve calculating key gene.
1g rhizosphere soils are mixed with 9mL aseptic deionized waters, 170rpm shakes 30min, and the soil supension of preparation is used
It is bacteriophage stoste after 0.22 μm of membrane filtration.The RS bacterium solutions and 6mL for taking 0.5mL exponential phases are cooled to 50 DEG C or so
NA semisolid culturemedium mixings, pour into immediately in the NA solid medium tablets solidified and double-layer plate be made, wait for upper layer train
It supports tablet subregion after base solidifies, puts the bacteriophage stoste for the gradient dilution for meeting 50 μ L, 30 DEG C of 24~48h of culture, statistics respectively
The quantity of plaque can be obtained the quantity of bacteriophage.
The control effect of bacteriophage NN-P42 is as shown in figure 8, inoculation bacteriophage NN-P42 shows good control effect.
Control group incidence is up to 83.33%, and the incidence of NN-P42 processing is 33.33%, substantially less than control group.Rhizosphere cause of disease
The quantity processing group of bacterium is also below control group (as shown in Figure 8 B).Test result illustrates that bacteriophage NN-P42 can significantly reduce blueness
The incidence of blight.One plant of Ralstonia solanacearum specific phage that the present invention obtains screening has carried out the analysis of biological characteristics, goods
The frame phase evaluates and the detection of protection effect.
Schonfeld,J.,Heuer,H.,van Elsas,J.D.&Smalla,K.(2003).Specific and
sensitive detection of Ralstonia solanacearum in soil on the basis of PCR
amplification of fliC fragments.Appl. Environ.Microbiol.,69,7248-7256.
Wei,Z.,Yang,X.M.,Yin,S.X.,Shen,Q.R.,Ran,W.&Xu,Y.C.(2011).Efficacy of
Bacillus- fortified organic fertiliser in controlling bacterial wilt of
tomato in the field.Appl.Soil Ecol., 48,152-159。
Claims (8)
1. one plant of Ralstonia solanacearum specific phage NN-P42 was preserved in China typical culture collection on March 6th, 2018
Center, deposit number are：CCTCC NO:M 2018102, Classification And Nomenclature are Podoviridae phage.
2. application of the Ralstonia solanacearum specific phage described in claim 1 in prevention soil passes bacterial wilt.
3. Ralstonia solanacearum specific phage described in claim 1 is in preparing the phage preparation for preventing soil biography bacterial wilt
Application.
4. a kind of prevention soil passes the phage preparation of bacterial wilt, it is characterised in that include claim 1 institute in the phage preparation
The Ralstonia solanacearum specific phage NN-P42 stated.
5. prevention soil according to claim 4 passes the phage preparation of bacterial wilt, it is characterised in that in the phage preparation
Content >=10 of bacteriophage6PFU/mL。
6. prevention soil according to claim 4 or 5 passes the phage preparation of bacterial wilt, it is characterised in that the phage preparation
In also be added with SM buffer solutions or 30% glycerine.
7. application of the phage preparation in prevention soil passes bacterial wilt described in claim 4,5 or 6.
8. a kind of method that prevention crop soil passes bacterial wilt, which is characterized in that by the phagocytosis system described in claim 4,5 or 6
Agent is inoculated into the plant rhizosphere of crop, and the additive amount of the phage preparation is >=106PFU/g dry ground.
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| CN109136194A (en) * | 2017-06-28 | 2019-01-04 | 菲吉乐科(南京)生物科技有限公司 | Novel Ralstonia solanacearum bacteriophage and combinations thereof and application |
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| CN115725512A (en) * | 2022-08-06 | 2023-03-03 | 菲吉乐科(南京)生物科技有限公司 | Ralstonia solanacearum bacteriophage and composition, kit and application thereof |
| CN115725512B (en) * | 2022-08-06 | 2024-07-26 | 菲吉乐科(南京)生物科技有限公司 | Ralstonia solanaceae phage and composition, kit and application thereof |
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