CN105103984A - Resistance identification method of cucumber corynespora leaf spot disease - Google Patents
Resistance identification method of cucumber corynespora leaf spot disease Download PDFInfo
- Publication number
- CN105103984A CN105103984A CN201510588060.4A CN201510588060A CN105103984A CN 105103984 A CN105103984 A CN 105103984A CN 201510588060 A CN201510588060 A CN 201510588060A CN 105103984 A CN105103984 A CN 105103984A
- Authority
- CN
- China
- Prior art keywords
- cucumber
- leaf
- filtrate
- leaf spot
- spore
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Cultivation Of Plants (AREA)
Abstract
The invention relates to a resistance identification method of the cucumber corynespora leaf spot disease. The method includes the steps that a fresh, healthy and complete cucumber true leaf serves as an infection identification material; an improved Czapek liquid culture medium is used for culturing corynespora cassiicola, and primary separation and extraction are performed on culture filtrate to obtain crude toxin filtrate of the corynespora cassiicola; the back of the cucumber detached leaf is inoculated with the crude toxin filtrate in a non-destructive hanging drop mode; then, moisturizing culture is performed in an illumination culture box; the leaf area infected by the toxin filtrate is inspected through a crossing method after the third day, and then the resistance of the cucumber variety to the corynespora leaf spot disease caused by the corynespora cassiicola is obtained. Through the method, the resistance of different varieties of cucumbers to the corynespora cassiicola can be rapidly and accurately identified, and the identification period is greatly shortened. The method is easy to implement, high in repeatability and applicable to resistance identification of the cucumber corynespora leaf spot disease.
Description
Technical field
The present invention is specifically related to the authentication method that content is the in vitro resistant material of cucumber rod spore leaf spot, belongs to technical field of plant protection.
Background technology
By how main excellent spore (
corynesporacassiicola) cucumber rod spore leaf spot (Corynesporaleafspot) that causes endangers comparatively serious a kind of disease in cucumber production in recent years, this disease mainly infects the blade of cucumber, stem and fruit.European reported first cucumber rod spore leaf spot in 1906, nineteen fifty-seven U.S. Bei Kalailuonazhou reports that this disease occurs, China's relative wore and female etc. reported the parasitism of this bacterium on China cucumber and cause harm nineteen sixty, started to break out at the many cucumber cultivations of China from 2005 subsequently, huge loss is brought to the production of peasant, become the Major Diseases in cucumber production, the economic loss caused peasant is according to incompletely statistics more than 200,000,000.
Control for cucumber rod spore leaf spot comprises the many-sides such as disease-resistant variety seed selection, chemical control and bionomic control, and disease-resistant variety seed selection is measure that is most economical, effective, environmental protection.Disease Resistance Identification, through the overall process of breeding for disease resistance, is an important ring in the work such as anti-source digging utilization, breeding for disease resistance, resistance monitoring.Therefore, method of resistance identification is the key determining breeding for disease resistance success or failure accurately and effectively.
To in the qualification of cucumber rod spore leaf spot resistant material, mainly adopt the method for artificial inoculation on seedling.Seedling age, inoculation method, the difference of culture environment and the uncontrollability of investigation method are inoculated in artificial inoculation on seedling qualification in application process, and qualification result often has difference.How main excellent spore bacterium can produce the toxin protein of host selection, and this toxin soiutions inoculation cucumber leaves direct infection can produce scab, therefore can it replace pathogen differentiate as selection pressure and screen disease-resistant variety.
At present, how main excellent spore toxin serves good application in the Disease Resistance Identification of rubber bar spore leaf spot, but applies less in the Disease Resistance Identification of cucumber rod spore leaf spot.At present, only Lu Haining, Wu Limin etc. affect the physical signs of cucumber resistant susceptible varieties at toxin, cell wall damage and the change of related resistance enzymic activity index had relevant report, and do not form the method being applied to the Disease Resistance Identification of cucumber rod spore leaf spot with it of system
The present invention obtains its specific Raw toxin filtrate to how main excellent spore bacteria liquid culture after extraction purification, sets up the in vitro resistant material rapid identification method of cucumber rod spore leaf spot on this basis.
Summary of the invention
In view of the deficiency of existing cucumber rod spore leaf spot seedling resistance authentication method, in rubber Disease Resistance Identification, reference is applied as with how main excellent spore toxin, the invention provides and a kind ofly operate qualification, cost is low, speed is fast, accuracy is strong, reproducible, the cucumber rod spore leaf spot method of resistance identification be adapted in cucumber breeding for disease resistance, to accelerate carrying out of cucumber rod spore leaf spot breeding for disease resistance work.
To achieve these goals, the present invention's " a kind of cucumber rod spore leaf spot Resistance Identification method ", comprises the following steps:
Using fresh, healthy, complete cucumber true leaf as infecting expert evidence, this expert evidence leaf age is the excised leaf of the 2nd true leaf on cucumber 3 true leaf plant in period;
With aseptic how main excellent spore (
corynesporacassiicola) toxin filtrate is inoculum, following steps are passed through in the acquisition of this inoculum:
How main excellent the spore of activation is inoculated in Czapek liquid nutrient medium, 120rpm/min, after 21d cultivated by shaking table, adopt double gauze broth filtrate, removing mycelium, reclaim filtrate, by after the filtrate centrifugal 5000rpm/min, the 30min that are recovered to, collect supernatant, adopt 0.45 μm of miillpore filter to carry out filtration sterilization, reclaim the inoculum of bacteria-free filtrate as Disease Resistance Identification;
The aseptic toxin filtrate not damaged hanging drop of joining is inoculated on fresh, healthy, complete Cucumber In Vitro blade, concrete operations are: on the excised leaf of each different cultivars, not damaged hanging drop inoculates 4 points, the toxin filtrate of often some inoculation 20ul, and each kind to be identified all inoculates 20ul sterile water for contrast with not damaged hanging drop;
After inoculation, blade is positioned over moisturizing in culture tray to cultivate, the filter paper that the two-layer 15ml sterile water that tiles bottom culture tray soaks, and the petiole base of the Cucumber In Vitro blade medical absorbent cotton soaked by sterile water is wrapped up, finally plastic pallet freshness protection package is covered moisturizing;
After cultivating 3d at 25 DEG C, postvaccinal blade, observe how main excellent spore toxin and situation is infected to excised leaf, right-angled intersection method is adopted to measure the lesion diameter inoculated on blade, by blade infecting the mean size qualification different cucumber varieties of scab to the resistance of how main excellent spore.
The Resistance Identification standard of cucumber rod spore leaf spot is as follows:
Immunity (I), infection court is without infecting;
High resistance (HR), infection court infects, and occur the point that chlorisis turns yellow, lesion diameter is less than 5mm;
In anti-(MR), infection court infects, and occurs the point that chlorisis turns yellow, lesion diameter 5 ~ 10mm;
Middle sense (MS), infection court infects, and occurs that chlorisis turns yellow circular scab, lesion diameter 10 ~ 15mm;
High sense (HS), infection court infects, and occur that chlorisis turns yellow circular scab, lesion diameter is greater than 15mm.
The method commonly used for cucumber rod spore leaf spot Resistance Identification is at present artificial inoculation on seedling qualification, in application process, the method is often because of inoculation seedling age, inoculation method, the difference of culture environment and the uncontrollability of investigation method, qualification result is caused to have certain difference, and the repeatability of qualification result is low, between different batches, otherness is larger.
Cucumber rod spore leaf spot Resistance Identification method involved in the present invention overcomes above deficiency, and qualification result only needs 3d to complete; And carry out in illumination box, all can control temperature, humidity, such environmental effects is little; Method of operating is identified, repeatable high.The method can realize high flux, Rapid identification different cultivars cucumber to the object of excellent spore leaf spot resistance.
Embodiment
Form is described in detail content of the present invention by the following examples, gives detailed embodiment and concrete operating process, and all technology realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1, different business cucumber variety is to the Resistance Identification of cucumber rod spore leaf spot
Invention is executed the anti-excellent spore leaf spot expert evidence of the cucumber selected in example and is: excellent No. 3 of Tianjin, and excellent No. 38 of Tianjin is business cucumber variety, is purchased from Tianjin Ke Run cucumber research institute; Middle peasant No. 5, middle peasant No. 8 is business cucumber variety, is purchased from Vegetable & Flower Inst., Chinese Academy of Agriculture Science.
Resistance Identification method is as follows:
1. the preparation of expert evidence is infected: be seeded in greenhouse nutritive cube (nutritive cube diameter 12cm, matrix components is the peat composed of rotten mosses: vermiculite=1:2) by 4 cucumber varieties for examination.When cucumber seedling grows to 3 true leaf periods, get every strain the 2nd true leaf, as the expert evidence of the inventive method application.Then, artificial inoculation on seedling qualification is carried out to the plant taking blade away.
2. with aseptic how main excellent spore (
corynesporacassiicola) preparation of toxin filtrate: how main excellent the spore of activation is inoculated in Czapek liquid nutrient medium, 120rpm/min, after 21d cultivated by shaking table, adopt double gauze broth filtrate, removing mycelium, reclaim filtrate, by after the filtrate centrifugal 5000rpm/min, the 30min that are recovered to, collect supernatant, adopt 0.45 μm of miillpore filter to carry out filtration sterilization, reclaim the inoculum of bacteria-free filtrate as Disease Resistance Identification.
3. aseptic toxin filtrate not damaged hanging drop inoculation identification method: not damaged hanging drop inoculates 4 points on the excised leaf of each different cultivars, the step of often some inoculation 20ul 2. in the toxin filtrate of joining, and each kind to be identified all inoculates 20ul sterile water for contrast with not damaged hanging drop, after inoculation, being positioned over by blade is covered with in the plastic pallet of two layers of filter paper, and at 25 DEG C, 3d is cultivated in moisturizing.
4. aseptic toxin filtrate inoculation " Invest, Then Investigate " authentication method: after inoculated and cultured 3d, observe how main excellent spore toxin and situation is infected to excised leaf, right-angled intersection method is adopted to measure the lesion diameter inoculated on blade, by blade infecting the mean size qualification different cucumber varieties of scab to the resistance of how main excellent spore.The standard of perfection of cucumber rod spore leaf spot resistance is as follows: immunity (I), and infection court is without infecting; High resistance (HR), infection court infects, and occur the point that chlorisis turns yellow, lesion diameter is less than 5mm; In anti-(MR), infection court infects, and occurs the point that chlorisis turns yellow, lesion diameter 5 ~ 10mm; Middle sense (MS), infection court infects, and occurs that chlorisis turns yellow circular scab, lesion diameter 10 ~ 15mm; High sense (HS), infection court infects, and occur that chlorisis turns yellow circular scab, lesion diameter is greater than 15mm.
5. artificial inoculation on seedling authentication method: head prepares spore concentration for (3 ~ 4) × 10
4the cucumber rod spore Leaf blotch pathogeny suspension of individual/mL, carry out spray inoculation to the plant taking the 2nd true leaf away, then by its moisturizing 24h under 25 ~ 28 DEG C of environmental conditions, only continue moisturizing at night after 24h, 7d after inoculation investigates incidence.During severity Scaling, standard used is: 0 grade, without illness; 1 grade, there is a small amount of scab in inoculation leaf; 2 grades, scab accounts for less than 1/3 of leaf area; 3 grades, scab accounts for 1/3 ~ 1/2 of leaf area; 4 grades, scab accounts for 1/2 ~ 2/3 of leaf area; 5 grades, scab accounts for more than 2/3 of leaf area.The Resistance Identification standard of cucumber rod spore leaf spot: immunity (I) DI=0; High resistance (HR), 0<DI≤15; In anti-(MR), 15<DI≤55; Middle sense (MS), 55<DI≤75; High sense (HS), DI >=75.
The comparison of the inoculation of toxin hanging drop and artificial inoculation on seedling qualification result
Result of the test shows, after business cucumber variety adopts the inoculation of toxin hanging drop and artificial inoculation on seedling, it shows the uniformity of height to the Resistance Identification result of cucumber rod spore leaf spot.Qualification result is as shown in table 1, and wherein excellent No. 38 of Tianjin is high resistance cucumber rod spore leaf spot, Tianjin excellent No. 3 be in anti-kind, middle peasant No. 5 and No. 8 are the middle sense kind of the excellent spore leaf spot of cucumber.
A table 14 commercial cucumber variety is through the inoculation of toxin hanging drop and artificial inoculation on seedling qualification result
How main excellent spore host-specific toxin can produce typical illness by rapid induction plant tissue.The resistance method of how main excellent spore toxin hanging drop inoculated identification different cultivars cucumber to excellent spore leaf spot is utilized to have by such environmental effects little, can manual control, qualification result can the advantage such as accurate quantification, can as the prefered method in the qualification of cucumber rod spore leaf spot disease resistance evaluation.
Claims (6)
1. Rapid identification cucumber is to a resistance method for the excellent spore leaf spot that how main excellent spore causes, it is characterized in that through the following step: 1. using fresh, healthy, complete cucumber true leaf as infecting expert evidence; 2. in this is identified with aseptic how main excellent spore toxin filtrate for inoculum; 3. the aseptic toxin filtrate not damaged hanging drop of 2. joining in step is inoculated on step Cucumber In Vitro blade 1.; 4., after inoculation, blade is positioned over moisturizing in the plastic pallet being covered with two layers of filter paper and cultivates; 5. by after cultivating 3d at 25 DEG C, postvaccinal blade, observe how main excellent spore toxin and situation is infected to excised leaf, right-angled intersection method is adopted to measure the lesion diameter inoculated on blade, by blade infecting the mean size qualification different cucumber varieties of scab to the resistance of how main excellent spore.
2. cucumber rod spore leaf spot Resistance Identification method according to claim 1, is characterized in that: the step expert evidence that 1. middle this authentication method introduced adopts is the excised leaf of the 2nd true leaf that cucumber 3 true leaf leaf age plant grow.
3. cucumber rod spore leaf spot Resistance Identification method according to claim 1, it is characterized in that: 2. step operates as follows: how main excellent the spore of activation is inoculated in Czapek liquid nutrient medium, 120rpm/min, after 21d cultivated by shaking table, adopt double gauze broth filtrate, removing mycelium, reclaim filtrate, by the centrifugal 5000rpm/min of filtrate be recovered to, after 30min, collect supernatant, adopt 0.45 μm of miillpore filter to carry out filtration sterilization, reclaim the inoculum of bacteria-free filtrate as Disease Resistance Identification.
4. cucumber rod spore leaf spot Resistance Identification method according to claim 1, it is characterized in that: step 3. in concrete operations be: on the excised leaf of each different cultivars, not damaged hanging drop inoculates 4 points, the step of often some inoculation 20ul 2. in the toxin filtrate of joining, and each kind to be identified all with not damaged hanging drop inoculation 20ul sterile water for contrast.
5. cucumber rod spore leaf spot Resistance Identification method according to claim 1, it is characterized in that: the Cucumber In Vitro blade moisturizing that step is 4. described, mainly realized by following steps: two layers of filter paper 15ml sterile water soaks, and the petiole base of the Cucumber In Vitro blade medical absorbent cotton soaked by sterile water is wrapped up, finally plastic pallet freshness protection package is covered moisturizing.
6. cucumber rod spore leaf spot Resistance Identification method according to claim 1, is characterized in that: the Resistance Identification standard of described step cucumber rod spore leaf spot is 5. as follows:
Immunity (I), infection court is without infecting;
High resistance (HR), infection court infects, and occur the point that chlorisis turns yellow, lesion diameter is less than 5mm;
In anti-(MR), infection court infects, and occurs the point that chlorisis turns yellow, lesion diameter 5 ~ 10mm;
Middle sense (MS), infection court infects, and occurs that chlorisis turns yellow circular scab, lesion diameter 10 ~ 15mm;
High sense (HS), infection court infects, and occur that chlorisis turns yellow circular scab, lesion diameter is greater than 15mm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510588060.4A CN105103984A (en) | 2015-09-16 | 2015-09-16 | Resistance identification method of cucumber corynespora leaf spot disease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510588060.4A CN105103984A (en) | 2015-09-16 | 2015-09-16 | Resistance identification method of cucumber corynespora leaf spot disease |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105103984A true CN105103984A (en) | 2015-12-02 |
Family
ID=54652331
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510588060.4A Pending CN105103984A (en) | 2015-09-16 | 2015-09-16 | Resistance identification method of cucumber corynespora leaf spot disease |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105103984A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105603119A (en) * | 2016-03-31 | 2016-05-25 | 天津科润农业科技股份有限公司 | SNP (single-nucleotide polymorphism) marker method for detecting cucumber Corynespora-cassiicola-resistant site |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101971754A (en) * | 2010-06-18 | 2011-02-16 | 唐兴龙 | Method for preventing and controlling physoderma maydismiyabe |
| CN103371163A (en) * | 2012-04-11 | 2013-10-30 | 中国农业科学院蔬菜花卉研究所 | Bactericide composition for control of corynespora cassiicola of crops |
| CN103931456A (en) * | 2014-03-22 | 2014-07-23 | 陈学好 | Method for planting apple trees |
-
2015
- 2015-09-16 CN CN201510588060.4A patent/CN105103984A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101971754A (en) * | 2010-06-18 | 2011-02-16 | 唐兴龙 | Method for preventing and controlling physoderma maydismiyabe |
| CN103371163A (en) * | 2012-04-11 | 2013-10-30 | 中国农业科学院蔬菜花卉研究所 | Bactericide composition for control of corynespora cassiicola of crops |
| CN103931456A (en) * | 2014-03-22 | 2014-07-23 | 陈学好 | Method for planting apple trees |
Non-Patent Citations (3)
| Title |
|---|
| 张贺等: "巴西橡胶树品系对棒孢霉落叶病的抗病性鉴定", 《植物保护》 * |
| 殷丽华: "苹果属资源对苹果褐斑病的抗性机理及抗性诱导研究", 《中国博士学位论文全文数据库电子期刊-农业科技辑》 * |
| 王惠哲: "黄瓜褐斑病抗病性鉴定技术及品种抗病性鉴定", 《中国蔬菜》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105603119A (en) * | 2016-03-31 | 2016-05-25 | 天津科润农业科技股份有限公司 | SNP (single-nucleotide polymorphism) marker method for detecting cucumber Corynespora-cassiicola-resistant site |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107090487B (en) | Method for identifying resistance of buckwheat black spot | |
| CN101712941B (en) | Paenibacillus polymyxa and application thereof | |
| CN106591418A (en) | Identification method for identifying resistance of tomatoes on gray leaf spot by utilizing in-vitro leaves | |
| CN105557347A (en) | Gray mold resistance identified seedling stage inoculation method for capsicum | |
| CN102021223B (en) | Quantitative dip in bacterial suspension method for identifying resistance to cotton verticillium wilt using vermiculite sandy soil bottomless bowl of paper | |
| CN105191682A (en) | Disease resistance identification method for phoma arachidicola | |
| CN103194524B (en) | Method for rapidly identifying peanut germplasm with bacterial wilt resistance | |
| CN103194421A (en) | Alternaria alternate spore suspension liquid and application thereof | |
| CN103181321A (en) | Breeding method for cold-resistant seedless grape | |
| CN104357333A (en) | Fusarium oxysporum single spore isolation method for soybean root rot | |
| CN106834419A (en) | A kind of method that use excised leaf identifies sugarcane toppers rot-resistant | |
| CN105103984A (en) | Resistance identification method of cucumber corynespora leaf spot disease | |
| CN112195217A (en) | Method for determining pathogenicity of pathogenic bacteria of weedy rice and identification device | |
| CN106957890B (en) | Method for improving indoor artificial inoculation identification accuracy of rice variety panicle blast | |
| CN101589677A (en) | The training quality detection method of hypsizygus marmoreus produced by applying liquid strains | |
| CN102960244A (en) | Method for introducing nitrogen-fixing bacteria into sugarcane tissue culture seedlings | |
| CN101268744B (en) | Method for innoculating soybean with phytophthora sojae | |
| CN113462740B (en) | Resistance identification method for gray leaf spot of buckwheat | |
| CN109628549A (en) | Using excised leaf Rapid identification capsicum single plant to the method for glue spore resistance toanthracnose | |
| CN104212896B (en) | The Molecular Identification primer of a kind of Tobacco Angular Leaf Spot Disease bacterium and authentication method | |
| CN101712942B (en) | Paenibacillus macerans for preventing and controlling plant bacterial wilt and application thereof | |
| CN104304016B (en) | A kind of method that utilization cultivating chamber prepares aweto strain material | |
| CN107488702A (en) | A kind of new identification sunflower method horizontal to resistance to sclerotinia sclerotiorum | |
| CN106222097A (en) | A kind of isolation and identification method of sorghum hybrid sudangrass Leaf blotch pathogeny | |
| CN107831150B (en) | A kind of method of quick diagnosis chamber crop physiological status |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20151202 |
|
| WD01 | Invention patent application deemed withdrawn after publication |