CN102808015A - Method for identifying resistance of sclerotinia by inoculating in-vitro stalk of plant - Google Patents
Method for identifying resistance of sclerotinia by inoculating in-vitro stalk of plant Download PDFInfo
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- CN102808015A CN102808015A CN2012102299521A CN201210229952A CN102808015A CN 102808015 A CN102808015 A CN 102808015A CN 2012102299521 A CN2012102299521 A CN 2012102299521A CN 201210229952 A CN201210229952 A CN 201210229952A CN 102808015 A CN102808015 A CN 102808015A
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Abstract
The invention relates to a method for identifying the resistance of sclerotinia by inoculating an in-vitro stalk of a plant. The method comprises the following steps of: cutting the stalk or branches of the plant down, and putting the stalk or the branches into closed environment in which temperature and humidity are constant to inoculate sclerotinia sclerotiorum; and recording the extension lengths of bacterial plaques to measure the strength of disease resistance of the material. Compared with the conventional method for identifying the resistance of the stalk with sclerotinia, the method has the advantages that the method is performed under the condition of controllable environment, the pathogenetic condition is consistent, and inoculating identification is stable; the method is simple, short in period and high in inoculating efficiency; and inoculated parts are flexible, and a single plant can be identified repeatedly, so the method is suitable for the large-scale inoculating identification of the material.
Description
Technical field
The invention belongs to plant pest resistance authentication method, be specifically related to the method that resistance to sclerotinia sclerotiorum is identified in the stripped cane inoculation of a kind of plant.
Background technology
Sclerotium disease can endanger 400 various plants, such as rape, and annual harm China rape underproduction 10 – 80% because of sclerotium disease.Cultivating disease-resistant variety is to prevent and treat the most economical approach of sclerotium disease harm.Simple, the resistance to sclerotinia sclerotiorum authentication method is the key of the disease-resistant material of screening accurately.
Resistance to sclerotinia sclerotiorum is identified at present has the blade resistance to identify and the evaluation of cane resistance that the blade resistance is identified and comprised excised leaf inoculation, axil inoculation, petiole inoculation, cotyledon inoculation etc.Though these methods are simple to operate, the resistance of blade can not reflect the resistance of cane fully.Sclerotium disease will cause output seriously to reduce after contaminating cane.The cane resistance is identified that field cane toothpick inoculation method and cane mycelia piece binding method are arranged, and two kinds of methods are simple, are fit to the large-scale inoculation evaluation, but receive the influence of land for growing field crops envrionment conditions easily, and inoculation is identified unstable.Such as running into the weather condition that drying etc. is unfavorable for the sclerotium disease morbidity, the failure that will cause the land for growing field crops inoculation to be identified.
Summary of the invention
The object of the present invention is to provide a kind of simple, stable cane inoculation to identify the method for resistance to sclerotinia sclerotiorum.Present method is created the temperature and humidity environment that suitable sclerotium disease is contaminated under indoor controlled condition, the cane that exsomatizes is inoculated evaluation, weighs the power of the anti-sclerotium disease of material according to the length of bacterial plaque.
Technical scheme of the present invention is following:
The method of resistance to sclerotinia sclerotiorum is identified in a kind of stripped cane inoculation, at first, with the edge punching of punch tool along the PDA substratum that is paved with the sclerotinite mycelia, obtains PDA mycelia piece of the same size, is used for stripped cane inoculation as inoculum; Then the intercepting cane and the cane that will exsomatize be transferred to indoor, make on the cane with the identical shaped wound of inoculum after, cover inoculum, in temperature and humidity constant indoor environment, carry out the sclerotinite inoculation; Write down the bacterial plaque extension length, weigh the power of anti-sclerotium disease.
Advantage of the present invention: this inoculation method is under the controllable environment condition of laboratory, and onset condition is consistent, and inoculation is identified stable; Method is simple, and inoculation efficient is high, and the cycle is short; Inoculation position is flexible, can carry out repetitive identified to individual plant, is fit to that material is carried out large-scale inoculation and identifies.
Description of drawings
Fig. 1 is the length of the 3rd day bacterial plaque behind the inoculation rape cane.
Embodiment
Below identifying that Btassica cane sclerotium disease disease resistance is a kind of embodiment of the inventive method, but be not qualification to the inventive method, any not ultra conversion from flesh and blood of the present invention must belong to protection scope of the present invention.
Embodiment 1
1. the preparation of inoculum
1) pathogenic bacteria is collected in experimental plot, rape engineering center, Chongqing City; Its purge process is: the sclerotium of gathering is inoculated in the potato glucose substratum (PDA); In 22 ℃ of dark culturing 2 days, the picking mycelia was inserted new PDA substratum from the edge, after 2 days once more with the mycelia switching at edge; So repeatedly, the mycelia that in substratum, grows does not contain till other assorted bacterium.Being prepared as of PDA substratum wherein: 200g potato, 20g sucrose and 15g agar powder, constant volume is to 1L.
2) isolated experiment inoculum (PDA mycelia piece) preparation: get bacterial classification one fritter after the activation; Be inoculated in the sterile petri dish central authorities that the PDA substratum is housed; Cultivated 2 days down in 22 ℃, 85% humidity condition; After treating the even confluent culture primary surface of mycelia, with punch tool punching along the petridish edge of diameter 6mm, the PDA mycelia piece of acquisition promptly can be used for stripped cane inoculation.
2. the cane that exsomatizes is seeded in the whole florescence, at the stem of the about 30cm length of 20cm place intercepting and 2 side shoots of the long lowermost end of the about 30cm of intercepting apart from ground, (scattering and disappearing to prevent that water from dividing) is twined with preservative film in the cane two ends.With 17 parts of brassica plants is that example is carried out sclerotium disease inoculation evaluation.These materials comprise 3 portions of Chinese cabbages (B.rapa), 2 parts of wild cabbages (B.oleracea), and 6 parts of swede type rapes (B.napus), 4 parts of mustard type rapes (B.juncea) and 2 parts of Ethiopia leaf mustard (B.carinata), its concrete material is seen table 1.
Inoculation is in the transfer room of about 20 square metres an of area, temperature controllable, to carry out.At first with 1 * 2m
2Three-ply-wood be positioned over and have a due proportion of, on the aluminum alloy frame of high 40cm, spread wet towel at three-ply-wood, above pad one deck filter paper.The cane of fetching is placed on the filter paper of getting ready, use diameter to make a call to 2 holes on the cane surface, thereby cause surface injury, be spaced apart 10cm between 2 holes as the punch tool of 4mm.With diameter is that the PDA mycelia piece face of carrying disease germs of 6mm is close to site of injury (Fig. 1).After inoculation finishes, above three-ply-wood, place the aluminum alloy frame of an identical size again, with plastic film that the aluminum alloy frame of three-ply-wood and top is together airtight at last, and keep airtight humidity to maintain a constant humidity in the 90-95%.Room temp is controlled at a steady temperature in 22-24 degree centigrade.Behind the in vitro inoculation 3 days, measure the length of bacterial plaque, find that the bacterial plaque size of storeroom is variant obviously, table 1 is seen in its concrete manifestation.The size of stem and side shoot bacterial plaque exist significant positive correlation (r=0.657, P=0.911).The bacterial plaque length of stem and side shoot and the diameter of cane do not exist significant correlation property (stem: r=0.056, P=0.824; Side shoot: r=-0.021, P=0.935).
Present embodiment result such as table 1.
The analytical results of this instance is consistent to be shown, but the cane inoculation of exsomatizing identifies it is a kind of large-scale operation, and envrionment conditions is controlled, good stability, and inoculation efficient is high, and flexibility ratio is high, can realize the method for the repetitive identified of individual plant.
Claims (5)
1. the method for resistance to sclerotinia sclerotiorum is identified in the stripped cane inoculation of a kind of plant, at first, with the edge punching of punch tool along the PDA substratum that is paved with the sclerotinite mycelia, obtains PDA mycelia piece of the same size, as inoculum; Then the intercepting plant stem and the cane that will exsomatize be transferred to indoor, make on the cane with the identical shaped wound of inoculum after, inoculum coverings in, the closed environment of constant in temperature and humidity, suitable sclerotium disease dip-dye carries out sclerotinite and inoculates; Write down the bacterial plaque extension length, weigh the power of anti-sclerotium disease.
2. inoculation is identified and it is characterized in that the method for resistance to sclerotinia sclerotiorum according to the stripped cane described in the claim 1: said plant is that cane such as rape infects the plant of sclerotium disease easily.
3. identify the method for resistance to sclerotinia sclerotiorum according to the stripped cane inoculation of the plant described in the claim 1, it is characterized in that: the cane of in vitro inoculation can be stem, side shoot, stem section.
4. identify the method for resistance to sclerotinia sclerotiorum according to the stripped cane inoculation of the plant described in claim 1 or 2; It is characterized in that: the environment that suitable sclerotium disease is contaminated does; In 22-24 ℃ of scope, set a steady temperature, in 90-95% humidity ranges, set a constant humidity.
5. the method for resistance to sclerotinia sclerotiorum is identified in stripped cane inoculation according to claim 1 and 2, it is characterized in that: the length of the 3rd to 4 day measurement bacterial plaque growth after inoculation.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201210229952.1A CN102808015B (en) | 2012-07-04 | 2012-07-04 | Method for identifying resistance of sclerotinia by inoculating in-vitro stalk of plant |
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| CN201210229952.1A CN102808015B (en) | 2012-07-04 | 2012-07-04 | Method for identifying resistance of sclerotinia by inoculating in-vitro stalk of plant |
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| CN102808015A true CN102808015A (en) | 2012-12-05 |
| CN102808015B CN102808015B (en) | 2014-10-15 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962605A (en) * | 2015-07-01 | 2015-10-07 | 中国农业科学院蔬菜花卉研究所 | Screening method of medicinal agents for preventing bacterial soft rot of vegetables |
| CN105624049A (en) * | 2016-03-23 | 2016-06-01 | 南京农业大学 | Method for separating Sclerotinia homoeocarpa |
| CN105734162A (en) * | 2016-05-05 | 2016-07-06 | 西南大学 | Use of Bo1024541 gene in identifying sclerotiniose resistance of plant |
| CN105861619A (en) * | 2016-04-29 | 2016-08-17 | 云南省烟草农业科学研究院 | Method for determining phytophthora nicotianae virulence through in-vitro shoots |
| CN108486214A (en) * | 2018-03-07 | 2018-09-04 | 云南省农业科学院生物技术与种质资源研究所 | A kind of buckwheat stem wilt Resistance Identification method |
| CN109182592A (en) * | 2018-11-08 | 2019-01-11 | 中国农业科学院油料作物研究所 | Chain SNP marker and application with rape multi-branched character main effect QTL site |
| CN110343739A (en) * | 2019-06-27 | 2019-10-18 | 武汉市农业科学院 | A kind of method of rape field stalk inoculation sclerotinite |
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| CN102433372A (en) * | 2011-12-05 | 2012-05-02 | 内蒙古农业大学 | Method for identifying resistance level of sunflower variety to sclerotiniose indoor |
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| CN102433372A (en) * | 2011-12-05 | 2012-05-02 | 内蒙古农业大学 | Method for identifying resistance level of sunflower variety to sclerotiniose indoor |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962605A (en) * | 2015-07-01 | 2015-10-07 | 中国农业科学院蔬菜花卉研究所 | Screening method of medicinal agents for preventing bacterial soft rot of vegetables |
| CN105624049A (en) * | 2016-03-23 | 2016-06-01 | 南京农业大学 | Method for separating Sclerotinia homoeocarpa |
| CN105624049B (en) * | 2016-03-23 | 2019-01-15 | 南京农业大学 | A kind of separation method of lawn coin pinta bacterium |
| CN105861619A (en) * | 2016-04-29 | 2016-08-17 | 云南省烟草农业科学研究院 | Method for determining phytophthora nicotianae virulence through in-vitro shoots |
| CN105734162A (en) * | 2016-05-05 | 2016-07-06 | 西南大学 | Use of Bo1024541 gene in identifying sclerotiniose resistance of plant |
| CN105734162B (en) * | 2016-05-05 | 2020-10-02 | 西南大学 | Application of Bol024541 gene in identifying plant sclerotinia sclerotiorum disease resistance |
| CN108486214A (en) * | 2018-03-07 | 2018-09-04 | 云南省农业科学院生物技术与种质资源研究所 | A kind of buckwheat stem wilt Resistance Identification method |
| CN108486214B (en) * | 2018-03-07 | 2022-03-11 | 云南省农业科学院生物技术与种质资源研究所 | Buckwheat stem blight resistance identification method |
| CN109182592A (en) * | 2018-11-08 | 2019-01-11 | 中国农业科学院油料作物研究所 | Chain SNP marker and application with rape multi-branched character main effect QTL site |
| CN109182592B (en) * | 2018-11-08 | 2021-06-29 | 中国农业科学院油料作物研究所 | SNP molecular marker linked with rape multi-branch character major QTL locus and application thereof |
| CN110343739A (en) * | 2019-06-27 | 2019-10-18 | 武汉市农业科学院 | A kind of method of rape field stalk inoculation sclerotinite |
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| CN102808015B (en) | 2014-10-15 |
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Granted publication date: 20141015 Termination date: 20170704 |