54 results about "Tomato spotted wilt virus" patented technology

The invention relates to a method for identifying tobacco resistance by use of a tomato spotted wilt virus NSm gene and belongs to the technical field of plant protection. The NSm gene contains a nucleotide sequence shown in SEQ ID No.1. The NSm gene is an avirulence gene which stimulates tobacco containing a resistant locus RTSW to produce disease resistance response. An agrobacterium tumefaciens transient expression system is adopted for transient expression of the NSm gene in tobacco leaves, and whether test tobacco contains the resistant locus RTSW is judged by detecting whether the test tobacco shows an allergic reaction. The resistance of the tobacco to the tomato spotted wilt virus disease can be rapidly and accurately identified with the method, and the method can be applied to disease-resistant breeding as well as positioning and cloning of disease-resistant genes.

A Solanum lycopersicum plant including within its genome at least one Tomato Spotted Wilt Virus (TSWV) resistance allele and at least one Phytophthora infestans resistance allele. The resistance alleles are present in the coupling phase at different loci on one chromosome and the plant is resistant against TSWV and resistant against at least Phytophthora infestans. A method for producing a hybrid Solanum lycopersicum plant including (a) obtaining a Solanum lycopersicum plant having within its genome at least one Tomato Spotted Wilt Virus (TSWV) resistance allele and at least one Phytophthora infestans resistance allele in the coupling phase; and (b) crossing the Solanum lycopersicum plant with a second Solanum lycopersicum plant bearing an additional resistance allele.

The invention discloses a biocontrol strain and agent capable of preventing and controlling plasmopara viticola and phytophthora capsici and an application of the biocontrol strain and agent. The biocontrol strain is characterized by being a streptomyces atratus py-1 strain in surface soil of a grapery and is collected with the number of China General Microbiological Culture Collection Center (CGMCC) No:7826 in CGMCC; and a secondary metabolite of streptomyces atratus py-1 is used as an active component of the biocontrol strain. The biocontrol agent is prepared through the steps of inoculating the streptomyces atratus py-1 into 200ml of psa culture solution in a 500ml triangular flask to culture for 5 days; centrifuging a fermentation solution for 30 minutes; filtering a supernatant solution by using a filter membrane to obtain a stock solution; diluting the stock solution, then, spraying the diluted stock solution on grapes, and irrigating the diluted stock solution into the roots of hot peppers. The biocontrol agent has a certain effect on inhibiting egg plant stem blight, egg plant root rot, tomato spotted wilt virus, tomato late blight, cucumber anthracnose, maize ear rot, sorghum curvularia lunata and wheat root rot.

The invention relates to a method for screening tobacco plants resistant to tomato spotted wilt virus, belonging to the field of plant protection technology. The method includes five steps: nicotianabenthamiana seedling raising, preparation of resistance identification for tobacco seedlings, preparation of inoculation buffer, inoculation of tobacco seedlings and identification of disease resistance. The principle of the invention is that before the tobacco disease resistance identification is carried out, the field tomato spotted wilt virus source is activated on the nicotianabenthamiana, and the diseased tissue of the tobacco is collected as a poison source to carry out disease resistance identification.

The invention discloses a method for detecting a tomato spotted wilf virus according to a pyrosequencing technology. The method comprises the following steps: firstly, designing a specific primer and a pyrosequencing primer according to the gene N of the tomato spotted wilf virus; secondly, extracting ribonucleic acid of a sample to be detected, and performing polymerase chain reaction (PCR) amplification by the specific primer of the gene N; thirdly, detecting an amplification product by agarose gel electrophoresis, preparing a pyrosequencing single-stranded template if the length of a primer amplification fragment is 143 bp, producing pyrosequencing reaction, and finally judging whether the sample contains the tomato spotted wilf virus or not according to a PCR amplification result and a pyrosequencing result. The method is suitable for quickly detecting and confirming the tomato spotted wilf virus, and can be widely applied to epidemic situation monitoring in agricultural production and environment and confirmation of the tomato spotted wilf virus in import and export trade; the operation is very easy and convenient, and the required sample amount is small.

The invention relates to a method for screening disease-resistant genes by the aid of tomato spotted wilt virus NSm (new smoking material) genes, and belongs to the technical field of plant gene cloning. Nucleotide sequences of the tomato spotted wilt virus NSm genes are shown as SEQ ID No.1. The method includes constructing plant expression vectors of the tomato spotted wilt virus NSm genes; acquiring agrobacterium tumefaciens by means of transformation; jointly infiltrating nicotiana benthamiana in the agrobacterium tumefaciens and candidate disease-resistant genes; observing nicotiana benthamiana leaves to determine whether the nicotiana benthamiana leaves have characters of hypersensitive necrosis or not; authenticating the candidate disease-resistant genes. The method has the advantages that the disease-resistant genes can be quickly authenticated by the method in a high-throughput manner, and accordingly important application value of the method developed on the basis of genes for screening disease-resistant genes of plants is indicated.

The invention discloses a liquid phase chip detection primer of a tomato spotted wilt virus, and a detection method thereof. The invention provides a kit used for the liquid phase chip detection or the assisted detection of the tomato spotted wilt virus. The kit comprises a primer group and a probe, and the primer group is composed of a primer 1 and a primer 2. Experiments prove that a sample can be rapidly detected through the detection method, and a process from sample treatment to result obtaining only costs about 4h; and there is no cross reaction of the primers designed in the invention and common plant viruses on tomatoes, so the false negative result caused by the non-specific amplification is reduced.

The invention discloses an RT-HDA primer, kit and method for detecting a tomato spotted wilt virus. The primer is an oligonucleotide sequence represented by SEQ ID NO:1 and SEQ ID NO:2 of a sequence table. The kit comprises the following components: RT-HDA amplified reaction liquid, deionized water without RNA enzyme, a positive control sample and a negative control sample, the deionized water is adopted as blank control, the positive control sample is an RNA template with the tomato spotted wilt virus, and the negative control sample is a healthy tomato tissue RNA template without the tomato spotted wilt virus. The detecting method comprises the following steps that (1) RNA extraction is carried out on a sample to be detected; (2) RT-HDA amplification is carried out on the RNA of the tomato spotted wilt virus; (3) an electrophoresis detecting result is described and judged. The HDA detecting method is faster, safe and reliable, and compared with a PCR, no PCR instrument is needed, the requirement for equipment is simple, the method is suitable for fast detecting and confirming the tomato spotted wilt virus, and the method can be widely applied to epidemic situation monitoring in agricultural production and environment and monitoring and detecting of this kind of viruses in import and export trade.

The invention relates to a method for inoculating tomato spotted wilt virus, which belongs to the field of plant protection. The method comprises the following steps: (1) configuring 50mM pH7.2 phosphate buffer solution and sterilizing; (2) sowing tomatoes, and growing to 4 true leaves for inoculation; (3) by 1:5 (mass:volume) Proportionally add phosphate buffer solution to grind the susceptible tomato leaves preserved at low temperature to homogenate; (4) take the homogenate and centrifuge at low temperature and high speed, and add the supernatant to a small plastic watering can; (5) immerse the tomato seedlings in the phosphate buffer in the ultrasonic cleaner After oscillating in the liquid for 10 seconds, the virus liquid in the watering can was sprayed onto the treated tomato seedling leaves; (6) after cultivating for 12 hours in a dark artificial climate box at 100% relative humidity, take it out and place it under conventional conditions nourish. The beneficial effect of the invention is that it can increase the virus inoculation speed of tomato seedlings, make the whole tomato leaves susceptible to the disease, quickly and efficiently identify the resistance of the tomato to the virus, and accelerate the selection of disease-resistant varieties.

The invention provides an SNP marker related to a pepper tomato spotted wilt virus disease resistance gene as well as specific primers and application of the SNP marker. A nucleotide sequence of the SNP marker related to the pepper and tomato spotted wilt virus disease resistance gene is as shown in SEQ ID NO.1, and the SNP marker is a 21st basic group of the sequence as shown in SEQ ID NO.1. Theinvention also provides the specific primers and a corresponding test kit for identifying the SNP marker, and provides an identification method for pepper resisting the tomato spotted wilt virus disease by using the specific primers or the test kit. By adopting the SNP molecular marker disclosed by the invention, the pepper TSWV (Tomato Spotted Wilt Virus) disease resistance gene can be quickly and accurately judged only by carrying out DNA detection on the pepper plant in a seedling stage without artificial inoculation identification of pepper TSWV disease resistance, and large-batch detection can be carried out on samples, so that the breeding efficiency is improved, and the identification time is shortened.

The invention discloses a preparation method of a tomato spotted wilt virus nucleic acid standard substance. The virus nucleic acid standard substance is prepared by the following steps of selecting agene conserved region as an amplification target region; designing primers and synthesizing; carrying out sequence amplification; constructing an expression vector; carrying out in-vitro transcription; measuring the concentration and diluting; performing split charging; and carrying out detection quantification. The standard substance prepared by the method has the characteristics of stability, no biological infectivity, wide application range and the like, and can be used for detecting the tomato spotted wilt virus by utilizing reverse transcription PCR and fluorescent quantitative PCR.

The invention discloses a hybridoma cell strain capable of secreting an anti-Pepino mosaic virus (PepMV) monoclonal antibody, and an application of the monoclonal antibody. Purified PepMV virus particles are used as an antigen to immunize BALB/c mice, the hybridoma cell strain 23D11 capable of secreting the anti-PepMV monoclonal antibody is obtained through a hybridoma technology, and the preservation number of the hybridoma cell strain 23D11 is CGMCC No.18845. The ascites indirect ELISA titer of the monoclonal antibody secreted by the cell strain reaches 10<-8>, the antibody type and subclassare IgG1 and kappa light chain, and the monoclonal antibody has specific immune reaction with PepMV, does not react with potato virus X belonging to the same genus, and does not have immune reactionwith tomato mottle mosaic virus, tomato mosaic virus, tomato spotted wilt virus, tomato yellow leaf curl virus, tomato black loop virus and healthy plant tissues. A variety of detection methods are established by using the 23D11 monoclonal antibody, and the sensitivity of ACP-ELISA and dot-ELISA methods for detecting diseased leaves reaches 1:40, 960 fold dilution and 1:20, 480 fold dilution (w/v,g/mL) respectively. The creation of the cell strain and the establishment of a virus serological detection method provide material and technical support for PepMV detection and diagnosis, epidemiological investigation and scientific prevention and control.

The invention discloses a method for reducing the morbidity of tomato spotted wilt of tobacco by using a green control technology. The method comprises the following steps: covering a tobacco field with silver black mulching films, and placing blue plates in the tobacco field cooperatively; and uniformly placing 5-15 pieces of blue plates per mu. The pheromone blue plate trapping and killing is a physical control technology which is adopted by utilizing the taxis of the thrips to the blue plates, and the pheromone blue plate trapping and killing is a green and environment-friendly technology which is free of pollution, convenient to use and remarkable in trapping and killing effect; and by covering with the silver black mulching films, the harm of the thrips can be effectively avoided, the growth of weeds can be inhibited, and the production cost is saved to a certain extent. According to the method, a sustainable green control measure for the tomato spotted wilt of the tobacco is provided safely, economically and effectively, the morbidity of the tomato spotted wilt in a demonstration area can be effectively reduced, the morbidity of plants is controlled within 5%, the economic loss of flue-cured tobacco is retrieved to 193.20 yuan/mu, and a scientific basis and a technical standard are provided for a comprehensive control technology of the tomato spotted wilt virus in a main diseased tobacco area.

The invention relates to a protein standard substance of tomato spotted wilt virus and an application of the protein standard substance. The protein standard substance is prepared by the following steps: firstly obtaining a whole-genome sequence for the tomato spotted wilt virus, and according to sequence comparison, obtaining a fusion protein expression vector containing a coat protein gene and a replicase gene, namely an XXX-CR vector; transferring the obtained eukaryotic expression vector of the coat protein gene and the replicase gene into a 293 cell strain, performing culturing and expressing, performing freezing and thawing at low temperature, performing centrifuging and performing purifying to obtain an expression product molecule; and performing DNase I digestion and purification, and performing quantifying to obtain the fusion expression protein standard substance of the coat protein and the replicase gene. The substance provided by the invention has the characteristics of good stability, no biological infectivity, ribonuclease resistance and the like, and is used as the standard substance in protein detection of the tomato spotted wilt virus.

The invention discloses a serological method for rapid detection of tomato spotted wilt virus carried by individual thrips and its application A hybridoma cell strain 1A5By capable of secreting monoclonal antibody against tomato spotted wilt virus and having a preservation number of CGMCC No.9343 is utilized to establish a dot-ELISA method for detection of tomato spotted wilt virus carried by individual thrips, to research and develop a rapid detection kit, and to rapidly detect the virus carried rate of the field thrips carrying tomato spotted wilt virus. The invention can be used in mass rapid investigation of virus carried rate of the thrips and in early prediction of tendency and outbreak of the tomato spotted wilt virus. The detection method is fast, efficient, sensitive and specific, and has high throughput and easy operation, and provides technical support for the rapid diagnosis, prediction, forecast and scientific prevention and control of the tomato spotted wilt virus disease.

The invention discloses nicotiana benthamiana TMP14 (Thylakoid Membrane Phosphoprotein 14) and application thereof in plant virus resistance. The amino acid sequence of the nicotiana benthamiana TMP14is as shown in the sequence table SEQ ID NO.1. The invention also discloses an optimized mutant of the nicotiana benthamiana TMP14. The nicotiana benthamiana TMP14 disclosed by the invention directlyinteracts with TSWV (Tomato Spotted Wilt Virus) NSm; the over-expression transgenic tobacco plant of the nicotiana benthamiana TMP14 has the function of resisting plant virus; the discovery providestheoretical and practical basis for researching transgenic anti-virus crops, and has a broad application prospect.

The invention discloses a hybridoma cell strain secreting monoclonal antibody against Iris yellow spot virus and application of the monoclonal antibody. Iris yellow spot virus particles purified by a differential centrifugation method are used as an antigen to immunize BALB / c mice; through cell fusion, screening and cloning, one strain of hybridoma cell strain 12C10 capable of stable passage and secreting anti-Iris yellow spot virus monoclonal antibodies is obtained; and the strain has a preservation number CGMCC No.9336. The monoclonal antibody secreted from the hybridoma cell strain has ascites indirect ELISA titer of 10 <-7>, and the antibody type and subtype are IgG1, kappa chain. The monoclonal antibody specifically and sensitively detect Iris yellow spot virus. The reaction with ZYMV. The monoclonal antibody secreted from 12C10 only has specific reaction with Iris yellow spot virus, but does not react with tomato spotted wilt virus, turnip mosaic virus and cucumber mosaic virus. The hybridoma cell strain 1C5 and monoclonal antibody secreted by the cell strain provide technical and material support for the diagnosis, inspection and quarantine, and scientific prevention and control of the virus.

The invention belongs to the technical field of drug synthesis and agricultural disease control, and particularly relates to an amide-containing ferulic acid derivative as well as a preparation method and application thereof. The synthesized amide-containing ferulic acid derivative compound can be applied to preparation of plant virus resisting agents and can effectively inhibit diseases such as tomato spotted wilf virus and cucumber mosaic virus disease. The compounds Y1, Y2, Y8, Z1 and Z2 in the amide-containing ferulic acid derivative disclosed by the invention show relatively good inhibitory activity on tomato spotted wilf virus and cucumber mosaic virus. Wherein the compound Y2 shows relatively good passivation activity on tomato spotted wilf virus and cucumber mosaic virus, and is superior to positive control medicaments such as ferulic acid, ningnanmycin and virazole. The compound can be used as a medicine or medicament for preventing and treating diseases such as tomato spotted wilf virus and cucumber mosaic virus. The structure of the material is derived from natural products, and the material is environment-friendly, easy to metabolize and degrade, simple in preparation process, relatively stable in physicochemical property and wide in application prospect.

The invention discloses a triple RT-PCR method capable of detecting three viruses causing potato tuber necrosis simultaneously and a primer combination thereof. Particularly, three pairs of primers and one RT-PCR are used for detecting a potato virus Y tuber necrosis strain (PVYNTN) injuring potato tubers, a tomato spotted wilt virus (TSWV) and a potato mop-top virus (PMTV) simultaneously. Due to the fact that the TSWV, the PVYNTN and the PMTV can all cause potato tuber necrosis, symptoms are difficult to distinguish. When a traditional single RT-PCR method is used, detection can only be completed through three reactions, and time and labor are consumed. Accordingly, the three viruses can be detected simply through a multiple RT-PCR reaction, the detection efficiency is greatly improved, and the detection cost is greatly lowered.

The invention provides crDNA, crRNA, a kit and a method for detecting plant tomato spotted wilt virus. The nucleotide sequence of the crDNA is as shown in SEQ ID NO.1; the nucleotide sequence of the crRNA is as shown in SEQ ID NO.2; the kit contains the crDNA or the crRNA, a Cas13a protein and a fluorescent probe. The method comprises the following steps of: extracting total RNA of a plant sample to be detected, and acquiring cDNA through reverse transcription; carrying out amplification by taking the cDNA as a template to obtain a DNA amplification product; and using a detection reaction system composed of the amplification product, crRNA, Cas13a protein, the fluorescent probe and enzyme-free water for detection. The crDNA, crRNA, a kit and a method for detecting plant tomato spotted wilt virus provided by the invention have the characteristics of low cost, repeated detection, simple method, high detection speed, sensitivity (the lowest detection limit reaches 10 copies/uL) and specificity.

The invention provides an application of C21 steroid compounds 1-8 separated from cynanchum paniculatum in preparation of anti-TSWV (tomato spotted wilt virus) active products. Tests prove that the C21 steroid compounds 1-8 in cynanchum paniculatum have anti-TSWV activity, and show different degrees of prevention effects on TSWV. Specifically, the invention also provides an application of the C21steroid compounds 1-8 of cynanchum paniculatum in preparation of a TSWV inhibitor. When the concentrations of the compound 4 and the compound 5 are 100 [mu]g/mL, the virus content is less than that ofningnanmycin, which indicates that the prevention effect on TSWV is superior to that of commercially available antiviral pesticide ningnanmycin.

The invention discloses a method for preventing and controlling tomato spotted wilt viruses. The method comprises the steps: cultivating tomatoes in a sunlight greenhouse according to large and smallrows, wherein a large row spacing is 80 cm, and a small row spacing is 60 cm; transplanting collards within 10-12 days before the tomatoes are planted, planting a row of collards between small rows ofa planting row, wherein the row spacing is 25 cm; carrying out plug seedling on the collards by using a substrate containing carbonized rice hulls, additionally applying the carbonized rice hulls into planting pits, and dipping plug trays and irrigating roots by using a dinetofuran liquid; and before transplanting the collards, sealing a ventilation opening of the sunlight greenhouse by using a 40-mesh insecticidal protection net. By using the method, the incidence rate of the tomato spotted wilt viruses can be effectively reduced on the premises that the dosage of a chemical pesticide is greatly reduced and the non-contact chemical prevention and control of main cultivated crops are realized, and generally, the incidence rate of the spotted wilt viruses of susceptible tomato varieties can be controlled within 5%.

The invention discloses quinolizidine alkaloid as well as a preparation method and application thereof, the quinolizidine alkaloid is selected from any one of new compounds 1-7, and the quinolizidine alkaloid is extracted and separated from seeds of wild cassia lanceolata or is obtained through artificial synthesis. The invention further discloses a preparation method of the quinolizidine alkaloid, and the quinolizidine alkaloid is verified to have a remarkable effect on prevention and treatment of tomato spotted wilt virus and pest killing.