CN104082055A - Method for high-flux identification of ralstonia solanacearum resistance - Google Patents
Method for high-flux identification of ralstonia solanacearum resistance Download PDFInfo
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- CN104082055A CN104082055A CN201410269005.4A CN201410269005A CN104082055A CN 104082055 A CN104082055 A CN 104082055A CN 201410269005 A CN201410269005 A CN 201410269005A CN 104082055 A CN104082055 A CN 104082055A
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Abstract
The invention discloses a method for high-flux identification of ralstonia solanacearum resistance. The method comprises the following steps: sterilizing quartz sand with particle size ranging from 2 mm to 4 mm at high temperature of 121 DEG C for 30 minutes, cooling, and spreading into an aseptic 12-pore cell culture plate, wherein each pore is filled with 10 g of the quartz sand and 4 ml of aseptic water; sowing flue-cured tobacco seeds to be tested onto the quartz sand of the 12-pore cell culture plate, after sowing, and culturing by the 12-pore cell culture plate under the conditions of 25 DEG C, RH of more than 80% and alternative 16-hour lighting and 8-hour shading; after the flue-cured tobacco seeds germinate, adding a Hoagland's nutrient solution into the 12-pore cell culture plate for culturing the flue-cured tobacco seeds under the aseptic condition, continuously culturing under the conditions of 25 DEG C, RH of more than 80% and alternative 16-hour lighting and 8-hour shading, and identifying the disease resistance of the flue-cured tobacco seeds each with 5-6 leaves, wherein each pore is filled with 2 ml of the Hoagland's nutrient solution; preparing inoculating liquid and inoculating; investigating and grading the disease states. According to the method, the ralstonia solanacearum resistance can be identified with high flux and high efficiency and a large amount of flue-cured tobacco varieties can be identified in one time.
Description
Technical field
The invention belongs to crop resistance authenticate technology field, be specifically related to a kind of authentication method of tobacco bacterial wilt resistance.
Background technology
By Ralstonia solanacearum (
ralstonia solanacearum(Smith) Yabuuchi et al) tobacco bacterial wilt that causes is that a kind of worldwide soil passes bacterial disease, very disruptive, and become the Major Diseases that restriction tobacco produces.Tobacco bacterial wilt all generally occurs in the main cigarette provinces and regions of producing.This disease is typical vascular bundle diseases, and Tobacco Root, stem, the each position of leaf all can be infected, but the root of main harm plant and stem, one of its cardinal symptom is the monolateral wilting of blade and flavescence too early.At present, breeding and application disease-resistant variety is to prevent and treat the most effective approach of bacterial wilt.But in breeding for disease resistance correlative study, need to evaluate quickly and accurately the resistance performance of lot of materials.Conventional authentication method has: 1) floating/potted plant cigarette the Miaos work inoculated identification method.The method cultivation temperature is unstable, is easily subject to the impact of variation of ambient temperature; Pathogen concentration is subject to the impact of factor such as water of the volume of water in nursery pond and cigarette basin; The incidence of disease and the disease index that closely affect cigarette seedling, affect the qualification of cigarette seedling resistance level, and qualification result is unstable, and repeatability is not high.The method requisite space is large, need to build green house, nursery pond, need to buy heater, and management cost is high, needs human cost high.The method flux is little, and that once can identify is of less types.In addition, the method is easily subject to the impact of other disease in assay period, as: black shank, damping off, gray mold, virus disease, powdery mildew etc., these diseases can affect the Resistance Identification result of cigarette seedling.2) sick garden, land for growing field crops Inoculation Method.The method can be reacted the resistance of kind preferably; But the method is grown, can only identify once every year qualification cycle, required sick garden area is large, for the identification of tobacco-containing material limited, Ralstonia solanacearum bacteria suspension demand large, it is inhomogeneous to fall ill, and exist and show between the time and intersite such as there are differences at the problem for the resistance of examination material.Potted plant, floating cigarette seedling and field resistance qualification environmental temperature are difficult to control.In the qualification of potted plant and field resistance, pathogen quantity is difficult to control.In order to screen better the anti-source of bacterial wilt, cultivate disease-resistant variety, to excavate disease-resistant gene, and study Hosts and make mutually mechanism etc., need a set of easy, quick and efficient tobacco bacterial wilt Resistance Identification method of setting up badly.
Summary of the invention
The object of the invention is to overcome above-mentioned shortcoming and a kind of energy high flux, high efficiency qualification tobacco bacterial wilt resistance are provided, and the method for the flue-cured tobacco cultivars that once can identify high throughput identification tobacco bacterial wilt resistance how.
Object of the present invention and solve its technical problem underlying and realize by the following technical solutions:
The method of a kind of high throughput identification tobacco bacterial wilt resistance the invention discloses, comprises the following steps:
(1) cultivate aseptic cigarette seedling
The quartz sand that is 2-4mm by granularity sterilizing 30 minutes under 121 DEG C of high temperature, is sown in aseptic 12 porocyte culture plates after cooling, every hole 10g quartz sand, and every hole adds 4ml sterile water; Tobacco seed to be measured is seeded on the quartz sand of 12 porocyte culture plates, after planting 12 porocyte culture plates is placed under the dark alternation condition of 25 DEG C, RH>80%, 16h illumination and 8h and cultivates;
(2) daily management of cigarette seedling
After cigarette kind is germinateed, under aseptic condition, in 12 hole microwell plates of cultivation cigarette seedling, add Huo Gelan (Hoglangd) nutrient solution, every hole 2ml, continue to be placed under the dark alternation condition of 25 DEG C, RH>80%, 16h illumination and 8h and cultivate, in the time of the cigarette seedling 5-6 leaf phase for Disease Resistance Identification;
(3) inoculation liquid preparation and inoculation
By tobacco Ralstonia solanacearum (
ralstonia solanacearum) bacterial strain use before at the upper activation of Raul Salmonella selective medium (SMSA) 48h, choose typical single bacterium colony to Raul Salmonella selective medium (SMSA) flat board, 30 DEG C of cultivation 48h.Lawn is scraped, be inoculated into beef extract-peptone liquid nutrient medium (NB) culture fluid, 30 DEG C of concussions cultivate 36 to 48h; Culture fluid is collected thalline in the centrifugal 20min of 4000 × g, is made into bacteria suspension with sterile water, and the concentration of adjusting bacterium is 1 × 10
8cfu/mL, is added into bacteria suspension in the micropore of 12 porocyte culture plates according to 0.5 ml/ hole;
(4) state of an illness investigation and classification
After connecing bacterium, 20d investigates incidence, later every an incidence of 3d investigation; Record the incidence of disease and disease index, the disease index that reaches 80% left and right using the susceptible contrast incidence of disease is as varietal resistance evaluation criterion.
The method of above-mentioned a kind of high throughput identification tobacco bacterial wilt resistance, wherein: beef extract-peptone solid culture medium (NA): beef extract 3g, peptone 10g, agar 16g, water 1000ml, pH 6.8～7.2.
The method of above-mentioned a kind of high throughput identification tobacco bacterial wilt resistance, wherein: beef extract-peptone liquid nutrient medium (NB): beef extract 3g, peptone 10g, water 1000ml, pH 6.8～7.2.
The method of above-mentioned a kind of high throughput identification tobacco bacterial wilt resistance, wherein: SMSA medium: peptone 10g, glycerine 5mL, casamino acids 1g, adjusts pH to 7.0, adds water and is assigned to 1L, adds agar powder 16g; When use, in the time that the medium having dissolved is cooled to 50 DEG C of left and right, every 250ml medium adds 1% polymyxin B 2.5ml, 1% crystal violet 125 μ L, 1%TTC 1.25ml, 1% bacitracin 625 μ L, 0.1% penicillin 125 μ L, 1% chloramphenicol 125 μ L.
The present invention compared with prior art has obvious advantage and beneficial effect.From above technical scheme: 1) the present invention cultivates cigarette seedling in 12 hole microwell plates, and required instrument is constant temperature illumination box, take up an area little, need space little; Thereby economical, convenient, can realize high throughput identification, the flue-cured tobacco cultivars that once can identify is many.2) tobacco growing and bacterial wilt onset temperature can thermostatic controls, and the incidence of disease and disease index result are stable, resistance level that can actual response flue-cured tobacco cultivars.3) can accurately control every kind and connect bacterium amount, make onset condition in full accord.4) in whole qualification process, in hole, Ralstonia solanacearum concentration and cultivation temperature are stable, are not subject to the variation of culture fluid volume and change, near and the incidence of disease and disease index are stable, have ensured to repeat the uniformity of intercurrent disease.5) pathogen is invaded from root, approaches disease field natural infection process, has ensured the reliability of Resistance Identification result.6) microwell plate quartz sand is cultivated cigarette seedling, possesses aseptic culture environment, avoids the impact of other microorganism on tobacco seedling growth.7) be conducive between year and different experiments chamber between result reappear and relatively.The method application potential in Resistance Identification, genetics of resistance law-analysing, disease-resistant gene excavation and the molecular pathology research of disease-resistant resource and breeding material is larger.
Embodiment
embodiment 1-8
A method for high throughput identification tobacco bacterial wilt resistance, comprises the following steps:
(1) cultivate aseptic cigarette seedling
The quartz sand that is 2-4mm by granularity sterilizing 30 minutes under 121 DEG C of high temperature, is sown in aseptic 12 porocyte culture plates after cooling, every hole 10g quartz sand, and every hole adds 4ml sterile water.Tobacco seed to be measured (8 kinds provide by breeding engineering center, Guizhou Province Tabacco Science and Technology Institute) is seeded on the quartz sand of 12 porocyte culture plates, after planting 12 porocyte culture plates is placed under the dark alternation condition of 25 DEG C, RH>80%, 16h illumination and 8h and cultivates.
(2) daily management of cigarette seedling
After cigarette kind is germinateed, under aseptic condition, in 12 hole microwell plates of cultivation cigarette seedling, add Huo Gelan (Hoglangd) nutrient solution, every hole 2ml, continue to be placed under the dark alternation condition of 25 DEG C, RH>80%, 16h illumination and 8h and cultivate, in the time of the cigarette seedling 5-6 leaf phase for Disease Resistance Identification.
(3) inoculation liquid preparation and inoculation
3 strains provide by Guizhou Province Tabacco Science and Technology Institute Microbiological Lab from the tobacco ralstonia solanacearum dominant strain of different regions, Guizhou Province, and all bacterial strains are No. 1 microspecies, biochemical type III, are Tobacco in Guizhou producing region tobacco ralstonia solanacearum dominant races.Before the tobacco Isolates of Pseudomonas Solanacearum Smith of preserving is used, at the upper activation of Raul Salmonella selective medium (SMSA) 48h, choose typical single bacterium colony to Raul Salmonella selective medium (SMSA) flat board, cultivate 48h for 30 DEG C.Lawn is scraped, be inoculated into beef extract-peptone liquid nutrient medium (NB) culture fluid, 30 DEG C of concussions cultivate 36 to 48h.Culture fluid is collected thalline in the centrifugal 20min of 4000 × g, is made into bacteria suspension with sterile water, and the concentration of adjusting bacterium is 1 × 10
8cfu/mL, is added into bacteria suspension in the micropore of 12 porocyte culture plates according to 0.5 ml/ hole.
Wherein: SMSA medium: peptone 10 g, glycerine 5 mL, casamino acids 1 g, adjusts pH to 7.0, adds water and is assigned to 1L, adds agar powder 16 g; When use, in the time that the medium having dissolved is cooled to 50 DEG C of left and right, every 250 ml medium add 1% polymyxin B 2.5 ml, 1% crystal violet 125 μ L, 1%TTC 1.25 ml, 1% bacitracin 625 μ L, 0.1% penicillin 125 μ L, 1% chloramphenicol 125 μ L.
Beef extract-peptone liquid nutrient medium (NB): beef extract 3g, peptone 10g, water 1000ml, pH 6.8～7.2.
(4) state of an illness investigation and classification
After connecing bacterium, 20d investigates incidence, later every an incidence of 3d investigation.Record the incidence of disease and disease index.The disease index that reaches 80% left and right using the susceptible contrast incidence of disease is as varietal resistance evaluation criterion.
Qualification result shows: in 8 kinds to be measured, the kind of tobacco bacterial wilt performance high resistance is had to 3, be respectively K346, No. 6, your cigarette and your cigarette No. 8; Anti-in performance have 3, is No. 14, rock cigarette 97, No. 1, your cigarette and your cigarette; Showing susceptible have 2, is the large gold dollar of safflower and long neck Huang.
The different authentication method acquired results contrasts of table 1
| Embodiment | Kind | Result |
| 1 | The large gold dollar of safflower | Sense |
| 2 | Long neck Huang | Sense |
| 3 | K346 | Anti- |
| 4 | Rock cigarette 97 | In anti- |
| 5 | No. 1, your cigarette | In anti- |
| 6 | No. 6, your cigarette | Anti- |
| 7 | No. 8, your cigarette | Anti- |
| 8 | No. 14, your cigarette | In anti- |
The above, it is only preferred embodiment of the present invention, not the present invention is done to any pro forma restriction, any technical solution of the present invention content that do not depart from, any simple modification, equivalent variations and the modification above embodiment done according to technical spirit of the present invention, all still belong in the scope of technical solution of the present invention.
Claims (3)
1. a method for high throughput identification tobacco bacterial wilt resistance, comprises the following steps:
(1) cultivate aseptic cigarette seedling
The quartz sand that is 2-4mm by granularity sterilizing 30 minutes under 121 DEG C of high temperature, is sown in aseptic 12 porocyte culture plates after cooling, every hole 10g quartz sand, and every hole adds 4ml sterile water; Tobacco seed to be measured is seeded on the quartz sand of 12 porocyte culture plates, after planting 12 porocyte culture plates is placed under the dark alternation condition of 25 DEG C, RH>80%, 16h illumination and 8h and cultivates;
(2) daily management of cigarette seedling
After cigarette kind is germinateed, under aseptic condition, in 12 hole microwell plates of cultivation cigarette seedling, add Huo Gelan nutrient solution, every hole 2ml, continues to be placed under the dark alternation condition of 25 DEG C, RH>80%, 16h illumination and 8h and cultivates, in the time of the cigarette seedling 5-6 leaf phase for Disease Resistance Identification;
(3) inoculation liquid preparation and inoculation
Before being used, tobacco Isolates of Pseudomonas Solanacearum Smith activates 48h on Raul Salmonella selective medium, choose typical single bacterium colony to Raul Salmonella selective medium flat board, cultivate 48h, lawn is scraped for 30 DEG C, be inoculated into beef extract-peptone liquid nutrient medium culture fluid, 30 DEG C of concussions cultivate 36 to 48h; Culture fluid is collected thalline in the centrifugal 20min of 4000 × g, is made into bacteria suspension with sterile water, and the concentration of adjusting bacterium is 1 × 10
8cfu/mL, is added into bacteria suspension in the micropore of 12 porocyte culture plates according to 0.5 ml/ hole;
(4) state of an illness investigation and classification
After connecing bacterium, 20d investigates incidence, later every an incidence of 3d investigation; Record the incidence of disease and disease index, the disease index that reaches 80% left and right using the susceptible contrast incidence of disease is as varietal resistance evaluation criterion.
2. the method for a kind of high throughput identification tobacco bacterial wilt resistance as claimed in claim 1, wherein: beef extract-peptone solid culture medium: beef extract 3g, peptone 10g, agar 16g, water 1000ml, pH 6.8～7.2.
3. the method for a kind of high throughput identification tobacco bacterial wilt resistance as claimed in claim 1 or 2, wherein: beef extract-peptone liquid nutrient medium: beef extract 3g, peptone 10g, water 1000ml, pH 6.8～7.2.
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Cited By (11)
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| CN105557343A (en) * | 2016-01-05 | 2016-05-11 | 中国烟草总公司福建省公司 | Method for quickly identifying tobacco variety resistance to bacterial wilt |
| CN105594340A (en) * | 2016-01-11 | 2016-05-25 | 河南省农业科学院烟草研究所 | Tobacco seed germination bed and production method thereof |
| CN106376329A (en) * | 2016-08-30 | 2017-02-08 | 中国烟草总公司广东省公司 | Field disease nursery identifying method for disease resisting strength of tobacco resisting bacterial wilt |
| CN106386205A (en) * | 2016-08-30 | 2017-02-15 | 中国烟草总公司广东省公司 | Indoor seedling-stage inoculation identification method for tobacco resistance to bacterial wilt |
| CN106576874A (en) * | 2017-01-06 | 2017-04-26 | 江西省农业科学院植物保护研究所 | Field high-flux identification method for bacterial wilt resistance of sesamum indicum varieties |
| CN106818326A (en) * | 2016-12-26 | 2017-06-13 | 新昌县钧国生物技术有限公司 | A kind of method for improving tobacco bacterial wilt resistance |
| CN107211861A (en) * | 2017-07-04 | 2017-09-29 | 四川省农业科学院经济作物育种栽培研究所 | The method of seedling stage Rapid identification tobacco bred Resistance to bacterial wilt |
| CN108085363A (en) * | 2017-12-21 | 2018-05-29 | 河南省农业科学院烟草研究所 | One identification method to grow tobacco to fusarium tabacinum Resistance To Root Rot Disease |
| CN108739130A (en) * | 2018-05-17 | 2018-11-06 | 广东省农业科学院植物保护研究所 | A kind of method of greenhouse seedling stage assay pumpkin Resistance to bacterial wilt kind |
| CN109321462A (en) * | 2018-10-17 | 2019-02-12 | 中国烟草总公司郑州烟草研究院 | A kind of method that separation ralstonia solanacearum is combined using resistant panel screening and test strips identification in field |
| CN113930373A (en) * | 2021-09-10 | 2022-01-14 | 南京工业大学 | Method for preserving pathogenic bacteria of tobacco bacterial wilt, namely ralstonia solanacearum |
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| CN105557343A (en) * | 2016-01-05 | 2016-05-11 | 中国烟草总公司福建省公司 | Method for quickly identifying tobacco variety resistance to bacterial wilt |
| CN105594340A (en) * | 2016-01-11 | 2016-05-25 | 河南省农业科学院烟草研究所 | Tobacco seed germination bed and production method thereof |
| CN106376329A (en) * | 2016-08-30 | 2017-02-08 | 中国烟草总公司广东省公司 | Field disease nursery identifying method for disease resisting strength of tobacco resisting bacterial wilt |
| CN106386205A (en) * | 2016-08-30 | 2017-02-15 | 中国烟草总公司广东省公司 | Indoor seedling-stage inoculation identification method for tobacco resistance to bacterial wilt |
| CN106818326A (en) * | 2016-12-26 | 2017-06-13 | 新昌县钧国生物技术有限公司 | A kind of method for improving tobacco bacterial wilt resistance |
| CN106576874A (en) * | 2017-01-06 | 2017-04-26 | 江西省农业科学院植物保护研究所 | Field high-flux identification method for bacterial wilt resistance of sesamum indicum varieties |
| CN106576874B (en) * | 2017-01-06 | 2018-04-17 | 江西省农业科学院植物保护研究所 | A kind of sesame variety resistance to bacterial wilt field high throughput identification method |
| CN107211861A (en) * | 2017-07-04 | 2017-09-29 | 四川省农业科学院经济作物育种栽培研究所 | The method of seedling stage Rapid identification tobacco bred Resistance to bacterial wilt |
| CN108085363A (en) * | 2017-12-21 | 2018-05-29 | 河南省农业科学院烟草研究所 | One identification method to grow tobacco to fusarium tabacinum Resistance To Root Rot Disease |
| CN108739130A (en) * | 2018-05-17 | 2018-11-06 | 广东省农业科学院植物保护研究所 | A kind of method of greenhouse seedling stage assay pumpkin Resistance to bacterial wilt kind |
| CN109321462A (en) * | 2018-10-17 | 2019-02-12 | 中国烟草总公司郑州烟草研究院 | A kind of method that separation ralstonia solanacearum is combined using resistant panel screening and test strips identification in field |
| CN113930373A (en) * | 2021-09-10 | 2022-01-14 | 南京工业大学 | Method for preserving pathogenic bacteria of tobacco bacterial wilt, namely ralstonia solanacearum |
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