CN101946607A - Method for identifying resistance of tomato to tomato yellow leaf curl virus by toothpick inoculation method - Google Patents
Method for identifying resistance of tomato to tomato yellow leaf curl virus by toothpick inoculation method Download PDFInfo
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- CN101946607A CN101946607A CN2010102512203A CN201010251220A CN101946607A CN 101946607 A CN101946607 A CN 101946607A CN 2010102512203 A CN2010102512203 A CN 2010102512203A CN 201010251220 A CN201010251220 A CN 201010251220A CN 101946607 A CN101946607 A CN 101946607A
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Abstract
The invention provides a fast, simple and effective method for identifying resistance to tomato yellow leaf curl virus, belonging to the field of plant protection. The tomato yellow leaf curl virus is a virus spread by bemisa babaci, the multi-sampling bemisa babaci inoculation identification method is applied to the traditional virus resistance identification; however, as affected by the bemisa babaci (such as inoculation quantity, virus-carrying rate and chow characteristic of the bemisa babaci) the bemisa babaci inoculation identification method is very difficult to provide uniform inoculation pressure and at the same time is interfered by other virus spread by the bemisa babaci, which leads to poor accuracy and repeatability of the identification result. However, the toothpick inoculation method well solves these problems, uses the constructed infective cloning, inoculates the tomato yellow leaf curl virus by puncturing the phloem of a tomato by a toothpick and identifies the resistance of the tomato material to the tomato yellow leaf curl virus. The method of the invention has the characteristics of no need to raise the bemisa babaci, simple operation, high efficiency, good repeatability, good controllability, accurate identification result and capability of providing relatively uniform inoculation pressure, thus being a simple and effective inoculation identification method.
Description
Technical field
The present invention relates to a kind of tomato yellow leaf curl virus resistance authentication method, platymiscium protection field.
Background technology
Tomato yellow leaf curl is a kind of destructive virus disease on the tomato, this disease is since reported first in 1964, the report that harm takes place is all arranged in areas such as Europe, Africa, Asia, the Caribbean and Oceanias, particularly in recent years, along with the change of global warming and cropping system and the increase of international trade, tomato yellow leaf curl is propagated in the world, has caused heavy losses to tomato production.1991-1992 should disease break out on the Fla. tomato, cause with a toll of 1.4 hundred million dollars.
In China, by tomato yellow leaf curl virus (Tomato yellow leaf curl virus, TYLCV) tomato yellow leaf curl that causes occurred the harm report early than 2006 in Shanghai and Zhejiang, can show the leaf margin yellow after tomato is infected by it in early days, go up volume, and blade diminishes, plant stunts; Whole strain top of later stage presents the shape that bunch contracts, and flower is wilted, and percentage of fertile fruit reduces greatly, has a strong impact on output and the quality of tomato, and the total crop failure total crop failure is caused in the heavy canopy chamber of morbidity more, and economic loss is huge.This disease in 2007 is imported Jiangsu, Anhui, Shandong harm into, subsequently in Hebei, harm also appears in provinces and cities such as Beijing, Tianjin, the Inner Mongol in succession, the expansion rate of propagation is very surprising, the harm that causes simultaneously is also heavy unusually.100,000,000 yuan nearby of the direct economic losses that cause because of tomato yellow leaf curl of Handan City, Hebei only at the beginning of the year ends to 2,009 2008.At present should disease become current tomato in China gradually and produced important restraining factors.
In the control of most of viroses of plant, resistant variety is the most cost-effective prophylactico-therapeutic measures, and in the excavation of resistance resource and utilizing, the resistance authentication method key link that is absolutely necessary.Because tomato yellow leaf curl virus is the louse-borne virus of a kind of tobacco powder, therefore resistance that for a long time should virus is identified the Bemisia tabaci inoculation survey methods that adopt more, pass poison but need Bemisia tabaci to finish in this method operating process, so problem that exists some to be difficult to overcome: 1, the inoculation worm amount of Bemisia tabaci is restive, because the life cycle of Bemisia tabaci is very short, the generation overlap phenomenon is very serious, cause its colony's multiplication capacity very strong, and the individuality of Bemisia tabaci is very little, therefore no matter be in the field or indoor, the Bemisia tabaci quantity of inoculating on the individual plant is difficult to control; 2, the inoculation quantity of band toxic smoke aleyrodid is restive, because the inoculation worm of Bemisia tabaci amount itself is restive, has also caused wherein measuring with the inoculation worm of toxic smoke aleyrodid being difficult to metering; 3, the biography toxic effect rate of Bemisia tabaci is restive, because Bemisia tabaci is a kind of infection insect, only in getting the process of food, it could realize passing poison, and it is got food and is subjected to all multifactor impacts, as temperature, humidity, illumination, host or the like, these factors can influence it and get food number of times and food ingestion, exert an influence to passing the toxic effect rate, and then the accuracy of qualification result is also exerted an influence; 4, because the louse-borne virus of tobacco powder is very many, when therefore using the Bemisia tabaci inoculating tomato yellow leaf curl virus, Bemisia tabaci may be carried other virus simultaneously, influence inoculation qualification result.
In order to solve the problem that exists on the Bemisia tabaci inoculation identification method, we have formulated a kind of method that inoculation is identified based on infectious clone, this method is easy and simple to handle, good reproducibility, controllability is good, the efficient height, the result is accurate, for the research and the utilization of the tomato material of anti-tomato yellow leaf curl virus provides a kind of new method.
Summary of the invention
The present invention has overcome shortcoming of the prior art, and identifying for the resistance of tomato yellow leaf curl virus provides a kind of quick, easy, effective method.
1, the material of resistance evaluation:
The Agrobacterium that contains PTYj01 is stored in disease chamber, Institute of Plant Protection, academy of agricultural sciences, Jiangsu Province (No. 50, Nanjing, Jiangsu Province Xiao Lingwei clock spirit street, 210014), and YEP medium and antibiotic preparation are with reference to " molecular cloning experiment guide (third edition) ".
2, resistance authentication method
A) the Agrobacterium inoculation that will contain PTYj01 contains in the YEP liquid nutrient medium of rifampin (Rifamycin) of the kanamycin (Kanamycin) of 50mg/ml and 50mg/ml, and 28 ℃ of 150rpm are cultured to OD
600Reach 0.6-0.8.
B) grow seedlings under the normal condition, move in the disposable nutritive cube during 2 true leaves, inoculate processing during 3-4 sheet leaf.
C) dip in the toothpick tip and get bacterium liquid and stab 3-4 place (whenever stab 1 place dip in get bacterium liquid 1 time) in 3-4 leaf phase tomato cane phloem, the tomato after the processing was placed 1-2 days in greenhouse (temperature is 25-28 ℃), and then incidence is observed in dislocation field or greenhouse.
D) 15-30 days statistics incidences calculate the incidence of disease, estimate the resistance of tomato material to tomato yellow leaf curl virus: the material performance that the incidence of disease is high is susceptible, and the material performance that the incidence of disease is low is disease-resistant.
The invention has the beneficial effects as follows only needs simple toothpick to stab the resistance evaluation that cane has just been realized tomato yellow leaf curl virus.Avoided conventional Bemisia tabaci inoculation identification method on defective, need not to raise Bemisia tabaci, need not loaded down with trivial details operation, simultaneously this method also have qualification result accurately, advantages such as good reproducibility, efficient height.
Description of drawings
Embodiment
Example one:
903 bright red with the cooperation of tomato material, 001,002,747 be research object, identify its resistance by the toothpick inoculation method to tomato yellow leaf curl virus.
1, experiment is handled
A) the Agrobacterium inoculation that will contain PTYj01 contains in the YEP liquid nutrient medium of rifampin (Rifamycin) of the kanamycin (Kanamycin) of 50mg/ml and 50mg/ml, and 28 ℃ of 150rpm are cultured to OD
600Reach 0.6-0.8.
B) grow seedlings under the normal condition, move in the disposable nutritive cube during 2 true leaves, inoculate processing during 3-4 sheet leaf.
C) dip in the toothpick tip and get bacterium liquid and stab 3-4 place (whenever stab 1 place dip in get bacterium liquid 1 time) in 3-4 leaf phase tomato cane phloem, the tomato after the processing was placed 1-2 days in greenhouse (temperature is 25-28 ℃), and then incidence is observed in dislocation field or greenhouse.
D) 15-30 days statistics field incidences calculate the incidence of disease, estimate the resistance of tomato material to tomato yellow leaf curl virus.
2, experimental result
Tomato after the processing occurred symptom (leaf margin yellow, last volume) in back about 15 days in inoculation, and is consistent with field tomato yellow leaf curl diseased plant symptom, the 20-25 days statistics incidences of disease:
Cooperate 903 bright red: 100% (30/30);
001：100％(24/24)；
002：100％(19/19)；
747：0％(0/22)；
As can be seen in 4 parts of tomato materials, cooperate 903 bright red, 001,002 all morbidities (incidence of disease is 100%) by the incidence of disease, be susceptible material; And 747 do not have a morbidity (incidence of disease is 0%), is disease-resistant material.
Claims (6)
1. one kind is utilized the toothpick inoculation method to identify the method for tomato to the tomato yellow leaf curl virus resistance, may further comprise the steps:
(1) the Agrobacterium inoculation that will contain PTYj01 contains in the YEP liquid nutrient medium of rifampin (Rifamycin) of the kanamycin (Kanamyein) of 50mg/ml and 50mg/ml, and 28 ℃ of 150rpm are cultured to OD
600Reach 0.6-0.8.
(2) grow seedlings under the normal condition, move in the disposable nutritive cube during 2 true leaves, inoculate processing during 3-4 sheet leaf.
(3) dip in the toothpick tip and get bacterium liquid and stab 3-4 place (whenever stab 1 place dip in get bacterium liquid 1 time) in 3-4 leaf phase tomato cane phloem, the tomato after the processing was placed 1-2 days in greenhouse (temperature is 25-28 ℃), and then incidence is observed in dislocation field or greenhouse.
(4) 15-30 days statistics incidences calculate the incidence of disease, estimate the resistance of tomato material to tomato yellow leaf curl virus: the material performance that the incidence of disease is high is susceptible, and the material performance that the incidence of disease is low is disease-resistant.
2. the method for tomato yellow leaf curl virus resistance is identified in toothpick inoculation according to claim 1, it is characterized in that the greenhouse temperature that place tomato inoculation and inoculation back is 25-28 ℃.
3. the method for tomato yellow leaf curl virus resistance is identified in toothpick inoculation according to claim 1, it is characterized in that the fate that place in the greenhouse tomato inoculation back is 1-2 days.
4. the method for tomato yellow leaf curl virus resistance is identified in toothpick according to claim 1 inoculation, and the instrument that it is characterized in that inoculating usefulness is a toothpick.
5. the method for tomato yellow leaf curl virus resistance is identified in toothpick inoculation according to claim 1, it is characterized in that inoculating the method for usefulness for stabbing.
6. the method for tomato yellow leaf curl virus resistance is identified in toothpick inoculation according to claim 1, and it is characterized in that inoculating the virus of identifying is tomato yellow leaf curl virus.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010102512203A CN101946607A (en) | 2010-08-12 | 2010-08-12 | Method for identifying resistance of tomato to tomato yellow leaf curl virus by toothpick inoculation method |
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| CN2010102512203A CN101946607A (en) | 2010-08-12 | 2010-08-12 | Method for identifying resistance of tomato to tomato yellow leaf curl virus by toothpick inoculation method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220437A (en) * | 2011-04-27 | 2011-10-19 | 贵州省烟草科学研究所 | Method for identifying viral disease resistance of tobacco through partial inoculation |
| CN102640642A (en) * | 2012-04-25 | 2012-08-22 | 浙江大学 | Method for pierce-inoculating geminivirus infectious clone-containing solid colony to plant, and application thereof |
| CN103026966A (en) * | 2012-12-17 | 2013-04-10 | 天津市农业生物技术研究中心 | Method for identifying tomato yellow leaf curl virus resistance by utilizing detached leaf |
| CN107410218A (en) * | 2017-05-19 | 2017-12-01 | 湖南省植物保护研究所 | A kind of rearing method of viral vector insect Bemisia tabaci |
-
2010
- 2010-08-12 CN CN2010102512203A patent/CN101946607A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220437A (en) * | 2011-04-27 | 2011-10-19 | 贵州省烟草科学研究所 | Method for identifying viral disease resistance of tobacco through partial inoculation |
| CN102220437B (en) * | 2011-04-27 | 2014-04-23 | 贵州省烟草科学研究所 | Method for identifying viral disease resistance of tobacco through partial inoculation |
| CN102640642A (en) * | 2012-04-25 | 2012-08-22 | 浙江大学 | Method for pierce-inoculating geminivirus infectious clone-containing solid colony to plant, and application thereof |
| CN102640642B (en) * | 2012-04-25 | 2013-11-06 | 浙江大学 | Method for pierce-inoculating geminivirus infectious clone-containing solid colony to plant, and application thereof |
| CN103026966A (en) * | 2012-12-17 | 2013-04-10 | 天津市农业生物技术研究中心 | Method for identifying tomato yellow leaf curl virus resistance by utilizing detached leaf |
| CN103026966B (en) * | 2012-12-17 | 2013-12-11 | 天津市农业生物技术研究中心 | Method for identifying tomato yellow leaf curl virus resistance by utilizing detached leaf |
| CN107410218A (en) * | 2017-05-19 | 2017-12-01 | 湖南省植物保护研究所 | A kind of rearing method of viral vector insect Bemisia tabaci |
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Application publication date: 20110119 |