CN109897882A - A method of infection bacterial canker of tomato is detected whether using single seed - Google Patents
A method of infection bacterial canker of tomato is detected whether using single seed Download PDFInfo
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- CN109897882A CN109897882A CN201910249435.2A CN201910249435A CN109897882A CN 109897882 A CN109897882 A CN 109897882A CN 201910249435 A CN201910249435 A CN 201910249435A CN 109897882 A CN109897882 A CN 109897882A
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Abstract
The invention discloses a kind of methods for detecting whether infection bacterial canker of tomato using single seed for belonging to Plant Pathology field.The method includes the following steps: that randomly selecting single seed from seed to be measured germinates, and the seedling after germination is ground, solution obtains extracting solution after extracting, the dilution of extracting solution is coated on selective medium, is cultivated, is then detected.The method that the present invention combines culture medium to be separately cultured using germination; realize the quantitative detection of active bacterial canker of tomato in simple grain tomato seeds; this method can be not only used for the basic microbiological research of bacterial canker of tomato; it can be applied in cause of disease analyte detection in agricultural production, disease screening and plant quarantine work, have a extensive future in microbiology and plant protection art again.
Description
Technical field
The invention belongs to Plant Pathology field, it is related to a kind of detecting whether infection bacterial canker of tomato using single seed
Method.
Background technique
Canker of tomato is one of tomato production destructive disease.The disease is in 1909 for the first time in Michigan, United States
Greenhouse tomato on find, be distributed widely in each tomato producing region in the world.Currently, in Africa, America, Asia, Europe and big
There are the report of canker of tomato occurrence injury in a countries and regions more than the 80 of foreign continent.The Symptoms of the disease be blade it is withered,
Vascular bundle browning etc., cause photosynthesis of plant efficiency reduction, moisture and nutrition transmission is obstructed, seriously affect tomato yield and
Quality, seriously ill field later period diseased plant rate is up to 100%, and production loss is 60% or more.Currently, multiple national by bacterial canker of tomato
Quarantine harmful organisms are classified as, bacterial canker of tomato was classified as A2 in 1975 by European and Mediterranean Plant Protection Organization (EPPO)
Class quarantine harmful organisms, it is Exit-Entry Quaratine harmful organism that the germ is also expressly recited in European Union's plant quarantine regulation, I
State was included in " People's Republic of China (PRC) enter the territory plant quarantine harmful organism register " in 2007.
Currently, commonly using copper agent or antibiotic in production to prevent and treat canker of tomato, but the effect is unsatisfactory.In field,
The main primary infection inoculum of canker of tomato morbidity is the seed or invalid body to carry disease germs, and seed is the main load propagated over long distances
Body, carries out seed health detection, and infected seed is controlled from source and enters production link, is the important means for preventing and treating the disease.Cause
This, it is the key that Seed-associated fungi that rapid sensitive, specific quantification, which detect active bacterial canker of tomato, and in production urgently
Problem to be solved.
In the detection that current seed carries active bacterial canker of tomato, commonly use culture medium isolated culture, i.e., it will kind
Son is directly put or seed cleaning solution is coated on ordinary culture medium or (partly) Selective agar medium plate, grows to colonies typical
Detection and identification is carried out after out, but this method cannot be used for detection in damage (injure) state or can not cultivate
The bacterial canker of tomato of (viable but non-culturable, VBNC) state, and lose the germ of state or VBNC state
Still keep the stability and activity of membrane structure, it is possible to restore extremely can cultivation conditions, so as to cause the generation of field diseases.Cause
This, the above-mentioned detection method based on culture may cause false negative as a result, bringing potential risk to production.Therefore, existing
By research and improvement on the basis of method, chooses seed to be measured and germinate, the extracting solution of seedling is coated on culture medium and is put down
On plate, realization detects active bacterial canker of tomato from simple grain tomato seeds, the sensitivity of detection is improved, in production
The spread in china and occurrence injury for controlling canker of tomato are of great significance.
Summary of the invention
In order to overcome the above problem, infection tomato is detected whether using single seed the purpose of the present invention is to provide a kind of
The method of ulcer bacteria.
For this purpose, the technical solution of the present invention is as follows:
A method of infection bacterial canker of tomato is detected whether using single seed, is included the following steps:
Single seed is randomly selected from seed to be measured to germinate, the seedling after germination is ground, and solution extracts
Extracting solution is obtained afterwards, and the dilution of extracting solution is coated on selective medium, is cultivated, is then detected.
In the above method, the method for the detection are as follows:
It has seen whether bacterium generation, has carried out qualitative detection；
Or the quantity of vibrant thallus is obtained using plate count method after growing out to colonies typical, into
Row quantitative detection.
In the above method, the seed is tomato seeds, and the bacterium in the microbiological contamination is bacterial canker of tomato.
In the above method, the bacterial canker of tomato is that stick nectar in Michigan holds Anya kind (Clavibacter
Michiganensis subsp.Michiganensis, abbreviation Cmm).
In the above method, the method for the grinding are as follows: seedling is subjected to ball-milling treatment
In the above method, the method for the ball milling includes: that the steel ball of seedling and sterilizing is put into centrifuge tube, with ball milling instrument
Handle 20s.
In the above method, the solution is 0.85%NaCl aqueous solution；The extracting solution is removal steel ball and plant tissue
Supernatant.
In the above method, the selective medium is culture medium mCNS culture medium semi-selective.
In the above method, the temperature of the culture is 28 DEG C, time 5-7d.
In the above method, the seedling is to be placed in conjunction on the sterile wet filter paper being placed in seed to be measured in culture dish
The seedling of suitable temperature incubation chamber culture 5-10d.
Advantages of the present invention:
The method that the present invention combines culture medium to be separately cultured using germination, realizes active in simple grain tomato seeds
The quantitative detection of bacterial canker of tomato, this method can be not only used for the basic microbiological research of bacterial canker of tomato, and can apply
In agricultural production in cause of disease analyte detection, disease screening and plant quarantine work, in microbiology and plant protection art application
It has a extensive future.
Method of the invention can specify potential bacteriosis First aggression with indirect application in the control of the source of disease
Corresponding prophylactic measures is formulated in source, to reduce spraying for chemical pesticide, has the work for reducing pesticide hazards and protecting ecology
With；And agriculture production cost is reduced, there is certain economic benefit；Society is served, there is significant ecological benefits and society
Benefit.
Detailed description of the invention
Fig. 1 is the quantity of bacterial canker of tomato in the infected seed of different vaccination bacterial concentration preparation.
Fig. 2 is negative after crimping kind 7d, the quantity of bacterial canker of tomato in the seedling in seed and after germination.
Fig. 3 is the standard curve of quantitative fluorescent PCR.
Fig. 4 is negative in crimping kind 70d, the quantity variation of bacterial canker of tomato in the seedling in seed and after germination.
Specific embodiment
Clear, complete description is carried out to technical solution of the present invention below in conjunction with specific embodiment, it is clear that described
Embodiment be a part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field
Those of ordinary skill's every other embodiment obtained without making creative work, belongs to guarantor of the present invention
The range of shield.Material used in the present invention and device are unless otherwise specified commercially available.
Below in conjunction with Figure of description and embodiment, the present invention is further illustrated, but is not the limitation present invention.
Bacterial canker of tomato (Clavibacter michiganensis subsp.michiganensis) is in document " sieve
Come prosperous, Li Jianqiang, research [J] Plant Pathology of Hasan Bolkan. Tomato Caused by Clavibacter michiganensis subsp. michiganensis Seedling Inoculation new method
Report, 2005,35 (2): being disclosed in 123-128. ", the public can from China Agricultural University, commercial sources or voluntarily acquisition obtain.
The method of the invention for detecting sub- bacterial canker of tomato using simple grain tomato species of embodiment 1
It randomly selects single seed from seed to be measured to germinate, the method for germination are as follows: seed to be measured is placed in 9cm
On sterile wet filter paper in culture dish (wetting of 2ml sterile water), 25 DEG C of incubator culture 7d are placed in, check the wet of filter paper daily
Degree adds sterile water moisturizing in time.
Seedling after germination is subjected to ball milling, selects ball milling instrument processing: the steel ball of the seedling that 1 plant is germinateed and 2 sterilizings
It is put into 2ml centrifuge tube, handles 20s with ball milling instrument.
After being handled with ball milling instrument, 400 μ l0.85%NaCl solution are added, are stored at room temperature 1h, remove steel ball and plant tissue
Supernatant obtain extracting solution.The dilution of extracting solution is coated on mCNS culture medium flat plate, incubator culture, the training are placed in
Supporting box temperature degree is 28 DEG C, and culture 5-7d can grow colonies typical, and specific gate time is the 5th day.It grows out to colonies typical
Plate count method is used later, obtains the quantity of active thallus.
The bacterium is bacterial canker of tomato, i.e. Michigan stick nectar holds Anya kind (Clavibacter
Michiganensis subsp.michiganensis, Cmm).
Embodiment 2 utilizes culture medium isolated culture and fluorescence quantitative PCR method detection bacterial canker of tomato one, healthy tomato
Seed inoculating tomato ulcer bacteria
1, the bacterial canker of tomato single bacterium of picking activation is fallen in 10ml LB liquid medium, and two repetitions are arranged, are placed in
28 DEG C, 120rpm shake training 22h to the logarithmic growth later period；
2, the bacterium solution for taking step 1 to obtain washes away culture medium with 0.85%NaCl solution, and adjusts concentration to OD600nm=
0.45 (concentration is about 108CFU/ml), taking appropriate volume to be diluted preparation concentration is 107CFU/ml、106 CFU/ml、105
CFU/ml、104 CFU/ml、103 CFU/ml、102 CFU/ml、101Each 20ml of the bacterial suspension of CFU/ml；
3, it takes 210 healthy tomato seeds to be put into 50ml centrifuge tube, 30ml70% ethyl alcohol, disinfection treatment is added
Seed is transferred on aseptic filter paper after sterile water wash 3 times and dries (2-3h) by 1min；
4,7 50ml centrifuge tubes are taken, the bacterium solution of the various concentration of 20ml step 2 preparation is separately added into, and are put into 30 steps
Rapid three dry after seed, place it in closed box, box is controlled to a vacuum pump, setting pressure value be 0.07MPa, take out
Vacuum 20min；
5, the bacterium solution in centrifuge tube is removed, the tomato seeds after inoculation are transferred on aseptic filter paper and are dried.
With in seed after seed culture medium isolated culture and fluorescence quantitative PCR method (being specifically shown in step 2) detection just inoculation
The quantity of bacterial canker of tomato.
To be inoculated with bacterial concentration as abscissa, Cmm cell quantity is ordinate mapping, as a result as shown in Figure 1.Fig. 1 shows
The tomato seeds of health can be made to carry disease germs by negative pressure inocalation method, and being inoculated with bacterial concentration is 107Closer to theoretical when CFU/ml
Inoculum concentration.The detected value of different vaccination bacterial concentration, culture medium isolated culture is below the detected value of fluorescence quantitative PCR method,
Illustrate that part thallus is dead after being inoculated with or loses Culturability.Two, bacterial canker of tomato in seedling after seed and germination
Culture medium isolated culture method
1,9cm culture dish is taken, aseptic filter paper is put into and the wetting of 2ml sterile water is added, the tomato after putting above-mentioned 6 inoculations
Seed is placed in 25 DEG C of incubator culture 7d, checks the humidity of filter paper daily, adds sterile water moisturizing in time；
2, the tomato seeds after taking 3 inoculations are put into 2ml centrifuge tube, and 1 plant of germination is taken from the culture dish of step 1
Seedling is put into another 2ml centrifuge tube, 2 repetitions of each processing, and 2 sterilizing steel balls are respectively added, and handles 20s with ball milling instrument
Afterwards, the 0.85%NaCl solution of 400 μ l sterilizing is added, is stored at room temperature 1h；
3, the supernatant for obtaining step 2 is transferred in new 2ml centrifuge tube, to remove steel ball and plant tissue, is prevented
Pipette tips are blocked when imbibition, and guarantee that bacterial canker of tomato is uniformly distributed, then prepare different extension rates with 0.85%NaCl solution
Bacteria suspension；
4, the 100 μ l of bacteria suspension of each extension rate is taken to be coated on mCNS culture medium flat plate semi-selective, each concentration 2 times
It repeats, the plate after coated plate is placed in 28 DEG C of incubator culture 5d, the plate for choosing suitable concentration is counted, and concentration conversion is public
Formula is as follows:
The tomato seeds that carry disease germs are prepared by the method for step 1, bacterial concentration used is OD600nm=0.05 (concentration is about
107CFU/ml), be put into 20ml bacterium solution 300 dry after seed, (20 DEG C) of room temperature storages are placed on after inoculation.After 7d, use
Canker of tomato in seedling in culture medium isolated culture and fluorescence quantitative PCR method the detection seed of step 2 and after germination
The quantity of bacterium.
Using sample ID as abscissa, Cmm cell quantity is ordinate mapping, as a result as shown in Figure 2.Fig. 2 shows tomato
It is about 10 that seed stores the content of molds after 7d under normal temperature conditions2CFU/seed, lower than the detected value of quantitative fluorescent PCR, explanation
Part thallus is dead in storage or loses Culturability.But after germination, bacterial canker of tomato in seedling
Quantity greatly improves, and content of molds is about 105CFU/seedling illustrates that in Seed Germination, bacterial canker of tomato is a large amount of
Culturability is bred or restored, detection sensitivity is substantially increased.In addition to this, tomato seeds are smaller, handled with ball milling instrument
Single seed error is larger, by can directly detect the quantity of bacterial canker of tomato in simple grain tomato seeds after germination, improves
Detection accuracy.
Wherein, the method for fluorescence quantitative PCR detection seed and the Cmm in seedling are as follows:
1. experimental material prepares
Instrument: centrifuge, vortex instrument, real-time fluorescence quantitative PCR instrument (ABI7500)；
Steel ball: steel ball is put into small beaker, spare after 121 DEG C of sterilizing 20min；
0.85%NaCl solution: 0.85g NaCl being added and is equipped in the deionized conical flask of 100ml, after completely dissolution,
121 DEG C of sterilizing 20min, it is spare；
Sterile water: deionized water is added in conical flask, spare after 121 DEG C of sterilizing 20min；
DNA kit: E.Z.N.A Bacterial DNA Kit D3350-1；
TaqMan Gene Expression Master Mix: Applied Biosystems, lot number 1509241 are purchased from；
Primer: commission Sangon Biotech (Shanghai) Co., Ltd.) limited liability company's synthesis, Spm4f:5 '-
TCAGGCGTCTGTTCTGGC-3 ', Spm2r:5 '-CCCACCACCATCCACAAC-3 ', Spm4f and Spm2r are applied in combination, and expand
Increasing obtains the segment of 223bp；
Probe:5 '-CCTTCTGGGTGTGTCTGGTTTC-3 ', 5 ' ends are marked with fluorescent reporter gene FAM, and 3 ' hold with glimmering
Optical quenching gene TAMRA label.
2 experimental methods
2.1 extract DNA
The DNA that Cmm is extracted using DNA kit, is modified slightly, is extracted according to the following steps:
1) tomato seeds (or seedling after 1 plant of germination) after taking 3 inoculations are put into 2ml centrifuge tube, each processing 2 times
It repeats, each steel ball that 2 sterilizings are added after handling 20s with ball milling instrument, is added 400 μ l sterile waters, is stored at room temperature 1h；
2) said extracted liquid (including plant tissue) is transferred in new 1.5ml centrifuge tube, steel ball is removed, with after an action of the bowels
Continuous centrifugally operated；
3) 100 μ l TE Buffer are added, vortex makes precipitating suspend, and 10 μ l Lysozyme, 37 DEG C of water-bath 10min are added；
4) after being cooled to room temperature, 100 μ l BTL Buffer and 20 μ l Proteinase K Solution are added, are vortexed
It mixes, 55 DEG C of water-bath 30min, every 10min oscillation is primary；
5) after being cooled to room temperature, 5 μ l RNase A are added, suction mixes, and is placed at room temperature for 5min；
6) after 13000rpm is centrifuged 2min, transfer supernatant to new 1.5ml centrifuge tube；
7) 220 μ l BDL Buffer are added, is vortexed and mixes, 65 DEG C of water-bath 10min；
8) 220 μ l100% ethyl alcohol are added, is vortexed with maximum speed and mixes 20s；
9) HiBind DNA Mini Column is inserted into 2ml Collection Tube, transfer all samples are extremely
In HiBind DNA Mini Column, 13000rpm is centrifuged 1min, removes the liquid in Collection Tube；
10) 500 μ l HBC Buffer, 13000rpm are added and are centrifuged 1min, remove the liquid in Collection Tube；
11) 700 μ l DNA Wash Buffer, 13000rpm are added and are centrifuged 1min, remove in Collection Tube
Liquid；
12) after repetitive operation (11), 13000rpm is centrifuged 2min, abandons Collection Tube；
13) HiBind DNA Mini Column is inserted into new 1.5ml centrifuge tube, 50 μ l Elution is added
Buffer (after 65 DEG C of water-baths), is placed at room temperature for 5min；
14) after 10000rpm is centrifuged 1min, the DNA after elution is saved at -20 DEG C.
2.2 quantitative fluorescent PCR
12 μ l DNA samples are shifted into PCR pipe, take 3 μ l carry out 4 times of gradient dilutions, choose DNA sample original content, 4
96 orifice plates are added by following reaction system (20 μ l) in times dilution and 16 times of dilutions, 3 repetitions of each concentration, setting it is negative and
Positive control.
Reaction system are as follows:
It after 96 orifice plates for having added reaction system are centrifuged 10s, is put into ABI7500 real-time fluorescence quantitative PCR instrument, opens
Experimental arrangement is arranged in 7500 softwares: the use of tool being 7500 Fast (96 Wells), experiment type Quantitation-
Standard Curve, detection target reagent areReagents, Standard (~2hours to complete a
run)；Title and the position of target and sample are arranged according to Loading sequence；Reaction condition is 95 DEG C of denaturation 10min；95 DEG C of denaturation
20s, 60 DEG C of annealing 1min carry out 40 circulations.Start quantitative fluorescent PCR after being provided with, to exporting after reaction as a result,
It saves and analyzes data.
3. the preparation of standard curve
Taking 1ml concentration is 107The bacterium solution of CFU/ml carries out 10 times of gradient dilutions, extracting concentration 102 CFU/ml、103
CFU/ml、104 CFU/ml、105 CFU/ml、106 CFU/ml、107The DNA of CFU/ml bacterium solution is simultaneously measured with quantitative fluorescent PCR
Ct value, obtains standard curve, as shown in figure 3, R2Value is 0.9949.The detection of the seed of 3 long term storage of embodiment
In the seed of long term storage, Pathogen detection can be carried out using the method for embodiment 1 at regular intervals.
The present embodiment is using the infected seed prepared in embodiment 2, every the method for 7 days repetition embodiments 1.It is with the time
For ordinate mapping, as a result as shown in Figure 4 abscissa can cultivate Cmm cell quantity.Fig. 4 shows tomato seeds in normal temperature condition
Content of molds in lower storage 70d maintains 1 × 102-1×103CFU/seed, tomato seeds carry disease germs in seedling after germination
Amount maintains 1 × 105-5×106CFU/seedling, detection sensitivity and accuracy are still very high, this method can be used for storing up for a long time
The detection for the seed deposited.
The above embodiments are merely examples for clarifying the description, and does not limit the embodiments.For institute
For the those of ordinary skill in category field, other various forms of variations or change can also be made on the basis of the above description
It is dynamic.There is no necessity and possibility to exhaust all the enbodiments.And obvious variation extended from this or change
It moves still within the protection scope of the invention.
Claims (10)
1. a kind of method for detecting whether infection bacterial canker of tomato using single seed, which comprises the steps of:
Single seed is randomly selected from seed to be measured to germinate, the seedling after germination is ground, and solution obtains after extracting
The dilution of extracting solution is coated on selective medium, is cultivated, then detected by extracting solution.
2. the method according to claim 1, wherein the method for the detection are as follows:
It has seen whether bacterium generation, has carried out qualitative detection；
Or the quantity of vibrant thallus is obtained, is determined using plate count method after growing out to colonies typical
Amount detection.
3. the method according to claim 1, wherein the seed is tomato seeds, the bacterium in the microbiological contamination
For bacterial canker of tomato.
4. according to the method described in claim 3, it is characterized in that, the bacterial canker of tomato is Michigan stick bacterium Michigan
Subspecies (Clavibacter michiganensis subsp.michiganensis).
5. the method according to claim 1, wherein the method for the grinding are as follows: seedling is carried out ball-milling treatment.
6. according to the method described in claim 5, it is characterized in that, the method for the ball milling includes: the steel by seedling and sterilizing
Pearl is put into centrifuge tube, handles 20s with ball milling instrument.
7. -6 any method according to claim 1, it is characterised in that: the solution is 0.85%NaCl aqueous solution；Institute
Stating extracting solution is the supernatant for removing steel ball and plant tissue.
8. the method according to claim 1, wherein the selective medium is culture medium mCNS semi-selective
Culture medium.
9. the method according to claim 1, wherein the temperature of the culture be 28 DEG C, time 5-7d.
10. according to the method described in claim 1, it is characterized by: the seedling is that seed to be measured is placed in culture dish
Sterile wet filter paper on, be placed in the seedling of suitable temperature incubator culture 5-10d.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103930562A (en) * | 2011-10-18 | 2014-07-16 | 格拉斯兰兹技术有限公司 | Detection of viable endophyte |
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2019
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103930562A (en) * | 2011-10-18 | 2014-07-16 | 格拉斯兰兹技术有限公司 | Detection of viable endophyte |
Non-Patent Citations (3)
| Title |
|---|
| SINING HAN ET AL.: "Detection of Clavibacter michiganensis subsp.michiganensis in viable but nonculturable state from tomato seed using improved qPCR", 《PLOS ONE》 * |
| 蒋娜 等: "植物病原细菌的VBNC状态研究进展", 《植物病理学报》 * |
| 蒋娜: "番茄溃疡病菌VBNC状态的诱导、复苏及机制研究", 《中国博士学位论文全文数据库 农业科技辑》 * |
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