CN102640642B - Method for pierce-inoculating geminivirus infectious clone-containing solid colony to plant, and application thereof - Google Patents

Abstract

The invention discloses a method for pricking and inoculating plants with a solid bacterium colony containing geminivirus infective clones and an application thereof. Spread Agrobacterium EHA105 containing geminivirus-infectious clones on YEP solid medium containing 100mg/l kanamycin and 50mg/l rifampicin and culture them at 28°C to form colonies, pick the colonies with toothpicks, and remove them from the roots At a distance of 1-2 cm, the phloem of the plants above the 3-leaf stage was pricked at 3-4 points, and the inoculated plants were cultivated in the greenhouse, and the symptoms of the virus were observed. The inoculation method of this invention patent does not need to raise Bemisia tabaci, has high inoculation efficiency, strong repeatability, simple operation, fast and easy large-scale promotion. The invention patent is applicable to 16 serious geminiviruses in my country, and the inoculated plants include Solanaceae, Cucurbitaceae and Convolvulaceae. The invention can be used for identification of geminivirus pathogenicity and virus gene function research, can also be used for determination of plant resistance varieties against geminivirus, and serves for plant disease-resistant breeding.

Description

Method for pricking and inoculating plants with a solid colony containing an infectious clone of geminivirus and its application

technical field

The invention relates to the field of biotechnology, in particular to a method for pricking and inoculating plants with a solid bacterium colony containing geminivirus infective clones and an application thereof.

Background technique

Geminiviruses are a class of plant viruses that occur widely. With global warming, changes in agricultural farming systems, the rapid strengthening of international trade activities and the unprecedented expansion of insect vectors around the world, geminiviruses are widely occurring around the world. At present, geminivirus has been widely distributed and prevalent in Asia, central America, central and northern South Africa, and the Mediterranean, and is further expanding to surrounding uninfected areas. According to preliminary statistics, cotton, cassava, tomato and other crops in at least 40 countries have suffered devastating damage from such viruses.

Since the 1990s, with the global warming and the increase of trade activities, the harm of geminivirus in our country has become more and more serious. In my country, the disease has been found in tomato, pumpkin, papaya, tobacco and other crops in more than 20 provinces and cities such as Beijing, Shanghai, Yunnan, Hainan, Zhejiang, Guangdong, Guangxi, Shandong, Xinjiang, and Inner Mongolia. A tendency to spread northwards. In Yunnan, oriental tobacco leaf curl disease caused by geminivirus has a diseased plant rate of more than 70% in some fields and 15-30% in some flue-cured tobacco in southern Yunnan tobacco areas, causing serious losses in tobacco yield and quality. And on the papaya and tomato in Guangxi, the incidence of geminivirus is also very common, and the rate of diseased plants in severe areas is as high as 30-50%, and 100% of the plants in some fields are infected with the disease, resulting in heavy losses. Tomato leaf curl disease caused by geminiviruses has shown a tendency to break out in my country, and has been reported in Shanghai, Zhejiang, Jiangsu, Anhui, Shandong, Shanxi, Shaanxi, Henan, Hebei, Beijing, Guangdong, Guangxi, Yunnan, Inner Mongolia, Liaoning, Fujian 18 provinces and cities including Taiwan, Hainan, and Taiwan have caused serious damage, and the rate of diseased plants in severely affected fields has reached more than 95%, causing devastating losses. According to incomplete statistics, the annual occurrence area of tomato yellow leaf curl virus disease in my country exceeds 1 million mu, and the annual economic loss is at least 2 billion yuan. At present, the disease has gradually become an important restrictive factor in tomato production in my country.

In plant virus research, virus inoculation is an indispensable key link. Because most of the geminiviruses that harm crops and cause important economic damage belong to the genus of bean golden mosaic virus, the virus of this genus cannot be transmitted by the method of juice friction inoculation, and is mainly transmitted by whitefly under natural conditions, so the virus of this genus is also Known as whitefly-borne geminivirus. For a long time, the research on geminivirus has mostly used the method of bemisia tabaci transmission and inoculation, but this method requires bemisia tabaci to complete the virus transmission, so there are many problems that are difficult to overcome: 1. Bemisia tabaci are difficult to raise; 2. Whether the whitefly is carrying the virus, whether the virus is pure and single, and the detection of the virus-carrying rate of the whitefly is difficult, etc., resulting in that the source of the virus in each test cannot be unified; Control; 4. The transmission efficiency of Bemisia tabaci is even more difficult to control, which affects the accuracy of the experimental results; 5. Because Bemisia tabaci can transmit a variety of geminiviruses and other diseases at the same time, the inoculation results are often disturbing. The recently developed Agrobacterium liquid injection method needs to use the inoculation buffer for post-processing the bacterial liquid, the steps are cumbersome, the dependence on the instrument is strong, the operation is inconvenient, and the experimental efficiency is reduced. In addition, the syringes and excess bacterial fluid consumed during the experiment caused environmental pollution, which was not conducive to environmental protection, and the injection inoculation method was poorly operable for plants with thick stems, which affected the efficiency of geminivirus inoculation.

In view of the defects in the methods of whitefly transmission inoculation and Agrobacterium liquid injection inoculation, the patent of the present invention provides a method of inoculation based on solid colonies of invasive clones, which is suitable for 16 species of plant twins that have seriously occurred in my country. Virus inoculation, the inoculation efficiency is as high as 100%, easy to operate, strong repeatability, good controllability, fast and effective, and easy to learn, does not rely on instruments, and is conducive to large-scale promotion and application in production practice. It provides a new method for the study of disease resistance determination and breeding of resistant plant materials.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for pricking and inoculating plants with a solid colony containing geminivirus infectious clones and its application.

The method for pricking and inoculating plants with solid colonies containing geminivirus infectious clones comprises the steps of:

1) Inoculate the source of the virus:

Collect geminivirus samples from the field and extract total genomic DNA; design geminivirus-specific primers, PCR amplify the full genome sequence of the virus, and clone the amplified product into T-Vector; perform PCR screening and sequence determination on the cloned product; use restrictions The 1.2-2 repeated sequences of the full-length viral genome were constructed on the improved plant expression vector pBinplus by the method of enzyme digestion, and the infectious clones with geminiviruses were obtained; the infectious clones of geminiviruses were passed through conventional triparental mating or electric shock method to introduce it into the Agrobacterium cell EHA105 to obtain the source of inoculation virus;

The geminiviruses are Chinese papaya leaf curl virus, Chinese tomato yellow leaf curl virus, Thai tomato yellow leaf curl virus, Chinese tomato leaf curl virus, Taiwan tomato leaf curl virus, Guangxi tomato leaf curl virus, and Chinese Shenghong virus. Thistle Yellow Vein Virus, China Daqing Golden Mosaic Virus, Jiangsu Daqing Golden Mosaic Virus, Poinsettia Leaf Curl Virus, Guangdong Saikui Leaf Curl Virus, Tobacco Stem Curl Virus, Yunnan Saikui Yellow Vein Virus, Saikui Yellow Vein Virus , Pseudohorax leaf curl virus, Yunnan tobacco leaf curl virus;

2) Cultivation of colonies on solid plate of poison source:

Inoculate Agrobacterium EHA105 containing geminivirus infectious clones frozen in the refrigerator into YEP solid medium containing 100 mg/l kanamycin and 50 mg/l rifampicin, streak inoculate or spread evenly on the plate, Incubate at 28°C for 24-48 hours until the colonies grow well or cover the entire plate;

3) Seedling cultivation:

The temperature is 25-28°C, the relative humidity is 75%, and the photoperiod is 16L: grow seedlings in an 8D greenhouse or plant incubator, transplant at the stage of 2 true leaves, and inoculate the virus at the stage of 3-4 true leaves; seedlings Including Solanaceae plants: tomato, pepper, eggplant, ordinary tobacco, benthamiana, heart leaf tobacco, Sansheng tobacco, Shanxi tobacco; Cucurbitaceae plants: cucumber, pumpkin, wax gourd, loofah, gourd; Convolvulaceae plants: dwarf morning glory.

4) Toothpick prick inoculation:

Use a toothpick to pick up the cultured solid plate colony, pierce the stem phloem of the plant at 1-2 cm away from the root at 3-4 points, and grow the inoculated plant in a greenhouse or a plant incubator under the same conditions as the seedling cultivation;

5) Symptom observation and incidence record and resistance analysis:

Record the symptoms of plant disease one week after inoculation, and count the disease in 15-30 days, calculate the disease rate, and evaluate the pathogenicity of the virus to different plants or the resistance of plant materials to geminiviruses: plants with high disease rates are susceptible, and the disease rate Plants with low or no disease are resistant to the disease.

The method of pricking and inoculating plants with solid colonies containing geminivirus infectious clones is used for the determination of pathogenicity of geminiviruses, the study of virus gene functions, the determination of resistant varieties against geminiviruses and the service of disease-resistant breeding.

The invention realizes geminivirus inoculation by using solid colonies to puncture plant stems with toothpicks. It avoids the defects of conventional whitefly inoculation or Agrobacterium inoculation method, does not need to raise whitefly, does not need cumbersome operations, has low dependence on instruments, and has little environmental pollution. At the same time, the method has good repeatability, The results are accurate, fast and efficient, easy to learn, and conducive to large-scale promotion and other advantages. The invention is suitable for the inoculation of 16 serious geminiviruses in my country, and the inoculated plants include Solanaceae, Cucurbitaceae and Convolvulaceae plants.

Description of drawings

Fig. 1 is the plate colony of streak culture;

Fig. 2 is that toothpick picks plate colony;

Fig. 3 is the plant seedling suitable for inoculation;

Fig. 4 is inoculation tool---toothpick;

Fig. 5 is plant inoculation site;

Figure 6 is the symptoms produced by ToLCTWV alone or co-inoculated with TYLCCNB;

Figure 7 Statistics of disease index after tomato inoculation with virus.

Detailed ways

The method for pricking and inoculating plants with solid colonies containing geminivirus infectious clones comprises the steps of:

1) Inoculate the source of the virus:

Collect geminivirus samples from the field and extract total genomic DNA; design geminivirus-specific primers, PCR amplify the full genome sequence of the virus, and clone the amplified product into T-Vector; perform PCR screening and sequence determination on the cloned product; use restrictions The 1.2-2 repeated sequences of the full-length viral genome were constructed on the improved plant expression vector pBinplus by the method of enzyme digestion, and the infectious clones with geminiviruses were obtained; the infectious clones of geminiviruses were passed through conventional triparental mating or electric shock method to introduce it into the Agrobacterium cell EHA105 to obtain the source of inoculation virus;

The geminiviruses are Chinese papaya leaf curl virus, Chinese tomato yellow leaf curl virus, Thai tomato yellow leaf curl virus, Chinese tomato leaf curl virus, Taiwan tomato leaf curl virus, Guangxi tomato leaf curl virus, and Chinese Shenghong virus. Thistle Yellow Vein Virus, China Daqing Golden Mosaic Virus, Jiangsu Daqing Golden Mosaic Virus, Poinsettia Leaf Curl Virus, Guangdong Saikui Leaf Curl Virus, Tobacco Stem Curl Virus, Yunnan Saikui Yellow Vein Virus, Saikui Yellow Vein Virus , Pseudohorax leaf curl virus, Yunnan tobacco leaf curl virus;

2) Cultivation of colonies on solid plate of poison source:

Inoculate Agrobacterium EHA105 containing geminivirus infectious clones frozen in the refrigerator into YEP solid medium containing 100 mg/l kanamycin and 50 mg/l rifampicin, streak inoculate or spread evenly on the plate, Incubate at 28°C for 24-48 hours until the colonies grow well or cover the entire plate;

3) Seedling cultivation:

The temperature is 25-28°C, the relative humidity is 75%, and the photoperiod is 16L: grow seedlings in an 8D greenhouse or plant incubator, transplant at the stage of 2 true leaves, and inoculate the virus at the stage of 3-4 true leaves; seedlings Including Solanaceae plants: tomato, pepper, eggplant, ordinary tobacco, benthamiana, heart leaf tobacco, Sansheng tobacco, Shanxi tobacco; Cucurbitaceae plants: cucumber, pumpkin, wax gourd, loofah, gourd; Convolvulaceae plants: dwarf morning glory

4) Toothpick prick inoculation:

Use a toothpick to pick up the cultured solid plate colony, pierce the stem phloem of the plant at 1-2 cm away from the root at 3-4 points, and grow the inoculated plant in a greenhouse or a plant incubator under the same conditions as the seedling cultivation;

5) Symptom observation and incidence record and resistance analysis:

Record the symptoms of plant disease one week after inoculation, and count the disease in 15-30 days, calculate the disease rate, and evaluate the pathogenicity of the virus to different plants or the resistance of plant materials to geminiviruses: plants with high disease rates are susceptible, and the disease rate Plants with low or no disease are resistant to the disease.

The method of pricking and inoculating plants with solid colonies containing geminivirus infectious clones is used for the determination of pathogenicity of geminiviruses, the study of virus gene functions, the determination of resistant varieties against geminiviruses and the service of disease-resistant breeding.

Example 1: Acquisition of Geminivirus Infectious Clone Inoculation Virus Source

Collect geminivirus samples from the field and extract total genomic DNA; design geminivirus-specific primers, PCR amplify the full genome sequence of the virus, and clone the amplified product into T-Vector; perform PCR screening and sequence determination on the cloned product; use restrictions The 1.2-2 repeated sequences of the full length of the viral genome were constructed on the improved plant expression vector pBinplus by the method of restriction enzyme digestion, and the infectious clones with geminiviruses were obtained; It was introduced into the Agrobacterium cell EHA105 by electric shock to obtain the source of inoculated virus.

The method that above-mentioned inoculation virus source obtains can specifically refer to the thesis published by the patent inventor and the graduation thesis of the guidance graduate student (Chinese papaya leaf curl virus source (specific method with reference to Journal of Zhejiang University-Science B 11 (2): 109- 114, 2010), source of tomato yellow leaf curl virus in China (refer to Journal of Virology 78(24):13966-13974, 2004 for specific methods), source of tomato yellow leaf curl virus in Thailand (refer to Virus Genes 38( 2): 323-328, 2009), Tomato Leaf Curl Virus Source in China (refer to Dr. source (for specific methods, refer to European Journal of Plant Pathology 118(3):287-294, 2007), the source of Chinese Shenghongji yellow vein virus virus (for specific methods, refer to Phytopathology 97(4):405-411, 2007), China University The source of cyan-golden mosaic virus (for specific methods, refer to Virus Genes 41(2):250-259, 2010), the source of Jiangsu Daqing golden mosaic virus (for specific methods, refer to Virus Genes 41(2):250-259, 2010 ), the virus source of poinsettia leaf curl virus (for specific methods, refer to Archives of Virology 156(3):517-521, 2011), the source of Guangdong saikui leaf curl virus (for specific methods, refer to Plant Pathology 56(5):771-776, 2007), the source of tobacco bent stem virus (for specific methods, refer to Phytopathology 95(8):902-908, 2005), the source of Yunnan Saikui yellow vein virus (for specific methods, refer to Dr. source (refer to Applied and Environmental Microbiology 74(6):1909-1913, 2008 for specific methods), virus source of false vermilion leaf curl virus (refer to Archives of Virology 150 (11):2257-2270, 2005 for specific methods), Yunnan tobacco koji Infectious clone of leaf virus source (for specific methods refer to European Journal of Plant Pathology 115(4):369-375, 2006).

Example 2: Pathogenicity determination of geminivirus and research on virus gene function (taking Taiwan tomato leaf curl virus as an example)

The Taiwan tomato leaf curl virus (Tomato leaf curl Taiwan virus, ToLCTWV) invasive clone Agrobacterium was inoculated into YEP liquid medium containing 100 mg/l kanamycin and 50 mg/l rifampicin for activation, 28°C 200rpm Cultivate until the OD ₆₀₀ reaches 0.8-1.0. Take 50ul of the above-mentioned activated bacterial solution into the YEP solid medium containing 100mg/l kanamycin and 50mg/l rifampicin, spread evenly on the plate, and culture at 28°C for 24-48h, until the colony covers the whole flat. Pick plate colonies, and inoculate the original host tomato, and other hosts Nicotiana benthamiana, Petunia, and Nicotiana cordata by pricking with a toothpick. ToLCTWV showed strong pathogenicity on its original host tomato, and all inoculated plants showed symptoms. Tomato plants exhibited leaf roll-down 14 days after inoculation, consistent with their field symptoms (Fig. 6a). However, the virus did not produce obvious symptoms in Nicotiana benthamiana, Petunia and Nicotiana cordis (Fig. 6c, 6e, 6g). The total leaf DNA of the tomato plant system with symptoms was extracted as a template, and ToLCTWV-specific full-length primers were used for PCR amplification, and the target fragment of about 2.7 kb could be obtained, but in the non-symptomatic N. benthamiana, N. No target fragment could be amplified on the bovine plants, indicating that ToLCTWV alone could not infect Nicotiana benthamiana, Nicotiana cordis and Petunia.

The ToLCTWV infectious clone and the satellite DNAβ (TYLCCNB) infectious clone associated with Tomato yellow leaf curl China virus (TYLCCNV) were mixed and inoculated with tomato, Nicotiana benthamiana, Petunia and Nicotiana cordis. After mixed inoculation, the symptoms of leaf vein yellowing and twisting of the top can be produced on tomato (Fig. 6b), which cannot be observed when the virus is inoculated alone; severe leaf downturning, leaf vein yellowing and leaf back surface can be produced on Nicotiana benthamiana Auricles appeared (Fig. 6d); on petunias there were also serious underturned leaves with auricles on the back (Fig. 6f); on the heart leaf tobacco there were also underturned leaves but no yellow veins and auricles (Fig. 6h). 100% of the above mixed inoculated plants showed symptoms. The total DNA of the diseased plants was extracted for PCR amplification, and the specific bands of ToLCTWV and TYLCCNB could be amplified respectively. This shows that ToLCTWV can indeed interact with TYLCCNB, which not only produces more severe symptoms, but also expands its host range.

Embodiment 3: the determination of the resistance variety of plant anti-geminivirus

Taking tomato varieties Zhe A, Zhe B, Zhe C and Ruby as the research objects, the phloem inoculation of plate colony toothpick was pricked to identify the resistance of each tomato variety to Chinese papaya leaf curl virus, tomato yellow leaf curl virus, and Chinese tomato yellow leaf curl virus. Leaf Curl Virus, Thai Tomato Leaf Curl Virus, and Taiwan Tomato Leaf Curl Virus.

1. Experimental processing

a) Inoculate Agrobacterium EHA105 containing infectious clones of Chinese papaya leaf curl virus, tomato yellow leaf curl virus, Chinese tomato yellow leaf curl virus, Thai tomato yellow leaf curl virus, and Taiwan tomato leaf curl virus containing 100mg/l kanamycin and 50mg/l rifampicin YEP liquid medium for activation, cultured at 28°C and 200rpm until OD ₆₀₀ reached 0.8-1.0.

Take 50ul of the above-mentioned activated bacterial solution into the YEP solid medium containing 100mg/l kanamycin and 50mg/l rifampicin, spread evenly on the plate, and culture at 28°C for 24-48h, until the colony covers the whole flat.

Tomato cultivars Zhe A, Zhe B, Zhe C, and Ruby were grown in a greenhouse at 25°C, moved to a disposable nutrient bowl at the stage of 2 true leaves, and inoculated at the stage of 3-4 true leaves.

Pick up cultured plate colonies with a toothpick, prick 3-4 spots on the phloem of tomato stems at the true leaf stage, and place the inoculated tomatoes in a greenhouse (temperature 20-30°C) to observe the disease.

Record the symptoms of plant disease one week after inoculation, count the disease situation 15-30 days, calculate the disease rate, and evaluate the resistance of different tomato varieties to Chinese papaya leaf curl virus, tomato yellow leaf curl virus, Chinese tomato yellow leaf curl virus, and Thai tomato yellow leaf curl virus. Resistance to Leaf Curl Virus, Taiwan Tomato Leaf Curl Virus. the

2. Experimental results

Symptoms appeared in the inoculated tomatoes around 15 days. Tomatoes inoculated with Chinese papaya leaf curl virus showed symptoms such as leaf curling, twisting, shrinkage, dwarfing, and incomplete new leaves stretching; tomatoes inoculated with tomato yellow leaf curl virus showed leaf shrinkage, thinning, and edge Rolling, protruding leaf veins, dwarfing of plants, incomplete stretching of new leaves and other symptoms; tomatoes inoculated with Chinese tomato yellow leaf curl virus showed severe leaf curling, protruding and thickening of veins on the back of leaves, yellow veins, dwarfing of plants, Symptoms such as ear bumps appeared in the later stage; tomatoes inoculated with Thai tomato yellow leaf curl virus showed symptoms such as leaf curling, shrinkage, and plant dwarfing; tomatoes inoculated with Taiwan tomato leaf curl virus showed leaf curling, edge yellowing, and leaf veins outside. Symptoms such as protrusion, thickening, and thinning of new leaves. Symptoms are consistent with those of diseased tomato plants in the field. The 30-day statistical incidence rate is shown in Table 1, and the disease index is shown in Figure 7.

Table 1 Determination of resistant varieties of tomato resistant to geminivirus

Calculate the disease index based on the incidence rate and severity of the disease (the disease index is a comprehensive index that fully considers the incidence and severity, and its calculation formula is: disease index = 100 × ∑ (number of diseased leaves at each level × representative value at each level) / ( The total number of leaves under investigation × the representative value of the highest level) ).

Inferred from the incidence rate and disease index, among the four tomato varieties, Ruby was highly susceptible to the above five geminiviruses and was a susceptible variety; Zhe A was susceptible to Chinese tomato yellow leaf curl virus, Thai tomato yellow leaf curl virus, Taiwan tomato leaf curl virus showed complete resistance, Chinese papaya leaf curl virus and tomato yellow leaf curl virus showed high resistance; Zhejiang B showed tomato yellow leaf curl virus and Thailand tomato yellow leaf curl virus showed resistance Complete resistance, Chinese papaya leaf curl virus, Chinese tomato yellow leaf curl virus, Taiwan tomato leaf curl virus showed high resistance; Zhejiang C was resistant to Chinese papaya leaf curl virus, tomato yellow leaf curl virus, Chinese tomato Yellow leaf curl virus, Thai tomato yellow leaf curl virus, and Taiwan tomato leaf curl virus all showed high resistance; Zhe A, B, and C were three resistant varieties with different resistances.

Claims (2)

1. the method with the solid bacterium colony thorn inoculation plant that contains the geminivirus infection infectious clone, is characterized in that comprising the steps:

1) inoculate malicious source:

Gather the sample of geminivirus infection from the field, extract genome DNA; Design geminivirus infection Auele Specific Primer, the pcr amplification virus genome complete sequence, extension amplification outcome is to T-Vector; Clone products is carried out PCR screening and sequencing; Utilize the method for restriction enzyme digestion that the 1.2-2 of a viral genome total length tumor-necrosis factor glycoproteins is building up on modified form plant expression vector pBinplus, obtain the infectious clone with geminivirus infection; The geminivirus infection infectious clone imports it in agrobatcerium cell EHA105 by the method for conventional triparental mating or electric shock, obtains to inoculate malicious source;

described geminivirus infection is Papaya leaf curl China virus, tomato yellow leaf curl china virus, Thailand's tomato yellow leaf curl virus, the Tomato in China curve leaf disease virus, Tomato leaf curl Taiwan virus, Guangxi tomato curve leaf disease virus, Ageratum yellow vein China virus, China's smalt golden mosaic virus, Jiangsu smalt golden mosaic virus, the poinsettia curve leaf disease virus, Guangdong match certain herbaceous plants with big flowers curve leaf disease virus, tobacco curly shoot virus, the Yunnan malvastrum yellow vein viruses, malvastrum yellow vein viruses, false horsewhip curve leaf disease virus, the Yunnan tobacco curve leaf disease virus,

2) cultivation of malicious source solid plate bacterium colony:

The Agrobacterium EHA105 that contains the geminivirus infection infectious clone that refrigerator is frozen is seeded in the YEP solid medium of Rifampin of the kantlex that contains 100mg/l and 50mg/l, streak inoculation or even coated plate, cultivate 24-48h for 28 ℃, grow good or cover with whole flat board to bacterium colony;

3) seedling culture:

Temperature is that 25～28 ℃, relative humidity are 75 %, photoperiod to be to grow seedlings in the greenhouse of 16L:8D or plant incubator, transplants during 2 leaf periods, carries out virus inoculation during 3-4 sheet leaf period; Seedling comprises plant of Solanaceae: tomato, capsicum, eggplant, common cigarette, Ben Shi cigarette, Nicotiana glutinosa, three lives cigarette, the western cigarette of coral; Cucurbitaceous plant: cucumber, pumpkin, wax gourd, sponge gourd, cucurbit; Convolvulaceous plant: petunia;

4) toothpick thorn inoculation:

With the cultured solid plate bacterium colony of toothpick picking, from root 1-2 centimeters thorn plant cane phloem 3-4 point, postvaccinal plant is incubation growth in the greenhouse identical with the seedling culture condition or plant incubator;

5) observation of symptoms and incidence record and Analysis of Resistance:

Record the plant disease symptom after inoculating a week, 15-30 days statistics incidences, calculate sickness rate, estimate virus to the virulencies of different plants or the vegetable material resistance to geminivirus infection: the plant performance that sickness rate is high is susceptible, and sickness rate is low or plant performance that do not fall ill is disease-resistant.

2. a use as claimed in claim 1 contains the application of method of the solid bacterium colony thorn inoculation plant of geminivirus infection infectious clone, it is characterized in that the Pathogenicity for geminivirus infection, the research of known viral gene function and the resistant variety of anti-geminivirus infection measure and the breeding for disease resistance service.

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