CN101236196A - Powdery mildew resisting green cucumber variety identification method - Google Patents
Powdery mildew resisting green cucumber variety identification method Download PDFInfo
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- CN101236196A CN101236196A CNA2008100339918A CN200810033991A CN101236196A CN 101236196 A CN101236196 A CN 101236196A CN A2008100339918 A CNA2008100339918 A CN A2008100339918A CN 200810033991 A CN200810033991 A CN 200810033991A CN 101236196 A CN101236196 A CN 101236196A
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Abstract
The invention discloses a powdery mildew immune cucumber variety identification method in the agricultural technical field. The invention comprises the following steps: step 1, collecting a leaf disc from cucumber seedling or adult plant through a stainless steel cork borer, and sterilizing the leaf disc; step 2, inoculating the leaf disc through a spray inoculation method; step 3, after inoculation, placing the leaf disc in a culture dish with two layers of filter paper; step 4, putting the culture dish into an illumination incubator, dropping sterilized water at intervals to keep the filter paper wet, investigating the disease state of the leaf disc, and making a disease resistance identification according to the measured average disease area of the leaf disc. The powdery mildew immune cucumber variety identification method which has more accurate identification results is capable of prolonging the disease resistance identification time and simultaneously identifying a plurality of physiological strains of the powdery mildew on the same variety material, thereby being more suitable for the characteristics of genetic study.
Description
Technical field
What the present invention relates to is a kind of authentication method of agricultural technology field, specifically is a kind of authentication method of powdery mildew resisting green cucumber variety.
Background technology
Powdery mildew of cucumber (PM, powdery mildew) causes by the biogenous powdery mildew of obligate, the leaf diseases that all generally takes place in greenhouse and land for growing field crops; The cause of disease powdery mildew mainly comprises two kinds of melon list softgel shell (Podosphaeraxanthii) and two spore powdery mildews (Golovinomyces cichoracearum); China is comparatively general with melon list softgel shell (P.xanthii) harm.Powdery mildew cause of disease host is extensive, and ripe spore can be propagated with air draught; Easily take place dry, nice and cool season, and the white powder germ can infect cucumber plant blade, stem and flower.According to report, infect powdery mildew in florescence and can reduce output 20～40%.
Spraying fungicide and plantation disease-resistant variety are this sick main method of defence.But use germifuge can endanger Environmental security for a long time, and cause that easily the variation of germ microspecies produces antagonism.The popularizing planting disease-resistant variety is safety, environmental protection and control strategy efficiently.
Find by prior art documents, " squash marble dust pathogen identification and the research of resistance authentication method " that Liu Xiubo etc. delivered on to 13 pages the 10th page of " Chinese melon dish " the 1st phase in 2006, the spore suspension spray inoculation method of powdery mildew is proposed in this article, its deficiency is: because powdery mildew of cucumber harm takes place greatly, the special normal mature that influences the cucumber fruits seed, the suspension spray inoculation method can only adopt the resistance of identifying the second generation to infer the resistance of contemporary material, this has not only delayed the progress of work of breeding for disease resistance, and has strengthened the workload of disease-resistant variety seed selection; Simultaneously some genetic stocks quantity seldom, scientific research needs a plurality of germ microspecies that genetic stocks is identified simultaneously if having time, the suspension spray inoculation method can not be finished such evaluation requirement.Find also in the retrieval that patent name is " primer sequence and an authentication method thereof of identifying cucumber powdery mildew resistance ", publication number: CN1482132.This patented technology discloses a kind of primer sequence and authentication method thereof of identifying cucumber powdery mildew resistance, primer sequence is by upstream primer: 5 '-CAG TAA ATG AAA GAA AAG AAG-3 ', downstream primer: 5 '-ATA CAT AGC CAT ACA AAA AT-3 ' composition, use the method for the evaluation cucumber powdery mildew resistance of this primer sequence, comprise the steps: the DNA of (1) extraction cucumber gene group; (2) carry out pcr amplification; (3) gel electrophoresis analysis of pcr amplification product; (4) identified the relative position of sample band on gel according to each, identified the resistance of powdery mildew of cucumber.But this patented technology is to need to use molecular marker data to carry out the evaluation of cucumber germplasm powdery mildew disease-resistant, and general production unit is difficult to satisfy the requirement of experiment of gathering molecular marker data, and the disease-resistant identical rate of qualification result and field is also not high.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of authentication method of powdery mildew resisting green cucumber variety is provided.The present invention carries out resistance according to the big characteristics of cucurbitaceous plant blade to identify under controlled condition, and according to the exsomatize resistance performance of leaf dish of blade, resistance to whole plant is identified, more original authentication method has the seed selection work that more is applicable to the cucumber powdery mildew resistance kind, the accuracy and the accuracy of qualification result are higher, widened and carried out the time that resistance is identified, and can on a kind material, identify a plurality of powdery mildew biological strains simultaneously simultaneously, be more suitable for the characteristics of genetic research.
The present invention is achieved by the following technical solutions, the present invention includes following steps:
The first step is got the leaf dish with the stainless steel card punch at cucumber seedling or one-tenth strain collection, and the leaf dish is carried out disinfection;
Described stainless steel card punch, its diameter are 2cm.
Described the leaf dish is carried out disinfection, be specially: distilled water rinsing 3s, 2% liquor natrii hypochloritis's rinsing 5s, 75% alcoholic solution rinsing 5s, distilled water rinsing 10s.
In second step, adopt the spray inoculation method that the leaf dish is inoculated;
Described employing spray inoculation method is inoculated the leaf dish, is meant: the pressure device of working pressure 0.05MP/mm (MPa/square millimeter), and spore suspension concentration is 5-7 * 10
4Spore/milliliter, seeded process is finished on the sterile working platform.
In the 3rd step, after the inoculation, the leaf dish is positioned in the double dish that is placed with two layers of filter paper;
Described filter paper soaks with 0.01% cephalo (mass ratio) solution.
The 4th step was placed on double dish in the illumination box, and every interval 4 days drips sterilized water and keeps filter paper moistening.Instituted an inquiry leaf dish incidence on the 12nd day, make the disease resistance evaluation according to the average onset area that the leaf dish is measured.
Described double dish is placed in the illumination box, is meant: double dish is placed on 23 ℃ of temperature, and illumination is 50umol/s/m
2Illumination box in 12 days.
The described average onset area of measuring according to the leaf dish is made disease resistance and identified, is meant: for auxiliary viewing tool, is gradient according to leaf dish onset area 10% by 40 times of anatomical lens, 0: not morbidity, 100%: be covered with the powdery mildew mycelia on the leaf dish.Convenient for genetic analysis, setting onset area is disease-resistant less than 20%, is susceptible greater than 20%.A kind can be got a plurality of leaf dishes, and the state of an illness gradient of the average onset area correspondence that the leaf dish is measured is the state of an illness gradient of this kind, and makes disease resistance according to this and identify.
The invention provides a kind of powdery mildew of cucumber leaf dish identification method, this method has the following advantages than spore suspension spray inoculation method:
(1) more is applicable to the seed selection work of cucumber powdery mildew resistance kind.Because adopted the leaf dish to identify, leaf dish diameter is 2cm only, and is damaging little to plant, plant can continued growth, satisfies other experiment and production purpose needs.Can select the present age, accelerated the process of breed breeding greatly, also reduced the workload of breeding.
(2) identify that can get a plurality of leaf dishes and carry out repetitive identified on each individual plant, the individual plant inoculation qualification result that utilizes mean value to inoculate as qualification result and suspension spray is compared owing to get the leaf dish, the appraising datum statistical significance is more accurate.
(3) can on same plant leaf, get a plurality of leaf dishes simultaneously, be used for the evaluation of a plurality of cause of disease microspecies; This can't realize for suspension spray inoculation method inoculation evaluation.
(4) in a season of growth, can carry out the resistance evaluation by separating for several times leaf dish to same material, increase the number of times of evaluation than the disposable evaluation of suspension spray inoculation; The time of identifying can guarantee that any time in 1 year carries out, thereby has widened the time that resistance is identified.
(5) be more suitable for genetic research.Science of heredity need make full use of the difference that disease-resistant material and susceptible storeroom exist in the genetic group, and the susceptible individual plant that carries out the resistance evaluation often is easy to generate dead seedling, both can't gather in the crops fruit, and also be unfavorable for gathering blade and carry out foranalysis of nucleic acids, and our rule can well address these problems.
Embodiment
Below embodiments of the invention are elaborated: present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
The inoculation of embodiment 1, Shen green No. 2 and Chang Chun Mi Ci is identified
Cucumber powdery mildew resistance kind Shen is commercial cucumber variety green No. 2, buys in Shanghai City seeds company.Powdery mildew of cucumber susceptible variety Chang Chun Mi Ci is also bought in Shanghai City seeds company for commercial cucumber variety.
The resistance authentication method is as follows:
One, the preparation of inoculation cucumber material
To be seeded in separately in the dish of cave, greenhouse (d=12cm, matrix components are 1 part of peat+1 part cinder+2 parts of vermiculites) for the examination cucumber variety.When seedling grows to 2 leaves wholeheartedly the time (20d approximately after planting), get leaf dish 1-2 every strain rough leaf, carry out indoor aseptic leaf dish and identify.After 7 days to taking the plant of leaf dish away, artificial spray inoculation germ spore suspension.
Two, inoculation is identified
Leaf dish inoculation: uses diameter to get leaf dish (diameter=2cm) in cucumber seedling or one-tenth strain collection as the stainless steel card punch of 2cm, the leaf dish is carried out following disinfecting process, distilled water rinsing 3s, 2% liquor natrii hypochloritis's rinsing 5s, 75% alcoholic solution rinsing 5s, distilled water rinsing 10s.Use inoculation method to be the spray inoculation method, utilize the pressure device of pressure 0.05MP/mm (MPa/square millimeter), use bacterial concentration to be 5-7 * 10
4Spore/milliliter; Process is finished on the sterile working platform.After the inoculation, the leaf dish is positioned in the double dish that is placed with two layers of filter paper, filter paper soaks with 0.01% cephalo solution.Double dish is placed on 23 ℃ of temperature, and illumination is 50umol/s/m
2Illumination box in 12 days, every interval 4 days drips the 10ml sterilized water and keeps filter paper moistening.Instituting an inquiry leaf dish incidence on the 12nd day, for auxiliary viewing tool, is gradient according to leaf dish onset area 10% by 40 times of anatomical lens, 0: not morbidity, 100%: be covered with the powdery mildew mycelia on the leaf dish.
The seedling artificial infection: adopt spore suspension spray inoculation method, spore suspension concentration is 5-7 * 10
4Spore/milliliter; In the greenhouse day temperature 22-28 ℃, at temperature 15-18 ℃ of night, about sunshine-duration 17h, in the 24h of inoculation back, keep relative humidity about 70% with humidifier atomizing.Institute an inquiry incidence behind the inoculation 12d, state of an illness statistical method is with reference to (Morishita M, Japan Agricultural Research Quarterly (Japanese agriculture research quarterly), 2003,37 (1): 7～14).
Four, the comparison of seedling artificial infection evaluation and leaf dish inoculation qualification result
Test findings shows the cucumber variety resistance performance basically identical when seedling stage, evaluation and leaf dish were identified that powdery mildew of cucumber is had different anti-sense reactions, shows that this method can be used for the cucumber powdery mildew resistance of identification of species.The result is as shown in table 1.
Table 1
| Title material | The seedling artificial infection is identified | The inoculation of leaf dish is identified | ||||
| Identify strain | The morbidity strain | The incidence of disease | Identify strain | The morbidity strain | The incidence of disease | |
| Green No. 2 Chang Chun Mi Cis in Shen | 100 100 | 9 64 | 9.0％ 64.0％ | 80 80 | 7 52 | 6.7％ 65.0％ |
Embodiment 2, Shen green No. 2 and Chang Chun Mi Ci F
2The genetic analysis of genetic group powder mildew resistance
Cucumber powdery mildew resistance kind Shen is commercial cucumber variety green No. 2, buys in Shanghai City seeds company.Powdery mildew of cucumber susceptible variety Chang Chun Mi Ci is also bought in Shanghai City seeds company for commercial cucumber variety.Make paternal hybrid with green No. 2 work female parents in Shen and Chang Chun Mi Ci, make up hereditary F
2Colony comprises 108 individual plants.
Inoculation identification method is as follows:
One, the preparation of inoculation cucumber material
Will be for the hereditary F of green No. 2 and Chang Chun Mi Ci assembly of examination Shen
2Colony is seeded in the dish of cave, greenhouse (d=12cm, matrix components are 1 part of peat+1 part cinder+2 parts of vermiculites).When seedling grows to 2 leaves wholeheartedly the time (20d approximately after planting), get leaf dish 1-2 every strain rough leaf, carry out indoor aseptic leaf dish and identify.After 7 days to taking the plant of leaf dish away, artificial spray inoculation germ spore suspension.
Two, inoculation is identified
Leaf dish inoculation: use diameter to get leaf dish (diameter=2cm) in the cucumber seedling collection as the stainless steel card punch of 2cm, the leaf dish is carried out following disinfecting process, distilled water rinsing 3s, 2% liquor natrii hypochloritis's rinsing 5s, 75% alcoholic solution rinsing 5s, distilled water rinsing 10s.Use inoculation method to be the spray inoculation method, utilize the pressure device of pressure 0.05MP/mm (MPa/square millimeter), use bacterial concentration to be 5-7 * 10
4Spore/milliliter; Process is finished on the sterile working platform.After the inoculation, the leaf dish is positioned in the double dish that is placed with two layers of filter paper, filter paper soaks with 0.01% cephalo solution.Double dish is placed on 23 ℃ of temperature, and illumination is 50umol/s/m
2Illumination box in 12 days, every interval 4 days drips the 10ml sterilized water and keeps filter paper moistening.Instituting an inquiry leaf dish incidence on the 12nd day, for auxiliary viewing tool, is gradient according to leaf dish onset area 10% by 40 times of anatomical lens, 0: not morbidity, 100%: be covered with the powdery mildew mycelia on the leaf dish.Setting onset area is disease-resistant less than 20%, is susceptible greater than 20%.Statistics Shen green No. 2 and Chang Chun Mi Ci heredity F
2Colony's individual plant powder mildew resistance.
The seedling artificial infection: adopt spore suspension spray inoculation method, spore suspension concentration is 5-7 * 10
4Spore/milliliter; In the greenhouse day temperature 22-28 ℃, at temperature 15-18 ℃ of night, about sunshine-duration 17h, in the 24h of inoculation back, keep relative humidity about 70% with humidifier atomizing.Institute an inquiry Shen green No. 2 and Chang Chun Mi Ci heredity F behind the inoculation 12d
2Colony's individual plant incidence, state of an illness statistical method is with reference to (Morishita M, JapanAgricultural Research Quarterly (Japanese agriculture research quarterly), 2003,37 (1): 7～14).
Four, Shen green No. 2 and Chang Chun Mi Ci heredity F are identified in the inoculation of seedling artificial infection evaluation and leaf dish
2The result's of colony comparison
Test findings shows Shen green No. 2 and Chang Chun Mi Ci heredity F
2Colony use that the leaf dish is identified and identify seedling stage and the performance of genetics of resistance rule in full accord, the Shen green No. 2 with the hereditary F of Chang Chun Mi Ci
2Colony's performance powder mildew resistance is controlled by recessive single-gene, and its anti-sense segregation ratio is 1: 3.The result is as shown in table 2.
Table 2
| Authentication method | Shen green No. 2 and Chang Chun Mi Ci heredity F 2Colony | |||
| Identify strain | Disease-resistant strain | Susceptible strain | Separate than row | |
| The inoculation of seedling inoculation leaf dish | 108 108 | 21 23 | 87 85 | 1∶3 1∶3 |
Claims (8)
1, a kind of authentication method of powdery mildew resisting green cucumber variety is characterized in that, comprises the steps:
The first step is got the leaf dish with the stainless steel card punch at cucumber seedling or one-tenth strain collection, and the leaf dish is carried out disinfection;
In second step, adopt the spray inoculation method that the leaf dish is inoculated;
In the 3rd step, after the inoculation, the leaf dish is positioned in the double dish that is placed with two layers of filter paper;
The 4th step was placed on double dish in the illumination box, dripped sterilized water at interval and kept filter paper moistening, instituted an inquiry leaf dish incidence, and made the disease resistance evaluation according to the average onset area that the leaf dish is measured in the 12nd day.
2, the authentication method of powdery mildew resisting green cucumber variety according to claim 1 is characterized in that, in the first step, described stainless steel card punch, its diameter are 2cm.
3, the authentication method of powdery mildew resisting green cucumber variety according to claim 1 and 2 is characterized in that, in the first step, described the leaf dish is carried out disinfection, be specially: distilled water rinsing 3s, 2% liquor natrii hypochloritis's rinsing 5s, 75% alcoholic solution rinsing 5s, distilled water rinsing 10s.
4, the authentication method of powdery mildew resisting green cucumber variety according to claim 1 is characterized in that, in second step, described employing spray inoculation method is inoculated the leaf dish, is meant: the pressure device of working pressure 0.05MP/mm, and spore suspension concentration is 5-7 * 10
4Spore/milliliter is seeded on the sterile working platform and finishes.
5, the authentication method of powdery mildew resisting green cucumber variety according to claim 1 is characterized in that, in the 3rd step, described filter paper mass percent is that 0.01% cephalo solution soaks.
6, the authentication method of powdery mildew resisting green cucumber variety according to claim 1 is characterized in that, in the 4th step, described double dish is placed in the illumination box, be meant: double dish is placed on 23 ℃ of temperature, and illumination is 50umol/s/m
2Illumination box in 12 days.
According to the authentication method of claim 1 or 6 described powdery mildew resisting green cucumber varieties, it is characterized in that 7, in the 4th step, described interval is meant every interval 4 days.
8, authentication method according to claim 1 or 6 described powdery mildew resisting green cucumber varieties, it is characterized in that, in the 4th step, the described average onset area of measuring according to the leaf dish is made disease resistance and is identified, be meant: be auxiliary viewing tool by 40 times of anatomical lens, according to leaf dish onset area 10% is gradient, 0: not morbidity, 100%: be covered with the powdery mildew mycelia on the leaf dish, setting onset area is disease-resistant less than 20%, greater than 20% being susceptible, and the desirable a plurality of leaf dishes of kind, the state of an illness gradient of the average onset area correspondence that the leaf dish is measured is the state of an illness gradient of this kind, and makes disease resistance according to this and identify.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103558141A (en) * | 2013-10-14 | 2014-02-05 | 西北农林科技大学 | Method for identifying heat resistance of capsicum by utilization of in vitro leaf disk |
| CN106031352A (en) * | 2015-03-11 | 2016-10-19 | 李保华 | A method for evaluating the prevention and control effects of fungicides on apple floral organ-infected diseases |
| CN109511415A (en) * | 2019-01-18 | 2019-03-26 | 江苏省农业科学院宿迁农科所 | A kind of inoculation cultural method of day lily rust |
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2008
- 2008-02-28 CN CNA2008100339918A patent/CN101236196A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103558141A (en) * | 2013-10-14 | 2014-02-05 | 西北农林科技大学 | Method for identifying heat resistance of capsicum by utilization of in vitro leaf disk |
| CN106031352A (en) * | 2015-03-11 | 2016-10-19 | 李保华 | A method for evaluating the prevention and control effects of fungicides on apple floral organ-infected diseases |
| CN106031352B (en) * | 2015-03-11 | 2018-12-04 | 李保华 | Method for evaluating control effect of bactericide on apple floral organ infected diseases |
| CN109511415A (en) * | 2019-01-18 | 2019-03-26 | 江苏省农业科学院宿迁农科所 | A kind of inoculation cultural method of day lily rust |
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