CN110923212A - Purification method and application for grafting tomato chlorosis virus by using tobacco as stock - Google Patents
Purification method and application for grafting tomato chlorosis virus by using tobacco as stock Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01G2/00—Vegetative propagation
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Abstract
The invention belongs to the technical field of plant virus purification, and discloses a purification method and application for grafting and inoculating tomato chlorosis virus by taking tobacco as a stock. When the scion tomato grows to a leaf stage, the scion tomato is cut off from the root of the tomato, the overground part of the scion tomato is reserved as a scion, the left and the right of the lower part of the scion tomato are respectively obliquely cut to form a part of phloem, the phloem is inserted into the cut of the Nicotiana benthamiana plant, and the grafting clamp is used for fixing the scion tomato. The ToCV infectious clone can successfully infect the Nicotiana benthamiana and has obvious disease symptoms, the ToCV infectious clone is directly inoculated to a tomato plant, the infection rate is extremely low, no symptoms are shown, and no virus fragment is detected in vivo after bemisia tabaci eats the tomato plant for 48 hours; the tomato plant grafted by the cleft grafting method has the morbidity as high as 79.08 percent, and is more suitable for grafting the tomato plant taking the burley tobacco as the stock.
Description
Technical Field
The invention belongs to the technical field of virus purification, and particularly relates to a purification method and application for grafting and inoculating tomato chlorosis virus to tobacco stocks.
Background
The tomato chlorosis virus is a new virus transmitted by whitefly pests, and infects host plants to cause severe yellowing imbalance, so that fruit ripening is delayed, yield is influenced, and huge economic loss is brought to growers. The disease was discovered in 1989 in the tomato growing area of florida, usa and was first identified in 1998 as a tomato chlorosis virus, which is then rapidly outbreak and epidemic worldwide. According to literature statistics, ToCV is reported in more than 20 other countries and regions besides Antarctica and oceania. China first detected the occurrence of ToCV in Taiwan in 2004, and detected the virus on tomato and pepper plants in great-rise facility in Beijing, China in 2012. At present, ToCV has been outbreak and popular in tomato growing areas such as Beijing, Shandong, Henan, Tianjin, Shanxi, Jiangsu, Liaoning, Guangdong and the like, which causes serious economic loss.
ToCV belongs to the family of the long linear virus Clostroviridae, genus Torovirus, Crinivirus, whose genome comprises two plus-sense single-stranded RNAs (+ ssRNA), each packaged in linear virions. RNA1 is 8594nt long, and contains 4 ORFs, and the encoded protein controls viral replication. RNA2 was 8242nt long and contained 9 ORFs, encoding proteins involved in viral encapsidation, motility, and mediator transmission. Previous studies found that both the 1 and 2 strands were present to ensure successful infection of the host cell. Like other filoviruses, ToCV is a phloem-restricted virus that cannot be inoculated by artificial mechanical abrasion, nor spread via seeds. Under natural conditions, ToCV can only transmit in a semi-persistent manner through whiteflies, which are classified as Bemisiatabaci, Bemisia tabaci, Trialeurodes vaporariorum and t. Although the toxic transmission efficiency of the several whiteflies is different, the whiteflies have effective transmission capacity.
The difficulty of solving the technical problems is as follows: in laboratories, whiteflies are generally used for taking virus-feeding plants for virus preservation, but the whiteflies are used as virus transmission media of various plant viruses, so that single infection of the viruses is difficult to guarantee (Andret-Link P and FuchsM, 2005; Hohn, 2007). Meanwhile, whitefly inoculation is also influenced by various factors such as sex, feeding, technology and the like, the operation is complex, and the inoculation rate is low. The conventional grafting inoculation uses a diseased tomato plant as a stock, and because the tomato plant can obtain various viruses through vector insects, the single source of the viruses is difficult to ensure.
The significance of solving the technical problems is as follows: aiming at the defects of the method existing in the medium insect virus-transferring inoculation and the conventional tomato grafting inoculation, the invention provides the method for grafting and inoculating the tomato chlorosis virus by taking tobacco as the rootstock, the method is suitable for grafting and inoculating the phloem plant virus, the inoculation rate is up to 80 percent, the operation is simple, quick and effective, the repeatability in a laboratory is strong, and the technical support is provided for the aspects of ToCV pathogenesis, medium insect-virus-host interaction mechanism, resistant variety breeding and the like.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a purification method and application for grafting and inoculating tomato chlorosis virus to a stock by tobacco.
The invention is realized by the method for purifying the tomato chlorosis virus grafted on the tobacco as the stock, and the method for purifying the tomato chlorosis virus grafted on the tobacco as the stock is characterized in that diseased Boyle tobacco is used as the stock, a tomato plant is used as a scion, a healthy tomato plant is used as the scion, and the scion tomato is grafted on the stock by the cleft grafting method. When the scion tomato grows to a leaf stage, the scion tomato is cut off from the root of the tomato, the overground part of the scion tomato is reserved as a scion, the left and the right of the lower part of the scion tomato are respectively obliquely cut to form a part of phloem, the phloem is inserted into the cut of the Nicotiana benthamiana plant, and the grafting clamp is used for fixing the scion tomato.
Further, the inoculated Nicotiana benthamiana plants are placed in an artificial climate box with the temperature of 26 +/-1 ℃, the relative humidity of 70 +/-5% and the photoperiod L: D of 16:8h for insect-proof seedling raising, and agrobacterium inoculation is carried out when the plants grow to the four-leaf stage. The inoculation steps were as follows: firstly, adding 30uL of activated positive clone agrobacterium liquid into 30mL of LB liquid culture medium containing antibiotics, carrying out shaking culture, centrifugally collecting thalli, resuspending MMA buffer solution for 4h, and adjusting the OD600 value of the thalli to 1.0 by using a spectrophotometer; and secondly, selecting plants with consistent healthy growth, infiltrating 0.5mL of thalli into plant leaves by using a sterilized syringe without a needle head and a 1mL syringe, placing the plant leaves in an intelligent greenhouse for culture, and taking pCY agrobacterium tumefaciens with no-load plasmid as negative control. And (3) detecting the disease susceptibility of the Nicotiana benthamiana plants by using RT-PCR after 21d of inoculation, and selecting the Nicotiana benthamiana plants expressing typical symptoms of ToCV as grafting stocks.
Further, the purification method for grafting and inoculating the tomato chlorosis virus on the tobacco as the stock is to cut off the tobacco in the middle of the third and fourth true leaves of the Nicotiana benthamiana parallel to the ground when the stem of the root of the Nicotiana benthamiana grows to 2mm, and vertically cut a 1.5cm cut from top to bottom for later use. The tomato plant is placed in an artificial climate box with the temperature of 26 +/-1 ℃, the relative humidity of 70 +/-5% and the photoperiod L: D of 16:8h for insect-isolated seedling raising, when the plant grows to a first heart stage, the tomato plant is cut off from the root of the plant, the overground part is reserved as a scion, the phloem of the left and right beveling parts at the lower part of the scion tomato is inserted into the cut of the Nicotiana benthamiana plant and is fixed by a grafting clip.
Further, the first step is shaking culture at 28 ℃ and 200r/min for 12 h.
Further, the second step is placed in a climatic chamber with the temperature of 26 +/-1 ℃, the relative humidity of 70 +/-5 percent and the photoperiod L: D of 16:8h for culture.
Further, plants grafted by the cleft grafting method are placed in an artificial climate box with the temperature of 26 +/-1 ℃ and the relative humidity of 70 +/-5% for dark treatment for 24 hours.
The invention also aims to provide an application of the purification method for grafting the tobacco into the rootstock with the tomato chlorosis virus in obtaining a single virus source of the tomato chlorosis virus.
The invention also aims to provide application of the purification method for grafting the tomato chlorosis virus to the tobacco as the rootstock in obtaining a single virus source of other phloem-restricted viruses.
The invention also aims to provide an application of the method for purifying the tomato chlorosis virus grafted on the rootstock by the tobacco in the detection of the pathogenesis of the tomato chlorosis virus.
The invention also aims to provide an application of the purification method for grafting the tomato chlorosis virus to the tobacco rootstock in an interaction mechanism of the tomato chlorosis virus and the host.
The invention also aims to provide the application of the purification method for grafting the tomato chlorosis virus on the tobacco as the rootstock in breeding the resistant varieties.
In summary, the advantages and positive effects of the invention are: in the experiment, the grafting and inoculation technology of the tomato chlorosis virus is established by taking diseased Benzenbachia as a stock and healthy tomato plants as scions, so that technical support is provided for the follow-up research of ToCV. Tomato chlorosis virus (ToCV) transmitted by bemisia tabaci brings serious economic losses to the tomato industry all over the world, and has attracted high attention from researchers. The lack of a high-efficiency and stable ToCV single-source acquisition technology causes the research on the pathogenic mechanism, the interaction with hosts, the breeding of resistant varieties and other related aspects to be greatly limited. Aiming at the problem, the invention takes tobacco as a rootstock to carry out the correlation analysis of grafting and inoculating the tomato chlorosis virus. The ToCV infectious clone is inoculated to a tomato plant which is a non-natural host and a tomato plant which is a natural host, and the result shows that the ToCV infectious clone can successfully infect the Benzenbach and has obvious disease symptoms, the infection rate in the tomato plant is extremely low and no symptom is shown, and no virus fragment is detected in vivo after the Bemisia tabaci eats the tomato plant for 48 hours; the tomato plant system infection can be caused by grafting the healthy tomato plant to the diseased tomato plant by using different grafting methods, the morbidity of the tomato plant grafted by the cleft grafting method is up to 79.08 percent, the tomato plant grafted by the cleft grafting method is more suitable for grafting the tomato plant with the tomato plant as the stock, and the virus can be spread by eating the grafted diseased tomato plant by bemisia tabaci.
Compared with the prior art, the invention has the advantages that:
according to the method, the tomato chlorosis virus is grafted and inoculated on the stock by using the diseased burley tobacco, the tobacco whitefly is not required to be fed and the virus transmission of the tobacco whitefly is completed through complex operation, the tobacco whitefly is a vector insect of various plant viruses, and the composite infection of the various plant viruses on tomato plants is easily caused by the virus transmission of the tobacco whitefly; the conventional grafting inoculation takes a diseased tomato plant as a rootstock, and because the tomato plant can obtain a plurality of viruses through vector insects, the single source of the viruses is difficult to ensure, and the subsequent analysis of the tomato chlorosis viruses is influenced; the grafting tomato is selected to grow seedlings for about 7d, the plants are directly cut off from the root and grafted to the Nicotiana benthamiana plants by the grafting method, and the seedling regeneration capacity is stronger, so that the grafting rate is higher than that of the traditional grafting in the 4-5 leaf stage. The grafting method has the advantages of good repeatability, short inoculation time, simplicity, easiness in learning and the like, is suitable for obtaining phloem-restricted virus sources, and has important significance on aspects of plant virus pathogenesis, vector insect-virus-host interaction research, resistant variety breeding and the like.
Drawings
FIG. 1 is a flow chart of a method for purifying a tomato chlorosis virus grafted on a rootstock by tobacco according to an embodiment of the present invention.
FIG. 2 is a schematic diagram of a target fragment of ToCV, provided by an embodiment of the present invention. And (3) carrying out electrophoresis detection on the ToCV infectious clone inoculated with the plant. In the figure: 1. 3: benshi tobacco; 2. 4: tomatoes; 5: healthy control.
FIG. 3 is a schematic diagram comparing Nicotiana benthamiana inoculated with a ToCV infectious clone and a tomato plant inoculated with a ToCV infectious clone provided in the examples of the present invention. In the figure: (A) (ii) inoculation of ToCV infectious clones of Nicotiana benthamiana; (B) tomato plants inoculated with ToCV infectious clones.
FIG. 4 is a schematic diagram of the survival rate of Bemisia tabaci inoculated into Nicotiana benthamiana and tomato plants according to the embodiment of the invention.
FIG. 5 is a schematic diagram of ToCV detection of Nicotiana benthamiana and tomato Bemisia tabaci.
FIG. 6 is a schematic illustration of the effect of different rootstocks on viral accumulation according to an embodiment of the present invention.
FIG. 7 is a schematic diagram of the grafting of healthy tomato plants to diseased Boyle's tobacco plants using 2 different grafting methods according to the present invention. Different grafting methods of tomato plants are as follows: a, approach grafting; b cleft grafting method.
FIG. 8 is a graph showing the cumulative results of ToCV on tomato plants according to different grafting methods provided by the present invention.
FIG. 9 is a schematic diagram showing the effect of different grafting methods on the incidence of ToCV.
Fig. 10 is a schematic diagram of the transmission of bemisia tabaci to grafted tomato plants according to the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a purification method and application for grafting and inoculating tomato chlorosis virus to a stock by using tobacco, and the invention is described in detail with reference to the attached drawings.
As shown in fig. 1, the method for purifying the tomato chlorosis virus grafted on the tobacco rootstock by the tobacco provided by the embodiment of the invention comprises the following steps:
s101: adding 30uL of activated positive clone agrobacterium liquid into 30mL of LB liquid culture medium containing antibiotics, carrying out shaking culture at 28 ℃ for 12h at 200r/min, centrifugally collecting thalli, resuspending MMA buffer solution for 4h, and adjusting the OD600 value of the thalli to 1.0 by using a spectrophotometer.
S102: selecting plants with consistent healthy growth, infiltrating 0.5mL of thalli into plant leaves by using a sterilized syringe without a needle head 1mL, placing the plants in an artificial climate box with the temperature of 26 +/-1 ℃, the relative humidity of 70 +/-5% and the light period L: D of 16:8h for culture, detecting the infection rate by using RT-PCR after inoculating for 21D, and taking agrobacterium with pCY no-load plasmid as a negative control.
S103: when the stem of the root of the Nicotiana benthamiana is 2mm thick, a sterilized blade is used for cutting the stem between the third and fourth true leaves of the Nicotiana benthamiana, a cut with the length of 1.5cm is vertically cut from top to bottom, a leaf of a tomato scion is reserved, the ground part is reserved as the scion, a part of phloem is obliquely cut on the left and right of the lower part of the tomato scion, the tomato scion is inserted into the cut of the Nicotiana benthamiana plant, and the tomato scion is fixed by.
The technical solution of the present invention will be further described with reference to the following experiments.
1. Materials and methods
Test method
1.1 ToCV infectious clonal inoculation
Adding 30uL of activated positive clone Agrobacterium liquid into 30mL LB liquid culture medium containing antibiotics, shaking and culturing at 28 deg.C for 12h at 200r/min, centrifuging, collecting thallus, MMA buffer solution (10 mmol. L)-1MgCl2，10mmol·L-1MES，200μmol·L-1As) was resuspended for 4h and the OD600 of the cells was adjusted to 1.0 with a spectrophotometer. Selecting plants with consistent healthy growth, infiltrating 0.5mL of thalli into plant leaves (ZHao et al, 2016) by using a sterilized syringe without a needle, placing the plants in an artificial climate box with the temperature of 26 +/-1 ℃, the relative humidity of 70 +/-5% and the light period L: D ═ 16:8h for culture, and detecting the infection rate by using RT-PCR after inoculating for 21D, wherein agrobacterium with pCY no-load plasmid is used as a negative control.
1.2 grafting method
The approach method comprises the following steps: when the stem of the root of the Shigella sonnei grows to 2mm, the stem is cut off from bottom to top between the third and fourth true leaves of the Shigella sonnei by a sterilized blade, the cut surface angle is about 45 degrees, the scion tomato is kept one heart and cut off at the position close to the stem thickness of the stock, the cut surface angle is consistent with the stock, the cut is smooth, the two cuts are aligned, and the grafting clip is used for fixing.
Cleft grafting: when the stem of the root of the Nicotiana benthamiana is 2mm thick, a sterilized blade is used for cutting the stem between the third and fourth true leaves of the Nicotiana benthamiana flat, a cut with the length of 1.5cm is vertically cut from top to bottom, a core is reserved for the grafting tomato, the root is cut at the position close to the stem of the root stock, the left side and the right side of the lower part of the grafting tomato are obliquely cut once, the grafting tomato is inserted into the cut of the Nicotiana benthamiana.
1.3 Effect of different grafting methods on tomato plant morbidity
The experiment was set up with 2 treatments: by adopting the grafting method (A) and the cleft grafting method (B), 30 diseased Bunsen plants and tomato plants with consistent growth vigor are selected for each treatment, tomatoes are grafted to the Bunsen plants according to different grafting methods, the insect-proof net cuts off pests, unified production management is carried out, and each treatment is repeated for 3 times. And (3) collecting tomato plant leaves after 21d of grafting, detecting the morbidity of the tomato plants in different grafting modes by RT-PCR, detecting the virus accumulation by qRT-PCR, and performing obvious analysis on differences among treatments.
1.4 Effect of different rootstocks on Virus accumulation
To determine the effect of different rootstocks on ToCV accumulation, a total of 2 treatments were set up: a, taking diseased Bosch tobacco as a stock, and grafting healthy tomato ears onto the tobacco; and B, taking the healthy tomato plants as stocks, and grafting the diseased Ben's ears onto the tomato plants. 30 plants were selected for each treatment and repeated 3 times. And (3) collecting tomato leaves after 7, 10, 14 and 21d of grafting, detecting the virus accumulation by utilizing qRT-PCR, and performing difference analysis among treatments.
2. Results and analysis
2.1 infection of ToCV-infectious clones in Nicotiana benthamiana and tomato plants
After infecting Nicotiana benthamiana 21d with ToCV infectious clone, the target fragments of about 751bp and 463bp can be detected by RT-PCR (FIG. 2), and the probability of detecting the two target fragments is 76.79%. After cloning, sequencing and BLAST comparison, the homology of the segment of the 2 items is found to be more than 99.0 percent compared with the registered RNA1 and RNA2 sequences in GenBank. Compared to healthy tobacco, burley tobacco inoculated with the ToCV infectious clone showed typical symptoms of ToCV onset, yellowing of the upper leaf, obvious chlorosis spots of the lower leaf, and leaf embrittlement (fig. 3-a), indicating that the ToCV infectious clone had successfully infected burley tobacco.
After the ToCV infectious clone infects tomato plants 21d, target fragments of about 751bp and 463bp can be detected by using an RT-PCR technology (figure 2), and the probability of detecting the two target fragments is 12.85 percent. After cloning, sequencing and BLAST comparison, the homology of the segment of the 2 items is found to be more than 99.0 percent compared with the registered RNA1 and RNA2 sequences in GenBank. However, tomato plants inoculated with ToCV infectious clones showed no signs of disease (FIG. 3-B).
2.2 Effect of Ben's tobacco and tomato on Bemisia tabaci survival and toxicity
And (3) inoculating the primarily emerged bemisia tabaci adults to ToCV-infected Bunge tobacco, healthy Bunge tobacco, ToCV-infected tomato plants and healthy tomato plants by using a micro-insect cage, and recording the survival condition of the bemisia tabaci. After 1h of feeding, the tobacco whiteflies inoculated on ToCV infected Nicotiana benthamiana plants and healthy Nicotiana benthamiana plants begin to die; the bemisia tabaci died peak appears after 2h, and the bemisia tabaci died rate on ToCV infected Ben tobacco is up to 59.37 percent and is obviously higher than that of healthy Ben tobacco and tomato plants; after 12h of feeding, all the bemisia tabaci infected on the ToCV Niger plants die, and all the bemisia tabaci on the healthy Niger plants die after 24 h; bemisia tabaci inoculated on tomato plants grew well (fig. 4).
Collecting bemisia tabaci eaten for 6 hours on ToCV infected Nicotiana benthamiana plants, and detecting by RT-PCR (reverse transcription-polymerase chain reaction) that the virus is not detected in the bemisia tabaci bodies; bemisia tabaci which is eaten for 24 hours is collected on ToCV infected tomato plants, and RT-PCR detection shows that the Bemisia tabaci does not carry the virus in vivo (figure 5).
2.3 Effect of different stocks on tomato plant morbidity
And (3) collecting leaves of tomato plants of treatment groups A and B after 7, 10, 14 and 21d of grafting, and detecting the accumulation condition of ToCV at different periods by utilizing qRT-PCR. As a result, it was found that ToCV was detected after 10 days in the treated group A plants, and the virus content increased with time. ToCV was detected in treated group B plants after 14d, and also increased in viral content with time (FIG. 6). These results indicate that the grafting technique can cause phloem-restricted virus system to infect healthy tomato plants, and that using burley tobacco as the rootstock is more conducive to accumulation of virus in tomato plants.
2.4 Effect of different grafting methods on tomato plant morbidity
The healthy tomato plants are grafted to diseased Bosch tobacco plants by 2 different grafting methods (figure 7), tomato plant leaves are collected after 21d for qPCR detection, and the result shows that the accumulation amounts of the viruses of tomato samples of a treatment group A and a treatment group B are not obviously different (figure 8), but the morbidity of the tomato plants by the different grafting methods is obviously different, and the morbidity of the cleft grafting method is as high as 79.08%, which is obviously higher than that of the inarching method (figure 9). Therefore, the cleft grafting method is more favorable for grafting and inoculation of tomato plants ToCV taking Nicotiana benthamiana as stocks.
2.5 toxin transfer study of Bemisia tabaci on grafted tomato plants
Inoculating the primarily emerged bemisia tabaci adults on the diseased grafted tomato leaves by using a micro-insect cage for eating for 48 hours, collecting single bemisia tabaci, and carrying out RT-PCR detection to find that 64.20% of bemisia tabaci bodies can detect ToCV fragments; the adult bemisia tabaci with toxicity is inoculated to healthy tomato plants at the 5-leaf stage for virus transmission for 48h, and after 21d, tomato leaves are collected for RT-PCR detection, and 85.98% of tomato plants can detect ToCV fragments and show obvious disease symptoms (figure 10).
3. Like other filoviruses, after infection of a plant by ToCV, virions conduct bidirectionally in the phloem of the plant. The grafting technique was used to transplant single leaves of ToCV tomato plants to the phloem of young tomato plants, and it was found that 87.8% of tomato plants were successfully infected with virus. Similarly, applying a stem patch of a ToCV tomato plant containing phloem tissue to the phloem of a healthy tomato plant also caused systemic infection of the tomato plant. In the experiment, healthy tomato plants are grafted to diseased Bunsen plants by using a grafting method and a cleft grafting method, so that the tomato plants can be diseased, which shows that the infectious clone has the capability of increasing the value in the tomato plants after being copied in the Bunsen plants and shows typical symptoms. Effective transmission of bemisia tabaci by inoculation onto engrafted tomato plants with virus also confirmed the integrity of the ToCV virions. The grafting inoculation method is simple, efficient and easy to implement, is not limited by seasons and temperature, is particularly suitable for grafting tomato plants taking the Shigella benthamiana as rootstocks, can be used as an effective method for obtaining single tomato chlorosis viruses, and has important significance for accelerating germplasm resource breeding and interaction mechanism research.
Information table of table detection primers
TABLE 1 primer information
Table1 Primer pairs used in paper
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. The method for purifying the grafted tomato chlorosis virus by taking tobacco as a stock is characterized in that the method for purifying the grafted tomato chlorosis virus by taking diseased Bosch tobacco as the stock and healthy tomato plants as scions is used for grafting the scion tomatoes onto the stock by a cleft grafting method;
when the scion tomato grows to a leaf stage, the scion tomato is cut off from the root of the tomato, the overground part of the scion tomato is reserved as a scion, the phloem is obliquely cut from the left and the right of the lower part of the scion tomato, the scion tomato is inserted into the cut of the Nicotiana benthamiana plant, and the scion tomato is fixed by a grafting clip.
2. The method for purifying the tomato chlorosis virus grafted on the tobacco rootstock according to claim 1, wherein the inoculated Nicotiana benthamiana plant is placed in an artificial climate box with the temperature of 26 +/-1 ℃, the relative humidity of 70 +/-5% and the photoperiod L: D ═ 16:8h for insect-isolated seedling raising, and the agrobacterium inoculation is carried out when the plant grows to the four-leaf stage, and the inoculation step is as follows:
firstly, adding 30uL of activated positive clone agrobacterium liquid into 30mL of LB liquid culture medium containing antibiotics, carrying out shaking culture, centrifugally collecting thalli, resuspending MMA buffer solution for 4h, and adjusting the OD600 value of the thalli to 1.0 by using a spectrophotometer;
secondly, selecting healthy and consistent Nicotiana benthamiana plants, infiltrating 0.5mL of thalli on the backs of plant leaves by using a sterilized 1mL needleless injector, carrying out dark treatment for 12h, then placing the plants in an artificial climate box with the temperature of 26 +/-1 ℃, the relative humidity of 70 +/-5% and the light cycle L: D of 16:8h for culture, and taking the agrobacterium with pCY no-load plasmid as negative control; and (3) detecting the disease susceptibility of the Nicotiana benthamiana plants by using RT-PCR after 21d of inoculation, and selecting the Nicotiana benthamiana plants expressing typical symptoms of ToCV as grafting stocks.
3. The method for purifying the tomato chlorosis virus grafted and inoculated on the tobacco rootstock according to claim 2, wherein in the first step, the temperature is 28 ℃ and the shaking culture is carried out at 200r/min for 12 h;
and in the second step, the seeds are placed in a climatic chamber for culture, wherein the temperature is 26 +/-1 ℃, the relative humidity is 70 +/-5%, and the photoperiod L: D is 16:8 h.
4. The method for purifying the grafted tomato chlorosis virus on the tobacco rootstock according to claim 1, wherein the grafted tomato chlorosis virus on the tobacco rootstock is purified by cutting off the root of the Nicotiana benthamiana from the middle of the third true leaf and the fourth true leaf of the Nicotiana benthamiana in parallel with the ground when the root of the Nicotiana benthamiana is 2mm thick, and vertically cutting off a 1.5cm cut from top to bottom for later use; placing the scion tomato plant in an artificial climate box with the temperature of 26 +/-1 ℃, the relative humidity of 70 +/-5% and the photoperiod L: D of 16:8h for insect-isolated seedling raising, cutting off the tomato plant root when the plant grows to a first heart stage, keeping the overground part as a scion, inserting the scion tomato plant cut into the cut of the Bosch tobacco plant by obliquely cutting the phloem on the left and right of the lower part of the scion tomato plant, and fixing the scion tomato plant cut by using a grafting clip; the scion tomato grows to a first heart stage for grafting.
5. The method for purifying the tomato chlorosis virus grafted on the tobacco rootstock according to claim 1, wherein the scion tomato grafted by the cleft grafting method is dark-treated in an artificial climate box with the temperature of 26 +/-1 ℃ and the relative humidity of 70 +/-5% for 24 hours.
6. The application of the purification method for grafting and inoculating the tomato chlorosis virus to the tobacco as the rootstock according to any one of claims 1 to 5 in obtaining a single virus source of the tomato chlorosis virus.
7. The application of the method for purifying the tomato chlorosis virus grafted and inoculated on the tobacco rootstock according to any one of claims 1 to 5 in the acquisition of single virus sources of other phloem-restricted viruses.
8. The application of the purification method of grafting the tobacco as the rootstock to the tomato chlorosis virus according to any one of claims 1 to 5 in the detection of the pathogenesis of the tomato chlorosis virus.
9. The application of the purification method of grafting and inoculating the tomato chlorosis virus to the tobacco as the rootstock according to any one of claims 1 to 5 in an interaction mechanism of the tomato chlorosis virus and a host.
10. Use of the method of any one of claims 1 to 5 for the purification of tomato chlorosis virus grafted onto tobacco stocks for breeding resistant varieties.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113396712A (en) * | 2021-06-23 | 2021-09-17 | 河北省农林科学院植物保护研究所 | Single-leaf grafting inoculation method for tomato yellow leaf curl virus and tomato chlorosis virus |
| CN115517121A (en) * | 2022-10-12 | 2022-12-27 | 浙江大学 | Application of Nicotiana benthamiana as trap-killing plant in preventing and controlling agricultural pests |
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