CN101884281A - Method for determining resistance of tomatoes to tomato yellow leaf curl viruses by injection inoculation - Google Patents
Method for determining resistance of tomatoes to tomato yellow leaf curl viruses by injection inoculation Download PDFInfo
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- CN101884281A CN101884281A CN2010102068852A CN201010206885A CN101884281A CN 101884281 A CN101884281 A CN 101884281A CN 2010102068852 A CN2010102068852 A CN 2010102068852A CN 201010206885 A CN201010206885 A CN 201010206885A CN 101884281 A CN101884281 A CN 101884281A
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Abstract
The invention discloses a quick, simple and effective tomato yellow leaf curl virus resistance determination method and belongs to the field of plant protection. Traditionally, the determination of the tomato yellow leaf curl virus resistance is performed by a Bemisia tabaci (Gennadius) inoculation determination method, while the Bemisia tabaci (Gennadius) inoculation determination method has difficulty in providing uniform and consistent inoculation pressure due to the influences (such as the number of the inoculated Bemisia tabaci (Gennadius), virus rate and feeding habit) of the Bemisia tabaci (Gennadius) and at the same time, suffers interference from other Bemisia tabaci (Gennadius) transmitted viruses, so the accuracy of the determination result is low. The agrobacterium rhizogenes injection inoculation method well solves the problem. The resistance of a tomato material to the tomato yellow leaf curl viruses can be determined by using constructed infectious clones and directly inoculating the tomato yellow leaf curl viruses by injection in stalks. The method avoids culturing Bemisia tabaci (Gennadius), ensures simple operation, high efficiency, high repeatability and high controllability, provides a relatively consistent pressure, obtains accurate result and is a simple and effective inoculation determination 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 of identifying based on the infectious clone injection inoculation, 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 (No. 50, Nanjing, Jiangsu Province Xiao Lingwei clock spirit street, disease chamber, Institute of Plant Protection, academy of agricultural sciences, Jiangsu Province, 210014), the syringe of inoculation usefulness adopts the disposable syringe (1ml) of standard, 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 sharp 50mg/ml of kanamycin (Kanamycin) of 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) get bacterium liquid in 3-4 leaf phase tomato cane phloem injection 3-4 point with the 1ml syringe, the tomato of injection treatment was placed 1-2 days in greenhouse (temperature is 23-30 ℃), 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, the material performance resistance that the incidence of disease is low.
Wherein said inoculation also can be 25-28 ℃ with 23-30 ℃ of greenhouse temperature.
The invention has the beneficial effects as follows the resistance evaluation that only needs simple cane injection inoculation just to realize 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
Accompanying drawing is to utilize injection inoculation method to identify the result (top be disease-resistant material, bottom be susceptible material) of tomato to the tomato yellow leaf curl virus resistance
Embodiment
Example one:
With tomato material east is research object holy No. 1,719,747,8849, identifies its resistance to tomato yellow leaf curl virus by injection inoculation.
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) get bacterium liquid in 3-4 leaf phase tomato cane phloem injection 3-4 point with the 1ml syringe, the tomato of injection treatment was placed 1-2 days in greenhouse (temperature is 23-30 ℃), 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:
Holy No. 1 of east: 100% (21/21);
719：100％(22/22)；
747：0％(0/22)；
8849：0％(0/19)；
As can be seen in 4 parts of tomato materials, holy No. 1, the 719 all morbidity (incidence of disease is 100%) in east is susceptible material by the incidence of disease; And 747,8849 do not have a morbidity (incidence of disease is 0%), is disease-resistant material.
Claims (4)
1. an injection inoculation is identified 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 (Kanamycin) 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) get bacterium liquid in 3-4 leaf phase tomato cane phloem injection 3-4 point with the 1ml syringe, the tomato of injection treatment was placed 1-2 days in greenhouse (temperature is 23-30 ℃), 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, the material performance resistance that the incidence of disease is low.
2. injection inoculation according to claim 1 is identified the method for tomato yellow leaf curl virus resistance, it is characterized in that the greenhouse temperature that place tomato inoculation and inoculation back is 23-30 ℃.
3. injection inoculation according to claim 1 is identified the method for tomato yellow leaf curl virus resistance, it is characterized in that the fate that place in the greenhouse tomato inoculation back is 1-2 days.
4. injection inoculation according to claim 1 is identified the method for tomato yellow leaf curl virus resistance, and it is characterized in that inoculating the virus of identifying is tomato yellow leaf curl virus.
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| CN2010102068852A CN101884281A (en) | 2010-06-23 | 2010-06-23 | Method for determining resistance of tomatoes to tomato yellow leaf curl viruses by injection inoculation |
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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 |
| CN102392080A (en) * | 2011-11-17 | 2012-03-28 | 天津市农业生物技术研究中心 | Method for identifying tomato yellow leaf curl virus resistance |
| 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 |
| CN102690776A (en) * | 2012-06-13 | 2012-09-26 | 浙江大学 | Agrobacterium strain carrying Yunnan tomato leaf curl virus infectious clones and application thereof |
| CN102948336A (en) * | 2012-11-12 | 2013-03-06 | 北京市农林科学院 | Method for inoculating tomatoes with tomato yellow leaf curl viruses |
| CN108841916A (en) * | 2018-07-30 | 2018-11-20 | 东北农业大学 | A kind of quick inoculation identification method of tomato stem rot root rot seedling stage |
| CN113125643A (en) * | 2021-04-02 | 2021-07-16 | 宁波市农业科学研究院 | Method for rapidly identifying tomato bacterial wilt seedling stage resistance by injection inoculation method |
-
2010
- 2010-06-23 CN CN2010102068852A patent/CN101884281A/en active Pending
Cited By (9)
| 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 |
| CN102392080A (en) * | 2011-11-17 | 2012-03-28 | 天津市农业生物技术研究中心 | Method for identifying tomato yellow leaf curl virus resistance |
| 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 |
| CN102690776A (en) * | 2012-06-13 | 2012-09-26 | 浙江大学 | Agrobacterium strain carrying Yunnan tomato leaf curl virus infectious clones and application thereof |
| CN102948336A (en) * | 2012-11-12 | 2013-03-06 | 北京市农林科学院 | Method for inoculating tomatoes with tomato yellow leaf curl viruses |
| CN108841916A (en) * | 2018-07-30 | 2018-11-20 | 东北农业大学 | A kind of quick inoculation identification method of tomato stem rot root rot seedling stage |
| CN113125643A (en) * | 2021-04-02 | 2021-07-16 | 宁波市农业科学研究院 | Method for rapidly identifying tomato bacterial wilt seedling stage resistance by injection inoculation method |
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Application publication date: 20101117 |