CN104152532A - Method for identifying bacterial wilt resistance of tobacco seedlings - Google Patents
Method for identifying bacterial wilt resistance of tobacco seedlings Download PDFInfo
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- CN104152532A CN104152532A CN201410385968.0A CN201410385968A CN104152532A CN 104152532 A CN104152532 A CN 104152532A CN 201410385968 A CN201410385968 A CN 201410385968A CN 104152532 A CN104152532 A CN 104152532A
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- tobacco
- seedlings
- bacterial wilt
- seedling
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- 241000208125 Nicotiana Species 0.000 title claims abstract description 27
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 22
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 16
- 241000589771 Ralstonia solanacearum Species 0.000 claims abstract description 10
- 238000005286 illumination Methods 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 4
- 230000003203 everyday effect Effects 0.000 claims abstract description 4
- 238000011835 investigation Methods 0.000 claims abstract description 4
- 239000003337 fertilizer Substances 0.000 claims abstract description 3
- 239000000618 nitrogen fertilizer Substances 0.000 claims abstract description 3
- 201000010099 disease Diseases 0.000 claims description 26
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 26
- 235000013882 gravy Nutrition 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 3
- 241000238631 Hexapoda Species 0.000 claims description 3
- 241000607479 Yersinia pestis Species 0.000 claims description 3
- 239000010451 perlite Substances 0.000 claims description 3
- 235000019362 perlite Nutrition 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 230000000050 nutritive effect Effects 0.000 claims description 2
- 238000009331 sowing Methods 0.000 claims 1
- 238000012360 testing method Methods 0.000 abstract description 7
- 238000004113 cell culture Methods 0.000 abstract 4
- 235000015097 nutrients Nutrition 0.000 abstract 2
- 230000003816 axenic effect Effects 0.000 abstract 1
- 239000007788 liquid Substances 0.000 abstract 1
- 239000000203 mixture Substances 0.000 abstract 1
- 239000004575 stone Substances 0.000 abstract 1
- 238000012797 qualification Methods 0.000 description 8
- 241000196324 Embryophyta Species 0.000 description 3
- 235000019504 cigarettes Nutrition 0.000 description 3
- 238000005192 partition Methods 0.000 description 3
- 235000003255 Carthamus tinctorius Nutrition 0.000 description 2
- 244000020518 Carthamus tinctorius Species 0.000 description 2
- 208000035240 Disease Resistance Diseases 0.000 description 2
- 206010061818 Disease progression Diseases 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 230000005750 disease progression Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000035558 fertility Effects 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000003595 mist Substances 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Cultivation Of Plants (AREA)
- Pretreatment Of Seeds And Plants (AREA)
Abstract
The invention discloses a method for identifying the bacterial wilt resistance of tobacco seedlings. A water retaining agent and pearl stone are mixed uniformly at the ratio of 2:1; the mixture is disinfected and then loaded into a 6-hole cell culture board; tobacco seeds are sowed on the cell culture board, wherein each kind of tobacco seeds is sowed in one hole, and 10-20 tobacco seeds are sowed in each hole; the cell culture board is placed in an axenic culture room to culture the tobacco seeds under the conditions that the temperature environment is 25 DEG C, the illumination time is 16 hours every day, the illumination intensity is 4,000 lux and the relative humidity is 70-80 %; after emergence of seedlings, 5 seedlings are reserved in each hole uniformly; a nutrient solution is prepared from special tobacco seedling raising fertilizers, wherein the concentration of a nitrogen fertilizer reaches 100 ppm; the nutrient solution is top-dressed into the culture board; when the seedlings send forth two true leaves, 1 ml of ralstonia solanacearum suspending liquid is vaccinated into each hole of the cell culture board through a liquid-transferring gun; investigation and recordation are carried out 15 days later. The method provided by the invention is shorter in identification period, can identify 25-day-old seedlings, is not limited by seasons and has high testing stability.
Description
Technical field
The invention belongs to disease resistance of plant authenticate technology field, be specifically related to the method that Seedling Stage is identified tobacco bacterial wilt resistance.
Background technology
By bacterial a kind of tobacco diseases, at a plurality of cigarettes of China, there is generation in district, and harm is serious.Bacterial wilt is lacked to special efficacy control medicament, once there is to be difficult to control.In bacterial wilt prone district, topmost method is selected resistance to bacterial wilt tobacco bred exactly.Bacterial wilt Disease Resistance Identification to tobacco bred, at present mainly relies on field Disease garden identification, but field Disease garden identification has that qualification time is long, cost is high, is subject to the shortcomings such as the impact in soil fertility, season is larger, and Resistance Identification is unstable.Chinese patent CN 200910094174 " a kind of sprout period authenticating method of tobacco bacterial wilt resistance " adopts floating pool constant temperature water planting inoculation identification method, have morbidity rapidly, reproducible, the result reliability advantages of higher of result, but this method complicated operation, time cycle is relatively still longer, needs the time of one month.
Summary of the invention
The technical problem to be solved in the present invention: grow, be subject to the problems such as the impact in soil fertility, season is larger, qualification cycle unstable for the Resistance Identification existing in prior art, the invention provides a kind of Seedling Stage and identify the method for tobacco bacterial wilt resistance, have qualification cycle short, be not subject to limit and the advantage such as qualification result is stable in season.
The technical solution used in the present invention: Seedling Stage is identified the method for tobacco bacterial wilt resistance, comprises the following steps:
(1) grow seedlings: water-holding agent and perlite are mixed in the ratio of 2:1, after sterilization, pack 6 porocyte culture plates into, tobacco seed is sowed on Tissue Culture Plate, each kind is respectively seeded in a hole, 10～20/hole, be placed in Sterile culture room and cultivate, 25 ℃ of temperature environments, illumination every day 16 hours, intensity of illumination 4000 luxs, relative temperature 70～80%, after emerging, in each hole, stay uniformly seedling 5 strains, with tobacco seedling special fertilizer preparation nutritive medium, make wherein nitrogenous fertilizer concentration reach 100ppm, impose in culture plate;
(2) preparation of Ralstonia solanacearum suspension: will be stored in the Ralstonia solanacearum in sterilized water at 20 ℃, at the flat lining out of bouillon media, be incubated at 28 ~ 30 ℃, after 24h, picking colonies typical dislocation is in gravy inclined-plane, after 28 ℃ of 24h, move to again gravy inclined-plane, after 28 ℃ of cultivation 24h, with sterilized water, be diluted to 10
8cfu/ml, the needed bacteria concentration that connects;
(3) inoculation: when seedling grows to two true leaves, Ralstonia solanacearum suspension is inoculated in each hole of Tissue Culture Plate to every hole inoculation 1ml, 15 days " Invest, Then Investigate " records with liquid-transfering gun.
Record method is carried out national standard " tobacco disease insect pest classification and investigation method (GB/T 23222-2008) ".Disease index (D) calculation formula:
D=∑ (diseased plant numbers at different levels * this disease progression) * 100/(investigates total strain number * highest number)
Disease severity grade scale is carried out by standard GB/T/T 23224-2008, and Partition in resistance is 6 ranks: high resistance or immunity (I): disease refers to be 0; Disease-resistant (R): disease-resistant (R); In anti-(MR): disease refers to be 20.1～40; Middle sense (MS): disease refers to be 40.1～60; Sense (S): disease refers to be 60.1～80; High sense (HS): disease refers to be 80.1～100.
The present invention is relative with prior art has following beneficial effect:
(1) qualification cycle shorter, 25 days seedlings can be for the identification of, the qualification cycle of comparing other prior art is short;
(2) take Tissue Culture Plate as carrier, the evaluation of growing seedlings under indoor constant environment, is not subject to seasonal restrictions, and test stability is strong;
(3) use aseptic seedling, be not subject to other microbiological effect, qualification result is stable.
Embodiment
embodiment 1
1. materials and methods
1.1 test materials
12 tobacco breds are carried out to bacterial wilt Resistance Identification.D101, long neck Huang, large brave ear, Ti448A, the large gold dollar of safflower, K326, No. 3, Nan Jiang, cloud and mist 85, rock cigarette 97, K346, RG17, Zhongyan-100
1.2 test method
Water-holding agent and perlite are mixed in the ratio of 2:1, after sterilization, pack 6 porocyte culture plates into, tobacco seed is sowed on Tissue Culture Plate.12 kinds of test are seeded into respectively in a hole to 10～20/hole.4 repetitions are established in test.Tissue Culture Plate is after planting placed in Sterile culture room and is cultivated, 25 ℃ of temperature environments, illumination every day 16 hours, intensity of illumination 4000 luxs (lux), relative temperature 70～80%.After emerging, in each hole, stay uniformly seedling 5 strains.When seedling grows to two true leaves (25～30 days), Ralstonia solanacearum suspension is inoculated in each hole of Tissue Culture Plate to every hole inoculation 1ml with liquid-transfering gun.15 days " Invest, Then Investigate " records.
The making of Ralstonia solanacearum suspension:
By being stored in the Ralstonia solanacearum in sterilized water at 20 ℃, at the flat lining out of bouillon media, be incubated at 28 ~ 30 ℃, after 24h, picking colonies typical dislocation, in gravy inclined-plane, after 28 ℃ of 24h, then moves to gravy inclined-plane, after 28 ℃ of cultivation 24h, with sterilized water, is diluted to 10
8cfu/ml, the needed bacteria concentration that connects.
Record method is carried out national standard " tobacco disease insect pest classification and investigation method (GB/T 23222-2008) ".Disease index (D) calculation formula:
D=∑ (diseased plant numbers at different levels * this disease progression) * 100/(investigates total strain number * highest number)
Disease severity grade scale is carried out by standard GB/T/T 23224-2008, and Partition in resistance is 6 ranks:
Table 1: national standard Partition in resistance table
| high resistance or immunity (I) | : | disease refers to be 0; |
| disease-resistant (R) | : | disease refers to be 0.1～20; |
| in anti-(MR) | : | disease refers to be 20.1～40; |
| middle sense (MS) | : | disease refers to be 40.1～60; |
| sense (S) | : | disease refers to be 60.1～80; |
| high sense (HS) | : | disease refers to be 80.1～100. |
Table 2: conventional Disease garden identification and seedling Rapid identification result
| Numbering/variety name | Disease refers to | Resistance |
| D101 | 19.0 | R |
| Long neck is yellow | 93.8 | HS |
| Large brave ear | 43.8 | MS |
| Ti448A | 54.2 | MS |
| The large gold dollar of safflower | 72.2 | S |
| K326 | 27.0 | MR |
| No. 3, river, south | 58.4 | MS |
| Cloud and mist 85 | 37.6 | MR |
| Rock cigarette 97 | 8.5 | R |
| K346 | 17.5 | R |
| RG17 | 21.4 | MR |
| Zhongyan-100 | 55.9 | MS |
Test the qualification result of selected kind, basically identical with the resistance in production test.Illustrate that the inventive method can be as a kind of quick method of Identification of Tobacco Bacterial Wilt Resistance.
Claims (2)
1. Seedling Stage is identified a method for tobacco bacterial wilt resistance, it is characterized in that: comprise the following steps:
(1) grow seedlings: water-holding agent and perlite are mixed in the ratio of 2:1, after sterilization, pack 6 porocyte culture plates into, by tobacco seed sowing, on Tissue Culture Plate, each kind is respectively seeded in a hole, 10～20/hole, be placed in Sterile culture room and cultivate, after emerging, in each hole, stay uniformly seedling 5 strains, with tobacco seedling special fertilizer preparation nutritive medium, make wherein nitrogenous fertilizer concentration reach 100ppm, impose in culture plate;
(2) preparation of Ralstonia solanacearum suspension: will be stored in the Ralstonia solanacearum in sterilized water at 20 ℃, at the flat lining out of bouillon media, be incubated at 28 ~ 30 ℃, after 24h, picking colonies typical dislocation is in gravy inclined-plane, 28 ℃, after 24h, then move to gravy inclined-plane, after 28 ℃ of cultivation 24h, with sterilized water, be diluted to 10
8cfu/ml, the needed bacteria concentration that connects;
(3) inoculation: when seedling grows to two true leaves, Ralstonia solanacearum suspension is inoculated in each hole of Tissue Culture Plate to every hole inoculation 1ml, 15 days " Invest, Then Investigate " records with liquid-transfering gun;
(4) record method is carried out national standard: GB/T 23222-2008, tobacco disease insect pest classification and investigation method.
2. Seedling Stage according to claim 1 is identified the method for tobacco bacterial wilt resistance, it is characterized in that: the culture condition of Sterile culture room is 25 ℃ of temperature environments, illumination every day 16 hours, intensity of illumination 4000lux, relative temperature 70～80%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410385968.0A CN104152532B (en) | 2014-08-07 | 2014-08-07 | A kind of method of Seedling Stage qualification tobacco bacterial wilt resistance |
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| CN201410385968.0A CN104152532B (en) | 2014-08-07 | 2014-08-07 | A kind of method of Seedling Stage qualification tobacco bacterial wilt resistance |
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| CN104152532A true CN104152532A (en) | 2014-11-19 |
| CN104152532B CN104152532B (en) | 2016-01-20 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN109566373A (en) * | 2018-12-26 | 2019-04-05 | 恩施土家族苗族自治州农业科学院 | A method of inoculation study bacterial wilt is impregnated by root |
| CN110317722A (en) * | 2019-08-01 | 2019-10-11 | 中国农业科学院烟草研究所 | A kind of tobacco black shank resistance identification apparatus and identification method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1287175A (en) * | 2000-09-08 | 2001-03-14 | 广东省农业科学院蔬菜研究所 | Water culture bacterination process to determine bacterial wilt resistance of plant |
| WO2002006447A2 (en) * | 2000-07-18 | 2002-01-24 | The General Hospital Corporation | Salicylic acid biosynthetic genes and uses thereof |
| CN101496481A (en) * | 2009-03-09 | 2009-08-05 | 云南省烟草科学研究所 | Method for identifying resistance to tobacco bacterial wilt during seedling stage |
| CN102771326A (en) * | 2012-08-08 | 2012-11-14 | 东莞市香蕉蔬菜研究所 | Method for rapidly identifying and screening anti-blight banana seedling |
-
2014
- 2014-08-07 CN CN201410385968.0A patent/CN104152532B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002006447A2 (en) * | 2000-07-18 | 2002-01-24 | The General Hospital Corporation | Salicylic acid biosynthetic genes and uses thereof |
| CN1287175A (en) * | 2000-09-08 | 2001-03-14 | 广东省农业科学院蔬菜研究所 | Water culture bacterination process to determine bacterial wilt resistance of plant |
| CN101496481A (en) * | 2009-03-09 | 2009-08-05 | 云南省烟草科学研究所 | Method for identifying resistance to tobacco bacterial wilt during seedling stage |
| CN102771326A (en) * | 2012-08-08 | 2012-11-14 | 东莞市香蕉蔬菜研究所 | Method for rapidly identifying and screening anti-blight banana seedling |
Non-Patent Citations (2)
| Title |
|---|
| 杨友才等: "烟草品种青枯病抗病性及抗性遗传研究", 《湖南农业大学学报》 * |
| 肖永生等: "对烟草青枯病苗期抗性鉴定接种方法的探讨", 《湖南农业科学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN109566373A (en) * | 2018-12-26 | 2019-04-05 | 恩施土家族苗族自治州农业科学院 | A method of inoculation study bacterial wilt is impregnated by root |
| CN110317722A (en) * | 2019-08-01 | 2019-10-11 | 中国农业科学院烟草研究所 | A kind of tobacco black shank resistance identification apparatus and identification method |
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| Publication number | Publication date |
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| CN104152532B (en) | 2016-01-20 |
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