CN104152532B - A kind of method of Seedling Stage qualification tobacco bacterial wilt resistance - Google Patents
A kind of method of Seedling Stage qualification tobacco bacterial wilt resistance Download PDFInfo
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- CN104152532B CN104152532B CN201410385968.0A CN201410385968A CN104152532B CN 104152532 B CN104152532 B CN 104152532B CN 201410385968 A CN201410385968 A CN 201410385968A CN 104152532 B CN104152532 B CN 104152532B
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- bacterial wilt
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- 241000208125 Nicotiana Species 0.000 title claims abstract description 24
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 24
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000012797 qualification Methods 0.000 title claims abstract description 19
- 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
- 239000000725 suspension Substances 0.000 claims abstract description 7
- 238000011081 inoculation Methods 0.000 claims abstract description 5
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 4
- 230000003203 everyday effect Effects 0.000 claims abstract description 4
- 239000010451 perlite Substances 0.000 claims abstract description 4
- 235000019362 perlite Nutrition 0.000 claims abstract description 4
- 238000009331 sowing Methods 0.000 claims abstract description 4
- 230000001954 sterilising effect Effects 0.000 claims abstract description 4
- 238000004659 sterilization and disinfection 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
- 230000000050 nutritive effect Effects 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
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 235000013882 gravy Nutrition 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
- 238000011835 investigation Methods 0.000 claims description 3
- 230000001932 seasonal effect Effects 0.000 abstract description 2
- 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
- 238000004364 calculation method Methods 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
- 239000002689 soil Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Cultivation Of Plants (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Pretreatment Of Seeds And Plants (AREA)
Abstract
The invention discloses the method for a kind of Seedling Stage qualification tobacco bacterial wilt resistance, water-holding agent and perlite are mixed in the ratio of 2:1, 6 porocyte culture plates are loaded after sterilization, 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, temperature environment 25 DEG C, illumination every day 16 hours, intensity of illumination 4000lux, relative humidity 70 ~ 80%, after emerging, seedling 5 strain is stayed uniformly in each hole, with tobacco seedling special fertilizer preparation nutritive medium, wherein nitrogenous fertilizer concentration is made to reach 100ppm, impose in culture plate, when seedling grows to two panels true leaf, Ralstonia solanacearum suspension liquid-transfering gun is inoculated in each hole of Tissue Culture Plate, every hole inoculation 1ml, 15 days " Invest, Then Investigate " records.Qualification cycle of the present invention is shorter, and 25 days seedlings can be for the identification of, and be not subject to seasonal restrictions, test stability is strong.
Description
Technical field
The invention belongs to disease resistance of plant authenticate technology field, be specifically related to the method for Seedling Stage qualification tobacco bacterial wilt resistance.
Background technology
By bacterial a kind of tobacco diseases, have generation in multiple cigarette district of China, harm is serious.Special efficacy pesticide control is lacked to bacterial wilt, once occur to be difficult to control.In bacterial wilt prone district, topmost method selects resistance to bacterial wilt tobacco bred exactly.To the bacterial wilt Disease Resistance Identification of tobacco bred, at present mainly rely on field Disease garden identification, but field Disease garden identification has that qualification time is long, cost is high, the impact by soil fertility, season is comparatively large, the shortcomings such as Resistance Identification is unstable.Chinese patent CN200910094174 " 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 relative still longer, needs the time of one month.
Summary of the invention
The technical problem to be solved in the present invention: unstable for the Resistance Identification that exists in prior art, qualification cycle long, by problems such as the impact in soil fertility, season are larger, the invention provides the method for a kind of Seedling Stage qualification tobacco bacterial wilt resistance, have qualification cycle short, by limit and the advantage such as qualification result is stable in season.
The technical solution used in the present invention: the method for Seedling Stage qualification tobacco bacterial wilt resistance, comprises the following steps:
(1) nursery: water-holding agent and perlite are mixed in the ratio of 2:1,6 porocyte culture plates are loaded after sterilization, 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, temperature environment 25 DEG C, illumination every day 16 hours, intensity of illumination 4000 lux, relative humidity 70 ~ 80%, after emerging, seedling 5 strain is stayed uniformly in each hole, 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: by the Ralstonia solanacearum be stored at 20 DEG C in sterilized water, at the flat lining out of bouillon media, at being incubated at 28 ~ 30 DEG C, after 24h, picking colonies typical dislocation is in gravy inclined-plane, after 28 DEG C of 24h, move to gravy inclined-plane again, after cultivating 24h at 28 DEG C, be diluted to 10 with sterilized water
8cfu/ml, required connect bacteria concentration;
(3) inoculate: when seedling grows to two panels true leaf, Ralstonia solanacearum suspension liquid-transfering gun is inoculated in each hole of Tissue Culture Plate, every hole inoculation 1ml, 15 days " Invest, Then Investigate " records.
Record method performs national standard " tobacco disease insect pest classification and investigation method (GB/T23222-2008) ".Disease index (D) calculation formula:
D=∑ (diseased plant number at different levels × this sick progression) × 100/(investigates total strain number × highest number)
Disease severity grade scale performs by standard GB/T/T23224-2008, 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 is shorter, and 25 days seedlings can be for the identification of, and the qualification cycle comparing other prior art is short;
(2) take Tissue Culture Plate as carrier, under indoor constant environment, nursery qualification, is not subject to seasonal restrictions, and test stability is strong;
(3) use aseptic seedling, not by other microbiological effect, qualification result is stablized.
Embodiment
embodiment 1
1. materials and methods
1.1 test materials
Resistance to bacterial wilt qualification is carried out to 12 tobacco breds.D101, long neck Huang, large brave ear, Ti448A, the large gold dollar of safflower, No. 3, K326, Nan Jiang, Yun yan85, 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, load 6 porocyte culture plates, by tobacco seed sowing on Tissue Culture Plate.12 kinds of test are seeded in a hole respectively, 10 ~ 20/hole.4 repetitions are established in test.Tissue Culture Plate is after planting placed in Sterile culture room and cultivates, temperature environment 25 DEG C, illumination every day 16 hours, intensity of illumination 4000 lux (lux), relative humidity 70 ~ 80%.After emerging, in each hole, stay seedling 5 strain uniformly.When seedling grows to two panels true leaf (25 ~ 30 days), Ralstonia solanacearum suspension liquid-transfering gun is inoculated in each hole of Tissue Culture Plate, every hole inoculation 1ml.15 days " Invest, Then Investigate " records.
The making of Ralstonia solanacearum suspension:
By the Ralstonia solanacearum be stored at 20 DEG C in sterilized water, at the flat lining out of bouillon media, at being incubated at 28 ~ 30 DEG C, after 24h, picking colonies typical dislocation is in gravy inclined-plane, after 28 DEG C of 24h, then moves to gravy inclined-plane, with sterilized water is diluted to 10 after cultivating 24h at 28 DEG C
8cfu/ml, required connect bacteria concentration.
Record method performs national standard " tobacco disease insect pest classification and investigation method (GB/T23222-2008) ".Disease index (D) calculation formula:
D=∑ (diseased plant number at different levels × this sick progression) × 100/(investigates total strain number × highest number)
Disease severity grade scale performs by standard GB/T/T23224-2008, 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 |
| Yun yan85 | 37.6 | MR |
| Rock cigarette 97 | 8.5 | R |
| K346 | 17.5 | R |
| RG17 | 21.4 | MR |
| Zhongyan-100 | 55.9 | MS |
The qualification result of the selected kind of test, basically identical with the resistance in production test.Illustrate that the inventive method can as a kind of quick method of Identification of Tobacco Bacterial Wilt Resistance.
Claims (2)
1. a method for Seedling Stage qualification tobacco bacterial wilt resistance, is characterized in that: comprise the following steps:
(1) nursery: water-holding agent and perlite are mixed in the ratio of 2:1,6 porocyte culture plates are loaded after sterilization, 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 seedling 5 strain uniformly, 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: by the Ralstonia solanacearum be stored at 20 DEG C in sterilized water, at the flat lining out of bouillon media, at being incubated at 28 ~ 30 DEG C, after 24h, picking colonies typical dislocation is in gravy inclined-plane, 28 DEG C, after 24h, then move to gravy inclined-plane, after cultivating 24h at 28 DEG C, be diluted to 10 with sterilized water
8cfu/ml, required connect bacteria concentration;
(3) inoculate: when seedling grows to two panels true leaf, Ralstonia solanacearum suspension liquid-transfering gun is inoculated in each hole of Tissue Culture Plate, every hole inoculation 1ml, 15 days " Invest, Then Investigate " records;
(4) record method performs national standard: GB/T23222-2008, tobacco disease insect pest classification and investigation method.
2. the method for Seedling Stage qualification tobacco bacterial wilt resistance according to claim 1, is characterized in that: the culture condition of Sterile culture room is temperature environment 25 DEG C, illumination every day 16 hours, intensity of illumination 4000lux, relative humidity 70 ~ 80%.
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Families Citing this family (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 |
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2014
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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 |
|---|
| 对烟草青枯病苗期抗性鉴定接种方法的探讨;肖永生等;《湖南农业科学》;20000331(第3期);第39-40页 * |
| 烟草品种青枯病抗病性及抗性遗传研究;杨友才等;《湖南农业大学学报》;20050831;第31卷(第4期);第381-383页 * |
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