CN101892201A

CN101892201A - Method for inoculating tomato yellow leaf curl virus by using injector to inject

Info

Publication number: CN101892201A
Application number: CN201010207066XA
Authority: CN
Inventors: 季英华; 周益军; 赵统敏; 余文贵; 程兆榜; 周彤; 孙海霞; 周晓伟
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2010-06-23
Filing date: 2010-06-23
Publication date: 2010-11-24

Abstract

The invention provides a method for fast inoculating a tomato yellow leaf curl virus and belongs to the field of plant protection. The tomato yellow leaf curl virus is fast inoculated by a stem injection method by using built infectious clone PTYj01. The tomato yellow leaf curl virus is frequently inoculated by the conventional bemisia tabaci gennadius inoculation method in which the bemisia tabaci gennadius needs to be specially fed, and the inoculation efficiency is influenced by the virus transmitting efficiency of the desmone insect and is interfered by the virus transmitting of other bemisia tabaci. The method effectively overcomes the defects of the conventional bemisia tabaci gennadius inoculation method, and has the advantages of no need of feeding the bemisia tabaci gennadius, simple operation, high efficiency, high repeatability, and high controllability, and is a simple and convenient and effective inoculation method.

Description

A kind of method of utilizing the injector to inject inoculating tomato yellow leaf curl virus

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 Bemisia tabaci inoculation method rice research 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 have some other inoculation method, as graft inoculation method, gene gun inoculation method (biolisticinoculation), but also there is complex operation in these inoculation methods, and cost is than problems such as height.

In order to solve the problem that exists on the inoculation methods such as Bemisia tabaci, we have formulated a kind of method based on the infectious clone injection inoculation, and this method is easy and simple to handle, good reproducibility, controllability is good, the efficient height, the result is accurate, for studying and utilizing the tomato yellowing tomato yellow leaf curl China virus that a kind of novel method is provided.

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 quick, easy, effective means.

1, the material of inoculation usefulness:

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 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 contain 50mg/ml kantlex (Kanamycin, Km) and the Streptomycin sulphate of 50mg/ml (Streptomycin, in YEP liquid nutrient medium SM), 28 ℃ of 150rpm are cultured to OD ₆₀₀Reach 0.6-0.8.

B) (temperature is 25-28 ℃) uses hole plate seedling growth in glasshouse, moves in the disposable nutrition pot during 2 true leaves, inoculates 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 after the injection treatment is placed in glasshouse (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 just can realize the inoculation of tomato yellow leaf curl virus by the injection of simple cane.

Description of drawings

Accompanying drawing is a tomato yellow leaf curl virus cane injection inoculation synoptic diagram

Embodiment

Example one:

With tomato variety east is material holy No. 1, by cane injecting method inoculating tomato yellow leaf curl virus.

1, experiment is handled

A) the Agrobacterium inoculation that will contain PTYj01 contain 50gm/ml kantlex (Kanamycin, Km) and the Streptomycin sulphate of 50mg/ml (Streptomycin, in YEP liquid nutrient medium SM), 28 ℃ of 150rpm are cultured to OD ₆₀₀Reach 0.6-0.8.

B) in glasshouse, use hole plate seedling growth, move in the disposable nutrition pot 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, inject tomato 21 strains altogether, the tomato after the injection treatment (25-28 ℃) in glasshouse is placed, and observes its incidence.

2, experimental result

Tomato after the processing occurred symptom in back 12 days in inoculation, and 16 days symptoms are typical case's (21 tomatoes all fall ill) very, shows tangible leaf margin yellow.Thereafter the diseased plant symptom continues to increase the weight of, and the performance blade diminishes, volume, plant symptom such as stunt on the leaf margin.Consistent with field tomato yellow leaf curl diseased plant symptom, illustrate that this inoculation method is effective.

Claims

1. method of utilizing the injector to inject inoculating tomato yellow leaf curl virus may further comprise the steps:

(1) the Agrobacterium inoculation that will contain PTYj01 contain 50mg/ml kantlex (Kanamycin, Km) and the Streptomycin sulphate of 50mg/ml (Streptomycin, in YEP liquid nutrient medium SM), 28 ℃ of 150rpm are cultured to OD ₆₀₀Reach 0.6-0.8;

(2) (temperature is 25-28 ℃) uses hole plate seedling growth in glasshouse, moves in the disposable nutrition pot during 2 true leaves, inoculates processing during 3-4 sheet leaf;

(3) get bacterium liquid in 3-4 leaf phase tomato stem phloem injection 3-4 point with the 1ml syringe, the tomato after the injection treatment is placed in glasshouse (temperature is 25-28 ℃), observes incidence.

2. the method for injection inoculation tomato yellow leaf curl virus according to claim 1 is characterized in that growing seedlings and the glasshouse temperature of inoculating usefulness is 25-28 ℃.

3. the method for injection inoculation tomato yellow leaf curl virus according to claim 1, the material that it is characterized in that inoculating usefulness is PTYj01.

4. the method for injection inoculation tomato yellow leaf curl virus according to claim 1 is characterized in that the virus of inoculating is tomato yellow leaf curl virus.