CN110343648A - A kind of method of simple separate tobacco ralstonia solanacearum - Google Patents
A kind of method of simple separate tobacco ralstonia solanacearum Download PDFInfo
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Abstract
A kind of method that the present invention discloses simple separate tobacco ralstonia solanacearum, feature is the following steps are included: 1 sick sample acquisition；2 pathogenicbacteria separations；3 Bacteria culturings；The preparation of 4 bacteria suspensions；5PCR amplification and identification.The present invention acquires field disease leaf, cut the vein vascular bundle at petiole, it is much higher than this feature of other microorganisms using the content of Ralstonia solanacearum in vein vascular bundle, isolate and purify tobacco ralstonia solanacearum, target selectivity of the present invention is strong, and purifying speed is fast, high-efficient, rate is isolated and purified 84% or more, and can ensure that as the disease pathogen.Using the present invention, target cause of disease fast can be accurately obtained, is laid the foundation for follow-up study.General scientific and technical personnel are also extremely easily mastered this method.
Description
Technical field
The invention belongs to plant disease technical fields, and in particular to a kind of method of simple separate tobacco ralstonia solanacearum.
Background technique
Tobacco bacterial wilt (Tobacco bacterial wilt) be tropical and subtropical region tobacco Major Diseases it
One.1880, tobacco bacterial wilt first discovery was in North Carolina Granville (Granville), also referred to as
" Granville wilt " then evolves as the weight on tobacco in states such as Indonesia, Japan, Australia and South Korea
Want disease.The disease China Yangtze river basin and its on the south each cigarette district generally occur, wherein Guangxi, Guangdong, Fujian, Hunan, Zhejiang
The harm of the provinces such as river, Anhui, Sichuan and Guizhou is serious.Yunnan Province just had the report of tobacco bacterial wilt early in 1987, but endanger compared with
Gently.Since 2002, Yunnan Province, nonirrigated farmland, south cigarette district bacterial wilt harm gradually aggravates, local plot disease incidence reach 80% with
On.Currently, the disease is in the mountain of papers in Yunnan Province, the Baoshan, Lincang, Red River, Kunming, Yuxi, Qujing, Zhaotong, Dali, Lijing, Chuxiong
There is generation with the city Dehong Deng Zhou, wherein some areas morbidity of mountain of papers, Lincang, the Baoshan and Dehong is more serious.Although tobacco
Bacterial wilt is more serious in Yunnan Province's morbidity, but to the research relative deficiency of the disease, the research especially in terms of aetology is still few.
Isolating and purifying the disease pathogen and obtaining pure culture is the basis for carrying out correlative study.
Tobacco bacterial wilt is that bacillary soil caused by Ralstonia solanacearum (Ralstonia solanacearum) infects passes
Disease.Pathogen thallus is rod-shaped, both ends blunt circle, 0.5-0.8 μm of μ m of size 0.9-2, has 1-3 root flagellum, more monopoles are raw, no pod
Film.Pathogen growth temperature limits 10 DEG C -37 DEG C, and 30-35 DEG C most suitable.Existing separation method is tissue isolation, with the knife after sterilizing
The epidermis that plant stem disease is good for intersection is pruned, disease is taken to be good for the vascular tissue of intersection, is placed in sterile water after impregnating and crosses
The method for diluting bacterium solution purifies pathogen.Since tobacco bacterial wilt often mixes generation with rhizomes diseases such as balck shanks, show in addition
Contain other a large amount of saprophytic bacterias in disease stem.Therefore, this method can often be separated to various other microorganisms, and cannot get the disease
Pathogen isolates and purifies low efficiency.General scientific and technical personnel use tissue isolation, and it is withered less than tobacco blueness often to will lead to separation
Disease gets target cause of disease wrong.
Summary of the invention
In view of the problems of the existing technology, the purpose of the present invention is to provide a kind of simple separate tobacco ralstonia solanacearums
Method, this method can quickly and accurately obtain the disease pathogen, effectively exclude other germs, and raising isolates and purifies efficiency.
A kind of method of simple separate tobacco ralstonia solanacearum, it is characterised in that the following steps are included:
1, sick sample acquisition
From the blade of field acquisition morbidity, whether observation vein vascular bundle has black splotch, is considered as bacterial wilt hair if having
Diseased plant；Integrated plate blade is put into sampler bag, takes back laboratory.
2, pathogenicbacteria separation
The tissue other than petiole is removed, with alcohol surface sterilization, cuts off the upper table other than vein vascular bundle with sterile razor blade
Skin, palisade tissue, spongy tissue and lower epidermis cut the vein vascular bundle of petiole base；By vein vascular bundle sterile razor blade
It is cut into several pieces of tissue block；It is placed in the sterilizes culture dish containing sterile water and impregnates 5-10min, it is milky white to have around tissue block
After color bacterium solution is overflowed, bacterium solution is mixed with sterile pipette tips.
3, Bacteria culturing
The side that bacterium solution is added drop-wise to TTC plate is drawn with pipettor, is crossed with sterile toothpick, superclean bench dries up postposition
The dark culture 24-48h in 28-30 DEG C of incubator.Form transparent petite after 22-25h, formed after 36-48h irregular shape or
Subcircular bacterium colony has wider white edge, more liquid, intermediate pinkiness or light red thin liquid shape.
4, prepared by bacteria suspension
5, PCR amplification and identification
The PCR identification of tobacco Ralstonia solanacearum uses universal primer Rs-759
(5'-GTCGCCGTCAACTCACTTTCC-3') and Rs-760
(5'-GTCGCCGTCAGCAATGCGGAATCG-3') amplifying specific segment, takes PCR product respectively after amplification
Each 3-7 μ L, loads in the glue hole of 2% Ago-Gel, is control with appropriate DNA standard specimen, separates 30- under 120V constant pressure
45min takes out soak 10min in the EB nucleic acid dye liquor containing 5 μ g/mL；It is placed in gel imager and obtains PCR product
Situation is separated, then having a size close at 300bp between 200 and 300bp if tobacco Ralstonia solanacearum is the band of 281bp.
Further, the sick leaf that step 1 acquires, performance shape are that one rim portion mesophyll tissue of blade is in marshy land shape, wilting, change
Yellow, plaster shape withered spot or sick leaf develop abnormal and bending deformation on one side.
Further, in step 2, when with alcohol surface sterilization, surface sterilization, time 3- are carried out using 70% alcohol
7min；When cutting the vein vascular bundle of petiole base, the length cut is 3-5cm；The tissue block size is 3-5mm × 3-
5mm；Sterile water in the sterilizes culture dish is 8-12mL；The sterile pipette tips are the sterile pipette tips of 10mL.
Further, in step 3, the bacterium solution content of absorption is 15-30 μ L；The production method of the TTC plate are as follows: claim respectively
Peptone 10.0g, glucose 10.0g, casein hydrolysate 1.0g and agar 15.0g are taken, distilled water is added to be settled to 1L, 121 DEG C of height
Pressure sterilizing 20min is placed on 55 DEG C of water-baths, and final concentration of 0.005% chlorination three is added when culture medium temperature is down to 55 DEG C
23-28mL culture medium is added in phenyl tetrazole (TTC) in each sterile petri dish after mixing, manufactured plate is after condensation
Plate containing TTC.
Further, in step 4, the preparation method of bacteria suspension is with the sterile pipette tips picking thallus of 10 μ L in sterile containing 10 μ L
In the PCR pipe of water, 1 μ L is taken to identify isolate for bacterium colony PCR after the concussion that is vortexed.
Further, in step 5, PCR reaction system is 25 μ L, and reaction system is by 12.5 μ L 2 × Es Taq MasterMix
(Dye)、10.5μL ddH2O, 0.5 μ L, 10 μM of Rs-759,0.5 μ L 10M Rs-760 and 1 μ L bacteria suspension composition；
PCR reaction condition is 94 DEG C of initial denaturation 2min, and amplification cycles include 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C
Extend 30s, expands 30 circulations, last 72 DEG C of extensions 2min, and PCR product is stored in 4 DEG C or is directly used in agarose and is coagulated
Gel electrophoresis.
Compared with the prior art, the present invention has the following beneficial effects: sample acquisition is easy, separating rate is fast, easy to be fast
Victory isolates and purifies high-efficient.Tobacco bacterial wilt acquires usually from the stalk of plant when acquiring sick sample at present, sample acquisition
Process is cumbersome, demand is big, and this method only needs acquisition morbidity tobacco leaf, greatly simplifies the operating procedure of sampling, reduces by every part of sample
Demand.Currently, often intersection separation is good for from stem disease in spite of illness, due to what is usually chosen when the separation of tobacco ralstonia solanacearum
Diseased tissues is the sick sample of typical case in morbidity later period, other saprophytic a large amount of microorganisms in sick sample, sample separates is separated to other carefully often
Bacterium and cause target pathogenic bacteria to mix up and mistake.And the present invention chooses blade in spite of illness from disease plant, takes from petiole base
Blade cuts intrapetiolar vein vascular bundle.Petiole vascular tissue is the tip that Ralstonia solanacearum is spread to above-ground plant parts, bacterium
Kind relatively single, aimed strain is selectively strong, and purifying speed is fast, high-efficient, isolates and purifies 84% or more rate, and can ensure that for
The disease pathogen.Using the present invention, target cause of disease fast can be accurately obtained, is laid the foundation for follow-up study.General science and technology
Personnel are also extremely easily mastered this method.
Detailed description of the invention
Fig. 1 is that the PCR of the doubtful pathogen of Yunnan Province's Dehong prefecture tobacco bacterial wilt is detected；
Fig. 2 is that the PCR of the doubtful pathogen of Yunnan Province Puer City tobacco bacterial wilt is detected；
Fig. 3 is that the PCR of the doubtful pathogen of Lincang City, Yunnan Province tobacco bacterial wilt is detected；
Fig. 4 is that the PCR of the doubtful pathogen of Wenshan Prefecture, Yunnan Province tobacco bacterial wilt is detected；
Wherein, A is Ralstonia solanacearum specific band.
Specific embodiment
Embodiment 1
A kind of method of simple separate tobacco ralstonia solanacearum, it is characterised in that the following steps are included:
1, from Yunnan Province's Dehong prefecture bacterial wilt morbidity field cigarette strain on choose one rim portion mesophyll tissue of blade in marshy land shape,
It wilts, turn yellow, plaster shape withered spot or sick leaf develop abnormal and bending deformation sick leaf on one side；Blade is taken from petiole, is seen
It examines whether vein vascular bundle has black splotch, is considered as bacterial wilt morbidity strain if having；Integrated plate blade is put into sampler bag, takes back reality
Room is tested, acquires 10 parts of sick samples altogether.
2, pathogenicbacteria separation
Remove the tissue other than petiole, 70% alcohol surface sterilization 5min；Other than cutting vein vascular bundle with sterile razor blade
Epicuticle, palisade tissue, spongy tissue and lower epidermis, and cut the vein vascular bundle of petiole base 4.0cm；Vein is tieed up and is managed
Beam is cut into the tissue block that several block sizes are 5mm × 5mm with sterile razor blade；It is placed in the sterilizes culture dish containing 10mL sterile water
Impregnate 5min；After having milky bacterium solution spilling around tissue block, bacterium solution is mixed with the sterile pipette tips of 10mL.
3, Bacteria culturing
20 μ L bacterium solutions are drawn in the plate containing TTC with pipettor, are crossed with sterile toothpick and are diluted bacterium solution, are placed in ultra-clean
Workbench drying；The production method of TTC plate are as follows: weigh peptone 10.0g, glucose 10.0g, casein hydrolysate 1.0g respectively
With agar 15.0g, distilled water is added to be settled to 1L, 121 DEG C of high pressure sterilization 20min are placed on 55 DEG C of water-baths, to culture medium temperature
Final concentration of 0.005% triphenyltetrazolium chloride (TTC) is added when being down to 55 DEG C, after mixing in each sterile petri dish
24mL culture medium is added, manufactured plate is the plate containing TTC after condensation.
It is placed in dark culture in 28 DEG C of incubators, in forming transparent petite afterwards for 24 hours, forms irregular shape or close after 36h
Circular colonies have wider white edge, more liquid, intermediate pinkiness or light red thin liquid shape.
4, prepared by bacteria suspension
The preparation method of bacteria suspension is the whirlpool with the sterile pipette tips picking thallus of 10 μ L in the PCR pipe containing 10 μ L sterile waters
1 μ L is taken to identify isolate for bacterium colony PCR after rotation concussion.
5, PCR amplification and identification
The PCR identification of Ralstonia solanacearum uses universal primer Rs-759
(5'-GTCGCCGTCAACTCACTTTCC-3') and Rs-760
(5'-GTCGCCGTCAGCAATGCGGAATCG-3') amplifying specific segment.PCR reaction system is 25 μ L, reactant
System is by 12.5 μ L 2 × Es Taq MasterMix (Dye), 10.5 μ L ddH2O、0.5μL 10μM Rs-759、0.5μL 10M
Rs-760 and 1 μ L bacteria suspension composition；
PCR reaction condition is 94 DEG C of initial denaturation 2min, and amplification cycles include 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C
Extend 30s, expands 30 circulations, last 72 DEG C of extensions 2min, and PCR product is stored in 4 DEG C or is directly used in agarose and is coagulated
Gel electrophoresis；
Appropriate DNA standard specimen and each 5 μ L of PCR product are taken respectively, are loaded in the glue hole of 2% Ago-Gel, in 120V
30min is separated under constant pressure, and impregnates 10min in the EB nucleic acid dye liquor containing 5 μ g/mL；
It is placed in gel imager and obtains PCR product separation situation, electrophoresis picture is shown between 200 and 300bp and approaches
Having the band that a size is 281bp at 300bp is tobacco Ralstonia solanacearum (partial results such as Fig. 1).This experiment is from Yunnan Province's moral
It is successfully separated in the sick sample of 10 parts of Hong Zhou acquisition and obtains 10 parts of tobacco ralstonia solanacearums, isolating and purifying success rate is 100.00%.
Embodiment 2
A kind of method of simple separate tobacco ralstonia solanacearum, it is characterised in that the following steps are included:
1,23 parts of tobacco bacterial wilt disease samples are acquired altogether from Yunnan Province Puer City, it is same as Example 1.
2, pathogenicbacteria separation
The tissue other than petiole is removed, with 70% alcohol surface sterilization 3min；With sterile razor blade cut vein vascular bundle with
Outer epicuticle, palisade tissue, spongy tissue and lower epidermis, and cut the vein vascular bundle of petiole base 3.0cm；Vein is tieed up
The tissue block that several block sizes are 3mm × 3mm is cut into tube bank with sterile razor blade；It is placed in the sterilizes culture dish containing 8mL sterile water
Middle immersion 7min；After having milky white liquid spilling around tissue block, bacterium solution is mixed with the sterile pipette tips of 10mL.
3, Bacteria culturing
15 μ L bacterium solutions are drawn in the plate containing TTC with pipettor, are crossed with sterile toothpick and are diluted bacterium solution, are placed in ultra-clean
Workbench drying；The production method of TTC plate are as follows: weigh peptone 10.0g, glucose 10.0g, casein hydrolysate 1.0g respectively
With agar 15.0g, distilled water is added to be settled to 1L, 121 DEG C of high pressure sterilization 20min are placed on 55 DEG C of water-baths, to culture medium temperature
Final concentration of 0.005% triphenyltetrazolium chloride (TTC) is added when being down to 55 DEG C, after mixing in each sterile petri dish
26mL culture medium is added, manufactured plate is the plate containing TTC after condensation.
It is placed in dark culture in 30 DEG C of incubators, transparent petite is formed after 22h, forms irregular shape or close after 42h
Circular colonies have wider white edge, more liquid, intermediate pinkiness or light red thin liquid shape.
4, prepared by bacteria suspension
The preparation method of bacteria suspension is the whirlpool with the sterile pipette tips picking thallus of 10 μ L in the PCR pipe containing 10 μ L sterile waters
1 μ L is taken to identify isolate for bacterium colony PCR after rotation concussion.
5, the PCR identification of Ralstonia solanacearum uses universal primer Rs-759
(5'-GTCGCCGTCAACTCACTTTCC-3') and Rs-760
(5'-GTCGCCGTCAGCAATGCGGAATCG-3') amplifying specific segment.PCR reaction system is 25 μ L, reactant
System is by 12.5 μ L 2 × Es Taq MasterMix (Dye), 10.5 μ L ddH2O、0.5μL 10μM Rs-759、0.5μL 10M
Rs-760 and 1 μ L bacteria suspension composition；
PCR reaction condition is 94 DEG C of initial denaturation 2min, and amplification cycles include 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C
Extend 30s, expands 30 circulations, last 72 DEG C of extensions 2min, and PCR product is stored in 4 DEG C or is directly used in agarose and is coagulated
Gel electrophoresis；
Appropriate DNA standard specimen and each 3 μ L of PCR product are taken respectively, are loaded in the glue hole of 2% Ago-Gel, in 120V
32min is separated under constant pressure, and impregnates 10min in the EB nucleic acid dye liquor containing 5 μ g/mL；
It is placed in gel imager and obtains PCR product separation situation, detection discovery is between 200 and 300bp close to 300bp
It is tobacco Ralstonia solanacearum (partial results such as Fig. 2) that, which there is the band that a size is 281bp at place,.This experiment is adopted from Yunnan Province Puer City
It is successfully separated in the sick sample of 23 parts of collection and obtains 21 plants of tobacco ralstonia solanacearums, isolating and purifying success rate is 91.30%.
Embodiment 3
A kind of method of simple separate tobacco ralstonia solanacearum, it is characterised in that the following steps are included:
1, from Lincang City, Yunnan Province, morbidity tobacco field acquires 13 parts of tobacco bacterial wilt disease samples, same as Example 1.
2, pathogenicbacteria separation
The tissue other than petiole is removed, with 70% alcohol surface sterilization 7min；Epicuticle, fence group are cut off with sterile razor blade
It knits, spongy tissue and lower epidermis, takes long 5cm vein vascular bundle；Vein vascular bundle, which is cut into several block sizes with sterile razor blade, is
The tissue block of 4mm × 4mm；It is placed in the sterilizes culture dish containing 12mL sterile water and impregnates 7min；It is milky white to have around tissue block
After color liquid overflows, bacterium solution is mixed with the sterile pipette tips of 10mL.
3, Bacteria culturing
30 μ L bacterium solutions are drawn in the plate containing TTC with pipettor, are crossed with sterile toothpick and are diluted bacterium solution, are placed in ultra-clean
Workbench drying；The production method of TTC plate are as follows: weigh peptone 10.0g, glucose 10.0g, casein hydrolysate 1.0g respectively
With agar 15.0g, distilled water is added to be settled to 1L, 121 DEG C of high pressure sterilization 20min are placed on 55 DEG C of water-baths, to culture medium temperature
Final concentration of 0.005% triphenyltetrazolium chloride (TTC) is added when being down to 55 DEG C, after mixing in each sterile petri dish
25mL culture medium is added, manufactured plate is the plate containing TTC after condensation.
It is placed in dark culture in 30 DEG C of incubators, transparent petite is formed after 25h, forms irregular shape or close after 48h
Circular colonies have wider white edge, more liquid, intermediate pinkiness or light red thin liquid shape.
4, prepared by bacteria suspension
The preparation method of bacteria suspension is the whirlpool with the sterile pipette tips picking thallus of 10 μ L in the PCR pipe containing 10 μ L sterile waters
1 μ L is taken to identify isolate for bacterium colony PCR after rotation concussion.
5, the PCR identification of Ralstonia solanacearum uses universal primer Rs-759
(5'-GTCGCCGTCAACTCACTTTCC-3') and Rs-760
(5'-GTCGCCGTCAGCAATGCGGAATCG-3') amplifying specific segment.PCR reaction system is 25 μ L, reactant
System is by 12.5 μ L 2 × Es Taq MasterMix (Dye), 10.5 μ L ddH2O、0.5μL 10μM Rs-759、0.5μL 10M
Rs-760 and 1 μ L bacteria suspension composition；
PCR reaction condition is 94 DEG C of initial denaturation 2min, and amplification cycles include 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C
Extend 30s, expands 30 circulations, last 72 DEG C of extensions 2min, and PCR product is stored in 4 DEG C or is directly used in agarose and is coagulated
Gel electrophoresis；
Appropriate DNA standard specimen and each 7 μ L of PCR product are taken respectively, are loaded in the glue hole of 2% Ago-Gel, in 120V
32min is separated under constant pressure, and impregnates 10min in the EB nucleic acid dye liquor containing 5 μ g/mL；
It is placed in gel imager and obtains PCR product separation situation, detection discovery is between 200 and 300bp close to 300bp
It is tobacco Ralstonia solanacearum that, which there is the band that a size is 281bp at place,.13 parts of tobacco blueness that this experiment is acquired from Lincang City, Yunnan Province
The pathogen for obtaining 11 parts of sick samples is successfully separated in blight disease sample, pathogen Success rate of virus isolation is 84.61%.
Comparative example
The method of conventional tobacco ralstonia solanacearum, comprising the following steps:
1, sick sample acquisition
The tobacco bacterial wilt band of stem side nigrescence, tobacco leaf yellow is chosen from Wenshan Prefecture, Yunnan Province tobacco bacterial wilt morbidity field
Sick cigarette strain, the stem in spite of illness for removing a length of 20cm are put into sample sack, take back use for laboratory and make pathogenicbacteria separation.Experiment acquires 18 altogether
The sick sample of part.
2, pathogenicbacteria separation
The epidermis that plant stem disease is good for intersection is pruned with the knife after sterilizing, disease is taken to be good for the vascular tissue of intersection about
1g is placed in 10mL sterile water, impregnates about 10min, mixes bacterium solution with the sterile pipette tips of 10mL.
3, Bacteria culturing
15 μ L bacterium solutions are drawn in the plate containing TTC with pipettor, are crossed with sterile toothpick and are diluted bacterium solution, are placed in ultra-clean
Workbench drying；The production method of TTC plate are as follows: weigh peptone 10.0g, glucose 10.0g, casein hydrolysate 1.0g respectively
With agar 15.0g, distilled water is added to be settled to 1L, 121 DEG C of high pressure sterilization 20min are placed on 55 DEG C of water-baths, to culture medium temperature
Final concentration of 0.005% triphenyltetrazolium chloride (TTC) is added when being down to 55 DEG C, after mixing in each sterile petri dish
25mL culture medium is added, manufactured plate is the plate containing TTC after condensation.
28 DEG C of cultures are placed in, in forming subcircular bacterium colony afterwards for 24 hours, centre is dark red, edge is transparent, colony diameter about 5mm, even
There is petite to be formed；Colony diameter is about 8mm after 48h, and intermediate pink, edge is transparent or white edge is relatively narrow, occasionally have irregular shape or
Subcircular, the bacterium colony with wider white edge, more liquid, intermediate pinkiness or light red thin liquid shape.
4, prepared by bacteria suspension
The preparation method of bacteria suspension is the whirlpool with the sterile pipette tips picking thallus of 10 μ L in the PCR pipe containing 10 μ L sterile waters
1 μ L is taken to identify isolate for bacterium colony PCR after rotation concussion.
5, the PCR identification of Ralstonia solanacearum uses universal primer Rs-759
(5'-GTCGCCGTCAACTCACTTTCC-3') and Rs-760
(5'-GTCGCCGTCAGCAATGCGGAATCG-3') amplifying specific segment.PCR reaction system is 25 μ L, reactant
System is by 12.5 μ L 2 × Es Taq MasterMix (Dye), 10.5 μ L ddH2O、0.5μL 10μM Rs-759、0.5μL 10M
Rs-760 and 1 μ L bacteria suspension composition；
PCR reaction condition is 94 DEG C of initial denaturation 2min, and amplification cycles include 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C
Extend 30s, expands 30 circulations, last 72 DEG C of extensions 2min, and PCR product is stored in 4 DEG C or is directly used in agarose and is coagulated
Gel electrophoresis；
Appropriate DNA standard specimen and each 3 μ L of PCR product are taken respectively, are loaded in the glue hole of 2% Ago-Gel, in 120V
32min is separated under constant pressure, and impregnates 10min in the EB nucleic acid dye liquor containing 5 μ g/mL；
It is placed in gel imager and obtains PCR product separation situation, testing result shows only have 4 parts of samples in 18 parts of samples
PCR product electrophoretic band have between 200 and 300bp close at 300bp a size be 281bp band be tobacco blueness
Withered bacterium (partial results such as Fig. 4).It is 22.22% with the success rate that the conventional method isolates and purifies tobacco bacterial wilt cause of disease.
Claims (7)
1. a kind of method of simple separate tobacco ralstonia solanacearum, feature the following steps are included:
(1) sick sample acquisition
From field, morbidity cigarette strain acquires sick leaf, and whether observation vein vascular bundle has black splotch, is considered as bacterial wilt morbidity if having
Strain；Full wafer tobacco leaf is put into sampler bag, takes back laboratory；
(2) pathogenicbacteria separation
The tissue other than petiole is removed, with alcohol surface sterilization, epicuticle, grid other than vein vascular bundle are cut off with sterile razor blade
Column tissue, spongy tissue and lower epidermis cut the vein vascular bundle of petiole base；If vein vascular bundle is cut into sterile razor blade
Stem organization's block；It is placed in the sterilizes culture dish containing sterile water and impregnates 5-10min, to there is milky bacterium solution spilling around tissue block
Afterwards, bacterium solution is mixed with sterile pipette tips；
(3) Bacteria culturing
Bacterium solution is drawn in the plate containing TTC with pipettor, is crossed with sterile toothpick and is diluted bacterium solution, is placed in superclean bench and blow
It is dry；
It is placed in dark culture 24-48h in 28-30 DEG C of incubator, forms transparent petite after 22-25h, bacterium colony is in after 36-48h
Irregular shape or subcircular have wider white edge, more liquid, intermediate pinkiness or light red thin liquid shape；
(4) prepared by bacteria suspension
(5) PCR amplification and identification
Universal primer Rs-759 (the 5'- that the PCR identification of tobacco Ralstonia solanacearum is detected using Ralstonia solanacearum
) and Rs-760 (5'-GTCGCCGTCAGCAATGCGGAATCG-3') amplifying specific piece GTCGCCGTCAACTCACTTTCC-3'
Section, takes each 3-7 μ L of PCR product, loads in the glue hole of 2% Ago-Gel, be with DNA standard specimen appropriate after amplification
Control, separates 30-45min under 120V constant pressure, by soak in the nucleic acid dye liquor 10min for containing 5 μ g/mL EB；It is placed in gel
PCR product is obtained on imager and separates situation, then has amplification close at 300bp between 200 and 300bp if tobacco Ralstonia solanacearum
Product band.
2. a kind of method of simple separate tobacco ralstonia solanacearum according to claim 1, it is characterised in that: step 1 acquisition
Blade, typical symptom be one rim portion mesophyll tissue of blade in marshy land shape, wilting, flavescence, plaster shape withered spot or sick leaf on one side
Develop abnormal and bending deformation.
3. a kind of method of simple separate tobacco ralstonia solanacearum according to claim 1, it is characterised in that: in step 2, use
When alcohol surface sterilization, surface sterilization, time 3-7min are carried out using 70% alcohol；When cutting vein vascular bundle, cut
Length is 3-5cm；The tissue block size is 3-5mm × 3-5mm；Sterile water in the sterilizes culture dish is 8-12mL；Institute
Stating sterile pipette tips is the sterile pipette tips of 10mL.
4. a kind of method of simple separate tobacco ralstonia solanacearum according to claim 1, it is characterised in that: in step 3, inhale
The bacterium solution content taken is 15-30 μ L；The production method of the TTC plate are as follows: weigh peptone 10.0g, glucose respectively
10.0g, casein hydrolysate 1.0g and agar 15.0g, add distilled water to be settled to 1L, and 121 DEG C of high pressure sterilization 20min are placed on 55 DEG C
Final concentration of 0.005% triphenyltetrazolium chloride is added, every after mixing in water-bath when culture medium temperature is down to 55 DEG C
23-28mL culture medium is added in a sterile petri dish, manufactured plate is the plate containing TTC after condensation.
5. a kind of method of simple separate tobacco ralstonia solanacearum according to claim 1, it is characterised in that: in step 4, bacterium
The preparation method of suspension is to take 1 after the concussion that is vortexed with the sterile pipette tips picking thallus of 10 μ L in the PCR pipe containing 10 μ L sterile waters
μ L identifies isolate for bacterium colony PCR.
6. a kind of method of simple separate tobacco ralstonia solanacearum according to claim 1, it is characterised in that: in step 5,
PCR reaction system is 25 μ L, and reaction system is by 12.5 μ L 2 × Es Taq MasterMix (Dye), 10.5 μ L ddH2O、0.5μ
10 μM of Rs-759 of L, 0.5 μ L 10M Rs-760 and 1 μ L bacteria suspension composition；
PCR reaction condition is 94 DEG C of initial denaturation 2min, and amplification cycles include 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extensions
30s expands 30 circulations, last 72 DEG C of extensions 2min, and PCR product is stored in 4 DEG C or is directly used in Ago-Gel electricity
Swimming.
7. a kind of method of simple separate tobacco ralstonia solanacearum according to claim 1, it is characterised in that: step (5) institute
Stating close to the band at 300bp is band at 281bp between 200 and 300bp.
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