CN102640642A - Method for pierce-inoculating geminivirus infectious clone-containing solid colony to plant, and application thereof - Google Patents
Method for pierce-inoculating geminivirus infectious clone-containing solid colony to plant, and application thereof Download PDFInfo
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Abstract
The invention discloses a method for pierce-inoculating geminivirus infectious clone-containing solid colony to plant, and an application of the method. The method comprises the steps of coating geminivirus infectious clone-containing Agrobacterium tumefaciens EHA105 on YEP solid culture medium containing 100 mg/L kanamycin and 50 mg/L rifampicin; culturing into colony at 28 DEG C; picking colony with toothpicks; piercing 3-4 points on phloem of plant after three-leaf stage, at position 1-2 cm from root; culturing inoculated plant in a greenhouse; and observing virus symptom. The inoculation method provided by the invention has the advantages of no need for raising Bemisia tabaci, high inoculation efficiency and repeatability, simple and rapid operation, and suitability for large-scale popularization. The method provided by the invention is applicable to 16 kinds of geminivirus with severe breakout in our country. The inoculated plants are in families of Solanaceae, Cucurbitaceae and Convolvulaceae. The method can be used for pathogenicity identification of geminivirus and virus gene function research, anti-geminivirus plant variety determination and its breeding.
Description
Technical field
The present invention relates to biological technical field, relate in particular to method and application thereof that a kind of usefulness contains the solid bacterium colony thorn inoculation plant of geminivirus infection infectious clone.
Background technology
Geminivirus infection is one type of plant virus of extensively taking place, and along with the rapid reinforcement and the insect amboceptor of the change of global warming, agricultural tillage system, international trade activity are unprecedentedly being expanded all over the world, geminivirus infection extensively takes place in the world.At present geminivirus infection in the Asia, areas such as the central and north in middle part, America, South Africa and Mediterranean extensively distribute and popular, and just further to infected zone expansion of periphery.According to the preliminary statistics, crops such as the cotton of existing at least more than 40 countries, cassava, tomato are endangered by the destructiveness of this viroids.
Since the last century the nineties, along with the increase of global warming and trade activity, geminivirus infection is also more and more serious in the harm of China.China has found this disease on various crop such as the tomato of more than 20 provinces and cities such as Beijing, Shanghai, Yunnan, Hainan, Zhejiang, Guangdong, Guangxi, Shandong, Xinjiang, the Inner Mongol, pumpkin, papaya papaw, tobacco, and has year by year northwards the trend that constantly expansion spreads.In Yunnan, the piece diseased plant rate reaches more than 70% the Turkish tobaccos leaf curl that geminivirus infection causes in local field, and diseased plant rate reaches 15-30% on the Yan Qu part flue-cured tobacco of the southern regions of the Yunnan Province, causes the heavy losses of tobacco production quality.And on the papaya papaw and tomato in Guangxi, the geminivirus infection morbidity is also very general, and the serious regional diseased plant rate of falling ill is up to 30-50%, and field piece 100% plant that has catches an illness, and suffers heavy losses.The tomato leaf curl that is caused by geminivirus infection presents the gesture of comprehensively breaking out in China; In Shanghai, 18 provinces and cities such as Zhejiang, Jiangsu, Anhui, Shandong, Shanxi, Shaanxi, Henan, Hebei, Beijing, Guangdong, Guangxi, Yunnan, the Inner Mongol, Liaoning, Fujian, Hainan, Taiwan cause serious harm; Serious morbidity field piece diseased plant rate reaches more than 95%, has caused crushing loss.According to incompletely statistics, the year of tomato in China tomato yellow leaf curl China viral disease area takes place and surpass 1,000,000 mu, year economic loss at least 20 hundred million RMB.Present this disease has become tomato in China gradually and has produced important restraining factors.
In plant virus research, the virus inoculation key link that is absolutely necessary.Because damage to crops and cause the geminivirus infection of important economic damage to belong to bean golden mosaic virus mostly to belong to virus; This genus virus can not be propagated through the method for juice frictional inoculation; Mainly rely on Bemisia tabaci to propagate under field conditions (factors), therefore this genus virus is called whitefly-transmitted geminivirus again.Adopt Bemisia tabaci to pass the method for poison inoculation for the research of geminivirus infection more for a long time, pass poison but need Bemisia tabaci to accomplish in this method operating process, therefore exist and much be difficult to the problem that overcomes: 1, Bemisia tabaci is difficult raises; Whether 2 Bemisia tabaci carry virus, and whether entrained virus is pure single, and the band poison rate detection difficult of Bemisia tabaci etc. cause the malicious source of each test to unify; 3 Bemisia tabaci individual minimum, inoculation worm amount is also restive; 4, the biography toxic effect rate of Bemisia tabaci is restive especially, influences the accuracy of experimental result; 5, because Bemisia tabaci can be propagated multiple geminivirus infection and other diseases simultaneously, often interference is big therefore to inoculate the result.The Agrobacterium bacterium liquid injecting method that latest developments are got up be owing to need carry out post-processed to bacterium liquid with the inoculation buffer solution, complex steps, and strong to the dependence of instrument, operation inconvenience has reduced conventional efficient.In addition, syringe that expends in the experimentation and unnecessary bacterium liquid have caused environmental pollution, are unfavorable for environmental protection, and injection inoculation method has influenced geminivirus infection inoculation efficient for the thick and stiff plant poor operability of cane.
Pass the defective that exists on poison inoculation and the Agrobacterium bacterium liquid injection inoculation method to Bemisia tabaci, patent of the present invention provides a kind of method based on infectious clone solid colony inoculation, and this method is applicable to that serious 16 plant species geminivirus infections inoculation takes place in China; Inoculation efficient is up to 100%, and is easy and simple to handle, and repeatability is strong; Controllability is good; Fast effectively, and easy to learn, do not rely on instrument; In production practices, be beneficial to large-scale promotion application, for the pathogenic mensuration of various plants geminivirus infection and the research of seed selection resistance plant material provide a kind of new method.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, method and the application thereof of plant inoculated in the solid bacterium colony thorn that provides a kind of usefulness to contain the geminivirus infection infectious clone.
Method with the solid bacterium colony thorn inoculation plant that contains the geminivirus infection infectious clone comprises 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's product is carried out PCR screening and sequencing; Utilize the method for restriction enzyme digestion to be building up to the 1.2-2 of a viral genome total length repetitive sequence on the modified form plant expression vector pBinplus, obtain to have the infectious clone of geminivirus infection; The geminivirus infection infectious clone imports it among agrobatcerium cell EHA105 through the method for conventional triparental mating or electric shock, obtains the malicious source of inoculation;
Described geminivirus infection is Chinese papaya papaw curve leaf disease virus; Tomato yellow leaf curl china virus; Thailand's tomato yellow leaf curl virus; China's tomato curve leaf disease virus; Taiwan tomato curve leaf disease virus; Guangxi tomato curve leaf disease virus; China's Ageratum conyzoides yellow vein 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 culture medium of rifampin of the kanamycin that contains 100mg/l and 50mg/l; Streak inoculation or even coated plate; Cultivate 24-48h for 28 ℃, long good or cover with whole flat board to bacterium colony;
3) seedling culture:
Temperature is that 25～28 ℃, relative moisture are 75 %, photoperiod to be to grow seedlings in greenhouse or the plant incubator of 16L:8D, 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, coral west cigarette; 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 greenhouse identical with the seedling culture condition or plant incubator;
5) observation of symptoms and incidence record and resistance analysis:
Record plant disease symptom after one week of inoculation; 15-30 days statistics incidences; Calculate the incidence of disease, estimate virus to the pathogenicities of different plants or the vegetable material resistance to geminivirus infection: the plant performance that the incidence of disease is high is susceptible, and incidence of disease plant performance low or that do not fall ill is disease-resistant.
Being used for the pathogenic mensuration of geminivirus infection, the research of known viral gene function and the resistant variety of anti-geminivirus infection with the method for the solid bacterium colony thorn inoculation plant that contains the geminivirus infection infectious clone measures and the breeding for disease resistance service.
The present invention utilizes the solid bacterium colony just to realize the inoculation of geminivirus infection through toothpick thorn plant stem.Avoided the defective on conventional Bemisia tabaci inoculation or the Agrobacterium bacterium liquid inoculation method; Need not to raise Bemisia tabaci; Need not loaded down with trivial details operation; Dependence to instrument is low, and the pollution of environment is little, simultaneously this method also have good reproducibility, result accurately, rapidly and efficiently, easy to learn, be beneficial to advantages such as large-scale promotion.The present invention is applicable to that the inoculation of 16 kinds of serious geminivirus infections takes place in China, and the inoculation plant comprises Solanaceae, Curcurbitaceae, convolvulaceous plant.
Description of drawings
Fig. 1 is the flat-plate bacterial colony that line is cultivated;
Fig. 2 is a toothpick picking flat-plate bacterial colony;
Fig. 3 is the plantling that is fit to inoculation;
Fig. 4 is an inoculating tool---toothpick;
Fig. 5 is the plant inoculation position;
Fig. 6 be ToLCTWV separately or the symptom that produces with the TYLCCNB co-inoculation;
Disease index statistics behind Fig. 7 tomato virus inoculation.
Embodiment
Method with the solid bacterium colony thorn inoculation plant that contains the geminivirus infection infectious clone comprises 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's product is carried out PCR screening and sequencing; Utilize the method for restriction enzyme digestion to be building up to the 1.2-2 of a viral genome total length repetitive sequence on the modified form plant expression vector pBinplus, obtain to have the infectious clone of geminivirus infection; The geminivirus infection infectious clone imports it among agrobatcerium cell EHA105 through the method for conventional triparental mating or electric shock, obtains the malicious source of inoculation;
Described geminivirus infection is Chinese papaya papaw curve leaf disease virus; Tomato yellow leaf curl china virus; Thailand's tomato yellow leaf curl virus; China's tomato curve leaf disease virus; Taiwan tomato curve leaf disease virus; Guangxi tomato curve leaf disease virus; China's Ageratum conyzoides yellow vein 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 culture medium of rifampin of the kanamycin that contains 100mg/l and 50mg/l; Streak inoculation or even coated plate; Cultivate 24-48h for 28 ℃, long good or cover with whole flat board to bacterium colony;
3) seedling culture:
Temperature is that 25～28 ℃, relative moisture are 75 %, photoperiod to be to grow seedlings in greenhouse or the plant incubator of 16L:8D, 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, coral west cigarette; 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 greenhouse identical with the seedling culture condition or plant incubator;
5) observation of symptoms and incidence record and resistance analysis:
Record plant disease symptom after one week of inoculation; 15-30 days statistics incidences; Calculate the incidence of disease, estimate virus to the pathogenicities of different plants or the vegetable material resistance to geminivirus infection: the plant performance that the incidence of disease is high is susceptible, and incidence of disease plant performance low or that do not fall ill is disease-resistant.
Being used for the pathogenic mensuration of geminivirus infection, the research of known viral gene function and the resistant variety of anti-geminivirus infection with the method for the solid bacterium colony thorn inoculation plant that contains the geminivirus infection infectious clone measures and the breeding for disease resistance service.
Embodiment 1: the geminivirus infection infectious clone is inoculated the acquisition in 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's product is carried out PCR screening and sequencing; Utilize the method for restriction enzyme digestion to be building up to the 1.2-2 of a viral genome total length repetitive sequence on the modified form plant expression vector pBinplus, obtain to have the geminivirus infection infectious clone; The geminivirus infection infectious clone imports it among agrobatcerium cell EHA105 through the method for conventional triparental mating or electric shock, obtains the malicious source of inoculation.
Paper and the living thesis that directs study (Chinese papaya papaw curve leaf disease virus poison source (concrete grammar reference thereof that the method that above-mentioned inoculation poison source obtains specifically can be delivered with reference to patented invention person
Journal of Zhejiang University-Science B11 (2): 109-114,2010), tomato yellow leaf curl china virus poison source (concrete grammar reference
Journal of Virology78 (24): 13966-13974,2004), Thailand's tomato yellow leaf curl virus poison source (concrete grammar reference
Virus Genes38 (2): 323-328,2009), the malicious source (concrete grammar is with reference to the Guo Wei thesis for the doctorate) of Chinese tomato curve leaf disease virus, Taiwan tomato curve leaf disease virus poison source (concrete grammar is with reference to opening a brightness thesis for the doctorate), source (the concrete grammar reference of Guangxi tomato curve leaf disease virus poison
European Journal of Plant Pathology118 (3): 287-294,2007), Chinese Ageratum conyzoides yellow vein virus poison source (concrete grammar reference
Phytopathology97 (4): 405-411,2007), Chinese smalt golden mosaic virus poison source (concrete grammar reference
Virus Genes41 (2): 250-259,2010), Jiangsu smalt golden mosaic virus poison source (concrete grammar reference
Virus Genes41 (2): 250-259,2010), poinsettia curve leaf disease virus poison source (concrete grammar reference
Archives of Virology156 (3): 517-521,2011), Guangdong match certain herbaceous plants with big flowers curve leaf disease virus poison source (concrete grammar reference
Plant Pathology56 (5): 771-776,2007), tobacco curly shoot virus poison source (concrete grammar reference
Phytopathology95 (8): 902-908,2005), Yunnan malvastrum yellow vein viruses poison source (concrete grammar is with reference to the red thesis for the doctorate in river), source (the concrete grammar reference of malvastrum yellow vein viruses poison
Applied and Environmental Microbiology74 (6): 1909-1913,2008), false horsewhip curve leaf disease virus poison source (concrete grammar reference
Archives of Virology150 (11): 2257-2270,2005), Yunnan tobacco curve leaf disease virus poison source (concrete grammar reference
European Journal of Plant Pathology115 (4): 369-375,2006) infectious clone).
Embodiment 2: the pathogenic mensuration of geminivirus infection and the research of known viral gene function (is example with Taiwan tomato curve leaf disease virus)
With Taiwan tomato curve leaf disease virus (Tomato leaf curl Taiwan virus, ToLCTWV) infectious clone Agrobacterium inoculation contains activation in the YEP liquid nutrient medium of rifampin of kanamycin and 50mg/l of 100mg/l, 28 ℃ of 200rpm are cultured to OD
600Reach 0.8-1.0.Bacterium liquid that above-mentioned activation is good is drawn 50ul to the YEP solid culture medium of the rifampin of the kanamycin that contains 100mg/l and 50mg/l, even coated plate, and 28 ℃ of cultivation 24-48h cover with whole flat board to bacterium colony.The picking flat-plate bacterial colony with toothpick thorn method inoculation original host tomato, reaches other host Ben Shi cigarettes, petunia, Nicotiana glutinosa.ToLCTWV shows extremely strong pathogenic on its original host tomato, the whole reveal any symptomses of inoculation plant.Inoculate that tomato plant shows the blade last volume after 14 days, this is consistent with its field symptom, and (Fig. 6 a).But should virus Ben Shi cigarette, petunia and Nicotiana glutinosa all can not produce manifest symptom (Fig. 6 c, 6e, 6g).The total DNA of Ye of tomato plant system that extracts reveal any symptoms is as template; Carry out pcr amplification with the special total length primer of ToLCTWV; All can obtain the purpose fragment of about 2.7kb; But on Ben Shi cigarette, Nicotiana glutinosa and the petunia plant of reveal any symptoms not, all expand, show that ToLCTWV can not infect Ben Shi cigarette, Nicotiana glutinosa and petunia separately less than the purpose fragment.
With the ToLCTWV infectious clone with follow Chinese tomato yellow leaf curl virus (Tomato yellow leaf curl China virus, TYLCCNV) satellite DNA β (TYLCCNB) infectious clone combined inoculation tomato, Ben Shi cigarette, petunia and Nicotiana glutinosa.On tomato, can produce the symptom (Fig. 6 b) of vein yellow and top distortion after the combined inoculation, this symptom does not observe when virus is inoculated separately; On the Ben Shi cigarette, produce serious blade last volume, ear prominent (Fig. 6 d) appears in vein yellow and leaf back; Serious blade last volume on petunia, also occurs, there is ear dash forward (Fig. 6 f) at the back side; On Nicotiana glutinosa, also produced the blade last volume but do not had yellow arteries and veins and ear to appear suddenly and resembled (Fig. 6 h).Equal 100% reveal any symptoms of the plant of above combined inoculation.The total DNA that extracts disease plant carries out pcr amplification, the specific band of can increase respectively ToLCTWV and TYLCCNB.This shows that ToLCTWV can do with TYLCCNB really mutually, not only produces more serious symptom, also makes its host range enlarge.
Embodiment 3: the resistant variety of the anti-geminivirus infection of plant is measured
With tomato variety Zhejiang A, Zhejiang B, Zhejiang C, ruby is research object; Through the inoculation of flat-plate bacterial colony toothpick thorn phloem, identify the resistance of each tomato variety to Chinese papaya papaw curve leaf disease virus, tomato yellow leaf curl virus, tomato yellow leaf curl china virus, Thailand's tomato curve leaf disease virus, Taiwan tomato curve leaf disease virus.
1, experiment is handled
A)The Agrobacterium EHA105 inoculation that will contain the infectious clone of Chinese papaya papaw curve leaf disease virus, tomato yellow leaf curl virus, tomato yellow leaf curl china virus, Thailand's tomato yellow leaf curl virus, Taiwan tomato curve leaf disease virus contains activation in the YEP liquid nutrient medium of rifampin of kanamycin and 50mg/l of 100mg/l, and 28 ℃ of 200rpm are cultured to OD
600Reach 0.8-1.0.
Bacterium liquid that above-mentioned activation is good is drawn 50ul to the YEP solid culture medium of the rifampin of the kanamycin that contains 100mg/l and 50mg/l, even coated plate, and 28 ℃ of cultivation 24-48h cover with whole flat board to bacterium colony.
Grow seedlings under tomato variety Zhejiang A, Zhejiang B, Zhejiang C, 25 ℃ of greenhouse experiments of ruby, move in the disposable nutritive cube during 2 leaf periods, inoculate processing during 3-4 sheet true leaf.
With the cultured flat-plate bacterial colony of toothpick picking, thorn 3-4 sheet leaf period tomato cane phloem 3-4 point, incidence is placed and observed to postvaccinal tomato in greenhouse (temperature is 20-30 ℃).
Record plant disease symptom after one week of inoculation; 15-30 days statistics incidences; Calculate the incidence of disease, estimate the resistance of different tomato varieties Chinese papaya papaw curve leaf disease virus, tomato yellow leaf curl virus, tomato yellow leaf curl china virus, Thailand's tomato yellow leaf curl virus, Taiwan tomato curve leaf disease virus.
2, experimental result
Postvaccinal tomato symptom occurs in about 15 days.The tomato that inoculates Chinese papaya papaw curve leaf disease virus presents blade last volume, distortion, shrinkage, and plant is downgraded, and young leaves is unfolded symptoms such as incomplete; Roll up on the tomato performance leaf-shrinkage of inoculating tomato yellow leaf curl virus, refinement, the edge, the vein evagination, plant is downgraded, and young leaves is unfolded symptoms such as incomplete; The serious last volume of tomato performance blade of inoculation tomato yellow leaf curl china virus, thickens blade back vein evagination, yellow arteries and veins, and plant is downgraded, and symptoms such as ear is prominent appear in the later stage; The tomato of inoculation Thailand tomato yellow leaf curl virus presents blade last volume, shrinkage, symptoms such as plant dwarfing; The tomato of inoculation Taiwan tomato curve leaf disease virus presents blade last volume, edge yellow, the vein evagination, thickens symptoms such as young leaves refinement.Consistent with field tomato diseased plant disease symptom, the statistics incidence of disease was as shown in table 1 in 30 days, and disease index is as shown in Figure 7.
The tomato resistant variety of the anti-geminivirus infection of table 1 is measured
(disease index is an overall target of comprehensively considering the incidence of disease and severity, and its computing formula is: disease index=100 * ∑ (the sick numbers of sheets at different levels * typical values at different levels)/(investigating total number of sheets * highest typical value)) to calculate disease index in conjunction with the incidence of disease and the morbidity order of severity.
Infer that by the incidence of disease and disease index in 4 tomato varieties, ruby all shows high sense to above-mentioned 5 kinds of geminivirus infections, is susceptible variety; Zhejiang A shows as complete resistance to tomato yellow leaf curl china virus, Thailand's tomato yellow leaf curl virus, Taiwan tomato curve leaf disease virus, and Chinese papaya papaw curve leaf disease virus and tomato yellow leaf curl virus show as higher resistance; Zhejiang B shows as complete resistance to tomato yellow leaf curl virus, Thailand's tomato yellow leaf curl virus, and Chinese papaya papaw curve leaf disease virus, tomato yellow leaf curl china virus, Taiwan tomato curve leaf disease virus show as higher resistance; Zhejiang C all shows as higher resistance to Chinese papaya papaw curve leaf disease virus, tomato yellow leaf curl virus, tomato yellow leaf curl china virus, Thailand's tomato yellow leaf curl virus, Taiwan tomato curve leaf disease virus; Zhejiang A, B, C are three disease-resistant varieties that resistance is different.
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's product is carried out PCR screening and sequencing; Utilize the method for restriction enzyme digestion to be building up to the 1.2-2 of a viral genome total length repetitive sequence on the modified form plant expression vector pBinplus, obtain to have the infectious clone of geminivirus infection; The geminivirus infection infectious clone imports it among agrobatcerium cell EHA105 through the method for conventional triparental mating or electric shock, obtains the malicious source of inoculation;
Described geminivirus infection is Chinese papaya papaw curve leaf disease virus; Tomato yellow leaf curl china virus; Thailand's tomato yellow leaf curl virus; China's tomato curve leaf disease virus; Taiwan tomato curve leaf disease virus; Guangxi tomato curve leaf disease virus; China's Ageratum conyzoides yellow vein 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 culture medium of rifampin of the kanamycin that contains 100mg/l and 50mg/l; Streak inoculation or even coated plate; Cultivate 24-48h for 28 ℃, long good or cover with whole flat board to bacterium colony;
3) seedling culture:
Temperature is that 25～28 ℃, relative moisture are 75 %, photoperiod to be to grow seedlings in greenhouse or the plant incubator of 16L:8D, 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, coral west cigarette; 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 greenhouse identical with the seedling culture condition or plant incubator;
5) observation of symptoms and incidence record and resistance analysis:
Record plant disease symptom after one week of inoculation; 15-30 days statistics incidences; Calculate the incidence of disease, estimate virus to the pathogenicities of different plants or the vegetable material resistance to geminivirus infection: the plant performance that the incidence of disease is high is susceptible, and incidence of disease plant performance low or that do not fall ill is disease-resistant.
2. a usefulness 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 being used for the pathogenic mensuration of geminivirus infection, the research of known viral gene function and the resistant variety of anti-geminivirus infection and measures and the breeding for disease resistance service.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102816730A (en) * | 2012-09-06 | 2012-12-12 | 浙江大学 | Agrobacterium strain containing cotton leaf curl Multan virus (CLCuMV) infectious clones and applications thereof |
| CN110904136A (en) * | 2019-10-31 | 2020-03-24 | 中国计量大学 | Construction method of infectious clone of solanum nigrum kojic virus |
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| CN110904136A (en) * | 2019-10-31 | 2020-03-24 | 中国计量大学 | Construction method of infectious clone of solanum nigrum kojic virus |
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