CN101948810A - Method for inoculating tomato yellow leaf curl virus by using toothpick puncture method - Google Patents
Method for inoculating tomato yellow leaf curl virus by using toothpick puncture method Download PDFInfo
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- CN101948810A CN101948810A CN2010102512330A CN201010251233A CN101948810A CN 101948810 A CN101948810 A CN 101948810A CN 2010102512330 A CN2010102512330 A CN 2010102512330A CN 201010251233 A CN201010251233 A CN 201010251233A CN 101948810 A CN101948810 A CN 101948810A
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Abstract
The invention provides a method for quickly inoculating tomato yellow leaf curl virus, and belongs to the field of plant protection. Quick inoculation of the tomato yellow leaf curl virus is realized by a toothpick puncture method by using constructed infective clone. Compared with the conventional bemisia tabaci inoculation method, the method has the advantages that the bemisia tabaci does not need to be bred and the influence of the bemisia tabaci on the virus spreading efficiency is avoided; and compared with the inoculation methods such as grafting, gene gun bombardment and the like reported in recent years, the method has the characteristics of simple operation, high efficiency, high repeatability, low cost and the like and is a simple, economic and effective inoculation method.
Description
Technical field
The present invention relates to a kind of inoculation method of tomato yellow leaf curl virus, 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 Sea 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 financial 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.
Because tomato yellow leaf curl virus is the louse-borne virus of a kind of tobacco powder, therefore conventional biological experiment usually adopts the Bemisia tabaci inoculation method to study tomato yellow leaf curl virus.But owing to need Bemisia tabaci to realize passing poison in this method operating process, therefore no matter be indoor or at field experiment, all there are some problems that are difficult to overcome in this method: 1, the inoculation worm of Bemisia tabaci amount 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, inoculation efficient is exerted an influence, and then the accuracy of experimental 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 result.
Except the Bemisia tabaci inoculation method, also has some other inoculation method, as Agrobacterium injection inoculation method, graft inoculation method, particle gun bombardment inoculation method (biolistic inoculation), but these inoculation methods also exist complex operation, cost higher, be difficult to be applicable to problem such as great amount of samples operation.
In order to solve the problem that exists on the inoculation methods such as Bemisia tabaci, we have formulated the method that a kind of toothpick is stabbed inoculation, this method is easy and simple to handle, good reproducibility, controllability is good, efficient is high, cost is low, the result is accurate, for research tomato yellowing tomato yellow leaf curl China virus provides a kind of novel method.
Summary of the invention
The present invention has overcome shortcoming of the prior art, for the inoculation of tomato yellow leaf curl virus provides a kind of easy, economy, effective means.
1, the material of inoculation usefulness:
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 substratum and antibiotic preparation are with reference to " molecular cloning experiment guide (third edition) ".
2, inoculation treatment process
A) the Agrobacterium inoculation that will contain PTYj01 contains the kantlex of 50mg/ml (Kanamycin, Km) and in the YEP liquid nutrient medium of the Rifampin (Rifamycin) of 50mg/ml, 28 ℃ of 150rpm is cultured to OD
600Reach 0.6-0.8.
B) (temperature is 25-28 ℃) uses hole plate seedling growth in the greenhouse, moves in the disposable nutrition pot during 2 true leaves, inoculates 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 is placed in greenhouse (temperature is 25-28 ℃), observes incidence.
The invention has the beneficial effects as follows avoided conventional Bemisia tabaci inoculation method on defective, need not to raise Bemisia tabaci, need not loaded down with trivial details operation, as long as stab the inoculation that cane just can be realized tomato yellow leaf curl virus by simple toothpick.
Description of drawings
Accompanying drawing is that toothpick is stabbed method inoculating tomato yellow leaf curl virus synoptic diagram
Embodiment
Example one:
To cooperate 903 big Fructus Lycopersici esculentis is material, stabs the method inoculating tomato yellow leaf curl virus by toothpick.
1, experiment is handled
A) the Agrobacterium inoculation that will contain PTYj01 contains the kantlex of 50mg/ml (Kanamycin, Km) and in the YEP liquid nutrient medium of the Rifampin (Rifamycin) of 50mg/ml, 28 ℃ of 150rpm is cultured to OD
600Reach 0.6-0.8.
B) (temperature is 25-28 ℃) uses hole plate seedling growth in the greenhouse, moves in the disposable nutrition pot during 2 true leaves, inoculates 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, inject tomato 30 strains altogether, the tomato after the processing is placed in greenhouse (temperature is 25-28 ℃), observes incidence.
2, experimental result
Tomato after the processing occurred symptom in back 13 days in inoculation, and 16 days symptoms are typical case's (30 tomatoes all fall ill) very, showed tangible leaf margin yellow, went up symptom (symptoms such as blade diminishes, plant stunts appear in the later stage) such as volume.Consistent with field tomato yellow leaf curl diseased plant symptom, illustrate that this inoculation method is effective.
Claims (5)
1. method of utilizing toothpick to stab the method inoculating tomato yellow leaf curl virus may further comprise the steps:
(1) the Agrobacterium inoculation that will contain PTYj01 contains the kantlex of 50mg/ml (Kanamycin, Km) and in the YEP liquid nutrient medium of the Rifampin (Rifamycin) of 50mg/ml, 28 ℃ of 150rpm is cultured to OD
600Reach 0.6-0.8.
(2) (temperature is 25-28 ℃) uses hole plate seedling growth in the greenhouse, moves in the disposable nutrition pot during 2 true leaves, inoculates 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 is placed in greenhouse (temperature is 25-28 ℃), observes incidence.
2. toothpick according to claim 1 is stabbed the method for method inoculating tomato yellow leaf curl virus, and the greenhouse temperature that it is characterized in that inoculating usefulness is 25-28 ℃.
3. toothpick according to claim 1 is stabbed the method for method inoculating tomato yellow leaf curl virus, and the instrument that it is characterized in that inoculating usefulness is a toothpick.
4. toothpick according to claim 1 is stabbed the method for method inoculating tomato yellow leaf curl virus, it is characterized in that inoculating the method for usefulness for stabbing.
5. toothpick according to claim 1 is stabbed the method for method inoculating tomato yellow leaf curl virus, it is characterized in that the virus of inoculating is tomato yellow leaf curl virus.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010102512330A CN101948810A (en) | 2010-08-12 | 2010-08-12 | Method for inoculating tomato yellow leaf curl virus by using toothpick puncture method |
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| CN2010102512330A CN101948810A (en) | 2010-08-12 | 2010-08-12 | Method for inoculating tomato yellow leaf curl virus by using toothpick puncture method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2010
- 2010-08-12 CN CN2010102512330A patent/CN101948810A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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Open date: 20110119 |