CN101671738A - Method for fast detection and identification of bacterial wilt caused by infection of ralstonia solanacearum - Google Patents
Method for fast detection and identification of bacterial wilt caused by infection of ralstonia solanacearum Download PDFInfo
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Abstract
The invention discloses a method for the fast detection and identification of bacterial wilt caused by the infection of ralstonia solanacearum. The method comprises three stages of preparing plant disease samples, observation with naked eyes and PCR verification, wherein the detection and the identification can be completed within four hours with low cost. The method makes a breakthrough on characteristics of traditional bacteriosis identification such as long period, high professional demand and higher cost and also overcomes the deficiencies of microbial identification system imported overseas, such as specific instruments and materials, high cost and longer identification period, and by means of the organic combination of special superficial symptoms of the disease sample and molecularbiotechnology, accurate and reliable results can be obtained in order to meet the demand of fast, accurate identification for plant diseases in actual production.
Description
Technical field
The invention belongs to Plant diseases authenticate technology field, particularly Solanaceae Ralstonia solanacearum (Ralstoniasolanacearum) infects the rapid detection authentication method of the bacterial wilt that causes.
Background technology
Infect the bacterial wilt that causes for the Solanaceae Ralstonia solanacearum and detect evaluation, prior art has three kinds: traditional plant pathogenetic bacteria isolation and identification method, BIOLOG microbial identification system and Mei Liai microorganism identification method.
Traditional vegetative bacteria bacterial wilt authentication method, mainly comprise steps such as bacterial isolate bacterium, the pure culture that obtains isolated strains, bacterium colony and morphological features observation, physio-biochemical characteristics mensuration and pathogenic mensuration from sick sample, this qualification process needs more than 1 month at least, workload is big, qualification time is long, and strongly professional.
As for the microbial identification system of the external import of selling on the market at present, the representational microbial identification system that U.S. BIOLOG company and the exploitation of French Mei Liai company are arranged.The former carries out the enzyme and tetrazolium class material generation color reaction and the differences in turbidity that produce in the metabolic processes to detect the different carbon sources of microorganism cells utilization, set up the database of finger printing and microbe species phase reaction, by collection of illustrative plates and the database reference of intelligence software, draw qualification result with microorganism to be identified.The latter is the physico-chemical property difference according to different microorganisms, adopt photoelectric colorimetry, measure the microbiological degradation substrate and cause pH to change and the different colours of generation, judge the result of reaction, compare with kind of the living model of database standard bacterium, obtain similar system evaluation value.The qualification process of above-mentioned these two kinds of identification systems all needed more than 18 hours, also need to buy special-purpose expert evidence of expensive import and plant and instrument, and this system mainly is applicable to people, animal and environmental microorganism evaluation, rarely be used for the evaluation of plant pathogenic microorganisms, document is not seen the report that is useful on the evaluation of Solanaceae bacterial wilt yet at present.
Summary of the invention
In order to solve above-mentioned the deficiencies in the prior art part, the object of the present invention is to provide a kind of easy and simple to handle, quick and precisely, Solanaceae bacterial wilt authentication method with low cost.The present invention organically combines sick sample special list disease and polymerase chain reaction (PCR) technology, with the distinctive FliC gene of Solanaceae Ralstonia solanacearum (one of oligogene of assembling Solanaceae Ralstonia solanacearum flagellum, the FliC gene in Solanaceae Ralstonia solanacearum flagellum assembling the position and effect as shown in Figure 1) the sequences Design Auele Specific Primer, set up Solanaceae Ralstonia solanacearum PCR reaction system, differentiate in conjunction with the spray feature that the phytopathy that infected by the Solanaceae Ralstonia solanacearum is organized in the water medium to be showed, reliable results, may be used in the actual production, Plant diseases is carried out rapid detection accurately and effectively.
Purpose of the present invention is achieved through the following technical solutions: a kind of rapid detection identifies that the Solanaceae Ralstonia solanacearum infects the method for the bacterial wilt that causes, comprises following operation steps:
(1) preparation of the sick sample of detection: water is rinsed the basal part of stem and the root of sick sample to be checked well, gets the vascular bundle tissue that a block sizes is 1.5-2mm * 2.5-3mm from basal part of stem;
(2) the sick sample of visual inspection is organized in phenotype in the slide glass water droplet: the aqua sterilisa of drawing 80～100 μ L, drop on the clean slide glass, vascular bundle tissue in the step (1) is put into the water droplet of slide glass, and slightly firmly be pressed in the water droplet, leave standstill, observation has or not the dense ejection from sick sample tissue of vaporific bacterium; If any, continue next step test, as not having, illustrate that this disease sample is not infected by the Solanaceae Ralstonia solanacearum to cause;
(3) the further detection validation of PCR: the vaporific bacterium that the sick sample tissue in above-mentioned water droplet sprays is dense as template, carries out the PCR reaction; Described upstream primer is 5 '-atgattctgcggcctacgcggcat-3 ', described downstream primer is 5 '-agtcgggtctggcgtcgaccatcaa-3 ';
(4) result differentiates: with reaction product electrophoresis on sepharose of step (3) gained; Observing in gel imaging system, if any the specific band of 517bp size, confirm promptly that the disease sample is infected by the Solanaceae Ralstonia solanacearum to cause, as do not have this specific band, then should the disease sample be that the Solanaceae Ralstonia solanacearum infects and causes.
Solanaceae Ralstonia solanacearum described in the step (2) infects the sick sample vascular bundle that causes and is organized in visible " spraying " phenomenon of naked eyes in the water.
PCR described in the step (3) reaction be the vaporific bacterium of the sick sample tissue ejection in above-mentioned water droplet dense absorption 1 μ L as template, join in the PCR pipe, add each 0.5 μ mol/L of the Taq of 1 unit (U) enzyme, 200 μ mol/L deoxyribonucleotides (dNTP), upstream primer and downstream primer, 1 times of PCR reaction buffer more respectively, complementing to cumulative volume with distilled water is 25 μ L, carries out the PCR reaction in the PCR instrument.
The described PCR reaction conditions of step (3) is 94 ℃ of pre-sex change 4min, 94 ℃ of sex change 1min, 52 ℃ of annealing 45s, 72 ℃ of extension 50s, and 10min is finally extended in 30 circulations.
The middle reaction product of step (4) is to be electrophoresis on the sepharose of (w/v) 0.8～1.5%, 50～75V constant voltage electrophoresis, 50～70min in the quality volume percent.
The sick sample tissues such as eggplant, tomato, peanut, potato, capsicum, tobacco, mater convolvulus, husky ginger, chrysanthemum or mulberry tree that described sick sample to be checked causes for the Solanaceae Ralstonia solanacearum infects.
The present invention has following advantage and beneficial effect with respect to prior art:
(1) fast and convenient: method of the present invention qualification cycle is in 4 hours, and the non-specialised staff all can operate, and authentication method adaptability is strong; And no matter be traditional plant pathogenetic bacteria authentication method or the microbial identification system of BIOLOG or Mei Liai, qualification cycle is all longer, and the former needed more than 1 month, and the latter at least also needs more than 18 hours.
(2) accurate: the present invention combines according to the reaction of diseased tissues characteristic and the special PCR detection method of Solanaceae Ralstonia solanacearum that are infected by the Solanaceae Ralstonia solanacearum, and the qualification result of acquisition more accurately and reliably; And biochemical reaction that microbial identification system is based on and color distortion, sometimes because of difference in operation and the not equal reason of check-out console, part is inconsistent with java standard library in the data that cause obtaining, and is difficult to final conclusion.
(3) low cost: the employed material of the invention is all cheaper, and slide glass not only price is low, but also can use repeatedly; Reagent such as used PCR instrument is very universal at home at present, enzyme, substrate and the agarose that PCR is used and medicine all are open, and the cost of identifying a sample is in 9.5 yuan; BIOLOG or Mei Liai microbial identification system, not only need specialized equipment equipment and finish by special certifying agency, but also need to buy the special-purpose consumptive material of identifying of the said firm, present professional certifying agency identifies that the expenses standard of a sample is 800 yuan, and traditional authentication method be also because will carry out that the complex physical biochemical characteristic is measured and operations such as pathogenic mensuration, and its cost is also wanted about 154 yuan.
Description of drawings
Fig. 1 is each gene sketch of Solanaceae Ralstonia solanacearum flagellum assembling.
Wherein the FliC genetic marker is red.This sketch derives from the KanehisaLaboratories KEGG of Kyoto Univ Japan (Kyoto Encyclopedia of Genes and Genomes) (http://www.genome.jp/kegg-bin/showpathway+rso02040).
Fig. 2 identifies that for detecting the Solanaceae Ralstonia solanacearum infects the process flow sheet of the bacterial wilt that causes.
Fig. 3 is organized in spraying phenomenon figure in the water droplet for the sick sample of embodiment 1.
Wherein, 3A is known eggplant bacterial wilt sample tissue sample; 3B is the healthy eggplant tissue sample of contrast.
Fig. 4 is the PCR product electrophoresis detection figure of embodiment 1.
Wherein, 1. standard molecular weight (100bp gradient); 2-9. different sick sample tissues; 10. negative control.
Fig. 5 is organized in spraying phenomenon figure in the water droplet for the sick sample of embodiment 2.
Wherein, 5A is No. 2 sick samples; 5B is No. 12 sick samples.
Fig. 6 is the PCR product electrophoresis detection figure of embodiment 2.
Wherein, 1. standard molecular weight (100bp gradient); 2-18. different sample to be checked.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
Embodiment 1: the detection to known sick sample is identified
One, experiment material: use the eggplant that known Solanaceae Ralstonia solanacearum infects the eggplant that causes and sick sample tissue of tomato and known health and be organized as material.
Two, experimental technique:
(1) water is rinsed the basal part of stem and the root of sick sample to be checked well, get the vascular bundle tissue that size is about 1.5～2mm * 2.5～3mm from the basal part of stem of sick sample to be checked, comprise the sick sample of 4 portions of eggplants and 4 parts of sick samples of tomato in 8 parts of samples to be checked, other gets a healthy eggplant sample and compares;
(2) draw the aqua sterilisa of 100 μ L with aseptic pipettor, drop on the clean slide glass, tweezers with sterilization are put into the sterilization water droplet of 100 μ L sizes on the clean slide glass with diseased tissues, note slightly firmly diseased tissues being pressed in the water droplet, leave standstill about 5 minutes; Observation has or not the dense ejection from sick sample tissue of vaporific bacterium, if any continuing next step test; As not having, illustrate that this disease sample is not infected by the Solanaceae Ralstonia solanacearum to cause;
(3) from the vaporific bacterium of sick sample tissue ejection is dense, get 1 μ L liquid in the PCR pipe, preparation polymerase chain reaction (PCR) mixture comprises each 0.5 μ mol/L of 1 Taq of unit enzyme, 4 kinds of deoxyribonucleotides (dNTP), 200 μ mol/L, upstream primer and downstream primer, 10 times of reaction buffers of 2.5 μ L and distilled water to 25 μ L; The PCR pipe is put into the PCR instrument, in the PCR instrument, carry out the PCR reaction.Reaction parameter is set: 94 ℃ of pre-sex change 4min, 94 ℃ of sex change 1min, 52 ℃ of annealing 45s, 72 ℃ of extension 50s, 10min is finally extended in 30 circulations;
(4) negate answers product to be electrophoresis on (w/v) 1% sepharose, 70V constant voltage electrophoresis 60min in the quality volume percent; In gel imaging system, observe, and take pictures; Make a determination according to electrophoresis result.
Detection evaluation Solanaceae Ralstonia solanacearum infects the process flow sheet of the bacterial wilt that causes and sees Fig. 2.
Three, experimental result and analysis:
As shown in Figures 3 and 4, use method of the present invention, the sick sample basal part of stem of the eggplant that the Solanaceae Ralstonia solanacearum causes and tomato vascular bundle is organized in and all occurs the spraying phenomenon in the slide glass water droplet (Fig. 3 only shows the spraying of the sick sample of one of them eggplant, other sick sample is identical with it), and with vaporific liquid be template can successfully increase the expection size be the special purpose fragment (see figure 4) of 517bp, and above spraying phenomenon and specific amplified band do not appear in healthy eggplant sample in contrast, illustrate that this detection method is accurately and reliably.
Embodiment 2: to the detection qualification test of the sick sample of the unknown
One, experiment material: 10 kinds of different plant-bacterial-wilt samples or the asymptomatic healthy sample of picking up from a plurality of regional fields detected.
Two, experimental technique:
10 kinds of different plant samples that collect from a plurality of areas, Guangdong are taken a sample, obtain 17 samples altogether and be numbered, information such as the pairing floristics of each number sample, collecting location and symptom performance are as shown in table 1.Detect to identify that the method that is adopted is identical with embodiment 1 method therefor, and with traditional plant pathogenetic bacteria isolation and identification method comparatively validate in addition.
Traditional plant pathogenetic bacteria isolation and identification method concrete steps comprise: 1. with 75% alcohol sick sample tissue surface to be checked is sterilized; 2. the scalper with sterilization cuts disease sample vascular bundle tissue and dilutes separation; 3. cultivated 48～36 hours containing on the TTC agar plate under line and 28～30 ℃ of conditions; 4. purifying list bacterium colony obtains bacterial strain; 5. bacterium colony and morphological features are observed; 6. the bacterial strain physio-biochemical characteristics are measured, and promptly measure utilization and decomposition to carbohydrate such as lactose, maltose, D (+)-cellobiose, dextrose plus saccharose, N.F,USP MANNITOL, sorbyl alcohol, melampyrins; 7. bacterial strain 16S rDNA gene clone and sequential analysis; 8. (25～35 ℃) carry out the artificial inoculation test in the greenhouse, and the mensuration isolated strains is pathogenic to former host plant; 9. inoculate the plant observation of symptoms; 10. from show the disease diseased plant, be separated to the pathogenic bacteria identical again with inoculating strain.
Three, experimental procedure:
(1) water is rinsed the basal part of stem and the root of sick sample to be checked well, get the vascular bundle tissue that size is about 1.5～2mm * 2.5～3mm from the basal part of stem of sample to be checked, from different sample to be checked (eggplant, tomato, potato, peanut, capsicum, tobacco, mater convolvulus, husky ginger, chrysanthemum, mulberry tree), get 17 duplicate samples tissues altogether;
(2) draw the aqua sterilisa of about 100 μ L with aseptic pipettor, drop on the clean slide glass, tissue is put into the about 100 μ L sterilization of clean slide glass water droplet, note slightly firmly sick sample tissue being pressed in the water droplet, leave standstill about 5 minutes with the tweezers of sterilization; Observation has or not that vaporific bacterium is dense to be sprayed from diseased tissues, if any continuing next step test; As not having, illustrate that this disease sample is not to be infected by the Solanaceae Ralstonia solanacearum to cause;
(3) from vaporific bacterium is dense, get 1 μ L liquid in the PCR pipe, preparation polymerase chain reaction (PCR) mixture comprises 1 Taq of unit enzyme, 4 kinds of each 0.5 μ mol/L of deoxyribonucleotide dNTP 200 μ mol/L, upstream primer and downstream primer, 10 times of reaction buffers of 2.5 μ L and distilled water to 25 μ L; The PCR pipe is put into the PCR instrument, in the PCR instrument, carry out the PCR reaction.Reaction parameter is set: 94 ℃ of pre-sex change 4min, 94 ℃ of sex change 1min, 52 ℃ of annealing 45s, 72 ℃ of extension 50s, 10min is finally extended in 30 circulations;
(4) product electrophoresis on 1% sepharose, 70V electrophoresis 60 minutes are answered in negate; In gel imaging system, observe, and take pictures; Make a determination according to electrophoresis result.
Four, experimental result and analysis:
Show as Fig. 5, Fig. 6 and table 1 result: 2,3,4,5,7,9,10,12,13,14,16,17 and No. 18 samples are all positive in 17 samples to be checked, be to contain the Solanaceae Ralstonia solanacearum in the disease sample, infect by the Solanaceae Ralstonia solanacearum and to cause, be consistent with its symptom performance; 6,8,11 and No. 15 samples are negative; be not contain the Solanaceae Ralstonia solanacearum in the sample; not to infect by the Solanaceae Ralstonia solanacearum to cause undesired or healthy plant; also consistent with its sign; and the result of the inventive method detected result and traditional plant pathogenetic bacteria isolation identification is in full accord, thereby has further verified the reliability of this detection authentication method.
The inventive method used by table 1 and traditional technique in measuring identifies that the result of different sources sample to be checked compares
| Sample number into spectrum | Host plant | The symptom performance | Gather ground | The inventive method | Traditional method |
| ??2 | Eggplant | Wilt | The Dongguan | ??+ | ??+ |
| ??3 | Eggplant | It is withered to wilt | Lianzhou City | ??+ | ??+ |
| ??4 | Tomato | Wilt | Foshan | ??+ | ??+ |
| ??5 | Tomato | The slight wilting | Guangzhou | ??+ | ??+ |
| ??6 * | Tomato | Healthy no illness | Guangzhou | ??- | ??- |
| ??7 | Potato | Wilt | Huizhou | ??+ | ??+ |
| ??8 * | Potato | Healthy no illness | Huizhou | ??- | ??- |
| ??9 | Peanut | Wilt | Guangzhou | ??+ | ??+ |
| ??10 | Capsicum | Wilt | High wanting | ??+ | ??+ |
| ??11* | Capsicum | Healthy no illness | Guangzhou | ??- | ??- |
| ??12 | Tobacco | Yellow, wilting | The Nanxiong | ??+ | ??+ |
| ??13 | Tobacco | The yellow of 1-2 sheet leaf | The Nanxiong | ??+ | ??+ |
| ??14 | Mater convolvulus | Wilt | Guangzhou | ??+ | ??+ |
| ??15* | Husky ginger | Healthy no illness | Spring | ??- | ??- |
| ??16 | Husky ginger | It is withered to wilt | Spring | ??+ | ??+ |
| ??17 | Chrysanthemum | Wilt | Guangzhou | ??+ | ??+ |
| ??18 | Mulberry tree | It is withered to wilt | Spring | ??+ | ??+ |
Annotate :+be expressed as the positive ,-be expressed as feminine gender
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
SEQUENCE?LISTING
＜110〉Inst. of Plant Protection, Guangdong Prov. Academy of Agricultural Sciences
＜120〉a kind of rapid detection identifies that the Solanaceae Ralstonia solanacearum infects the bacterial wilt method that causes
<130>66
<160>2
<170>PatentIn?version?3.2
<210>1
<211>24
<212>DNA
<213>artificial?sequence
<220>
＜223〉upstream primer
<400>1
atgattctgc?ggcctacgcg?gcat????????????????????????????24
<210>2
<211>25
<212>DNA
<213>artificial?sequence
<220>
＜223〉downstream primer
<400>2
agtcgggtct?ggcgtcgacc?atcaa???????????????????????????25
Claims (5)
1, a kind of rapid detection identifies that Solanaceae Ralstonia solanacearum (Ralstonia solanacearum) infects the method for the bacterial wilt that causes, comprises following operation steps:
(1) preparation of the sick sample of detection: water is rinsed the basal part of stem and the root of sick sample to be checked well, gets the vascular bundle tissue that a block sizes is 1.5～2mm * 2.5～3mm from basal part of stem;
(2) the sick sample of visual inspection is organized in phenotype in the slide glass water droplet: the aqua sterilisa of drawing 80～100 μ L, drop on the clean slide glass, vascular bundle tissue in the step (1) is put into the water droplet of slide glass, and be pressed in the water droplet, leave standstill, observation has or not the dense ejection from sick sample tissue of vaporific bacterium; If any, continue next step test, as not having, illustrate that this disease sample is not infected by the Solanaceae Ralstonia solanacearum to cause;
(3) the further detection validation of PCR: the vaporific bacterium of getting sick sample tissue ejection in the water droplet of step (2) is dense as template, carries out the PCR reaction; Described upstream primer is 5 '-atgattctgcggcctacgcggcat-3 ', described downstream primer is 5 '-agtcgggtctggcgtcgaccatcaa-3 ';
(4) result differentiates: with reaction product electrophoresis on sepharose of step (3) gained; Observing in gel imaging system, if any the specific band of 517bp size, confirm promptly that the disease sample is infected by the Solanaceae Ralstonia solanacearum to cause, as do not have this specific band, then should the disease sample be that the Solanaceae Ralstonia solanacearum infects and causes.
2, a kind of rapid detection according to claim 1 identifies that the Solanaceae Ralstonia solanacearum infects the method for the bacterial wilt that causes, it is characterized in that, PCR described in the step (3) reaction be the vaporific bacterium of the sick sample tissue ejection in the water droplet of step (2) dense absorption 1 μ L as template, join in the PCR pipe, add each 0.5 μ mol/L of 1 Taq of unit enzyme, 200 μ mol/L deoxyribonucleotides, upstream primer and downstream primer, 1 times of PCR reaction buffer more respectively, complementing to cumulative volume with distilled water is 25 μ L, carries out the PCR reaction in the PCR instrument.
3, a kind of rapid detection according to claim 1 identifies that the Solanaceae Ralstonia solanacearum infects the method for the bacterial wilt that causes, it is characterized in that, the described PCR reaction conditions of step (3) is 94 ℃ of pre-sex change 4min, 94 ℃ of sex change 1min, 52 ℃ of annealing 45s, 72 ℃ of extension 50s, 10min is finally extended in 30 circulations.
4, a kind of rapid detection according to claim 1 identifies that the Solanaceae Ralstonia solanacearum infects the method for the bacterial wilt that causes, it is characterized in that, reaction product is to be electrophoresis on 0.8～1.5% the sepharose in the quality volume percent in the step (4), 50～75V constant voltage electrophoresis, 50～70min.
5, a kind of rapid detection according to claim 1 identifies that the Solanaceae Ralstonia solanacearum infects the method for the bacterial wilt that causes, it is characterized in that the sick sample tissue of eggplant, tomato, peanut, potato, capsicum, tobacco, mater convolvulus, husky ginger, chrysanthemum or mulberry tree that described sick sample to be checked causes for the Solanaceae Ralstonia solanacearum infects.
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| CN102559923A (en) * | 2012-03-20 | 2012-07-11 | 南京农业大学 | Method of quickly identifying category of pathogenic bacteria of fungal diseases in chrysanthemum |
| CN103614474A (en) * | 2013-11-27 | 2014-03-05 | 福建省农业科学院植物保护研究所 | Loop-mediated isothermal amplification detection primer for ralstonia solanacearu and detection method for ralstonia solanacearu |
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| CN102559923A (en) * | 2012-03-20 | 2012-07-11 | 南京农业大学 | Method of quickly identifying category of pathogenic bacteria of fungal diseases in chrysanthemum |
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| CN114511157B (en) * | 2022-04-18 | 2022-07-05 | 广东省农业科学院植物保护研究所 | Microecological regulation and control method and system based on vegetable bacterial wilt prevention and control |
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