To develop a rapid and high-sensitivity method for detection of grapevine virus E(GVE),a SYBR Green based real-time fluorescence quantitative RT-PCR method(RT-qPCR)was established.This method could be used to detect G...To develop a rapid and high-sensitivity method for detection of grapevine virus E(GVE),a SYBR Green based real-time fluorescence quantitative RT-PCR method(RT-qPCR)was established.This method could be used to detect GVE specifically,and the sensitivity was about 100 times greater than conventional RT-PCR.An excellent linear correlation(R=0.997)and a high amplification efficiency(E=97.5%)were obtained from the standard curve of this method.Reproducibility tests revealed that the coefficients of variation in the intra-and inter-assay results were 0.31-1.03%and 0.82--262%,respectively,indicating a good reproduiblity.The RT-qPCR method could be used to detect GVE in a wide range of grapevine sample types.The detection rates of RT-qPCR for nearly all sample types from different positions and seasons were higher than conventional RT-PCR.The detection rates in spring,summer,autumn and winter increased gradually.Samples in autumn and winter were best for detection,and the detection rates of most samples were 80-100%,which were 10 to 40%higher than conventional RT-PCR.In general,old petioles and branches were the best tissues for GVE detection.The detection rates of these samples in each season were all 100%,which were 20 to 40%higher than conventional RT-PCR.The second highest rates were in the old leaf,with detection rates for RT-qPCR of 80-100%in all seasons,which were 20 to 40%higher than conventional RT-PCR.GVE could be difficultly detected in young leaves by conventional RT-PCR,and the detection rates were only 0-50%,while by RT-qPCR the rates could increase to 0--80%.A total of 33 out of 363 samples(belonging to 68 cultivars)from 20 regions in China were detected to be positive by RT-qPCR(9.1%),which was more than twice the rate of the conventional RT-PCR(3.9%).展开更多
Vitis vinifera 87-1 plants infected by grapevine fleck virus(GFkV) and grapevine rupestris stem pitting-associated virus(GRSPaV) were used as the plant materials for virus elimination treatment. This study evaluated t...Vitis vinifera 87-1 plants infected by grapevine fleck virus(GFkV) and grapevine rupestris stem pitting-associated virus(GRSPaV) were used as the plant materials for virus elimination treatment. This study evaluated the effects of ribavirin at different concentrations(15 and 25 μg mL^(–1);R15 and R25, respectively), thermotherapy(37°C;T), and the combination of ribavirin and thermotherapy(R15+T and R25+T) on eliminating viruses from grapevine plants in vitro. Both R15 and R25 had phytotoxic effects and weakened plant growth. Thermotherapy positively affected the growth of grapevine plants. Plant height was significantly greater in T, R15+T, and R25+T than in CK, R15 and R25. The proportion of dead plants after T, R15+T, and R25+T was 51.4, 11.4, and 8.6%, respectively. The survival rates of regenerated plants after all treatments were >68.0%. Ribavirin concentration and treatment time were related to the regeneration of shoot tips and elimination efficiencies of the two viruses. The survival rates of plants after R15+T for 30, 40, and 50 days were 97.3, 90.7, and 74.4%, respectively. The elimination rates of GRSPaV from plants in the three time quantum were 55.6, 84.6, and 93.8%, respectively. The elimination rate of GFkV was 23.9% higher in R25(35/44) than in R15(25/45), and that of GRSPaV was 7.0% higher in R25 than in R15. The combination of thermotherapy and chemotherapy was found to have a positive effect on the eradication of GFkV and GRSPaV, and R25+T for 50 days was able to completely eliminate the two viruses from in vitro grapevines.展开更多
Hungarian isolates of Grapevine leafroll associated virus 1 and 3 (GLRaV-1, GLRaV-3) were tested using serological (DAS-ELISA) and molecular (RT-PCR) methods. Five hundred bp long PCR products of the part of HSP70 gen...Hungarian isolates of Grapevine leafroll associated virus 1 and 3 (GLRaV-1, GLRaV-3) were tested using serological (DAS-ELISA) and molecular (RT-PCR) methods. Five hundred bp long PCR products of the part of HSP70 gene of one serologically positive GLRaV-1 and four GLRaV-3 isolates were sequenced. These sequences were applied for phylogenetic analysis and compared to foreign virus isolates of NCBI GenBank. Phylogenetic analysis of GLRaV-1 HSP70 gene supported the earlier results that it could be divided into two clusters: E and A. The Hungarian isolate 6.4.1 belonged to the group E. This isolate showed the highest homology with the AY754914 isolate from the Czech Republic. GLRaV-3 sequence data could cluster five groups. Hungarian 2.2;3.5 and 4.2 isolates were estimated belonging to the group II. The 1.4 isolate from the same vineyard as 2.2 varied in sequence data so it belonged to the other, IV. variant group with two South African, two Austrian and a Syrah isolate. According to the phylogenetic analysis, two variant groups occurred in Hungary. These isolates related with each other, but showed higher similarity of foreign counties. In some cases, they were similar to isolates of the neighbour countries such as Slovakia and Austria. It could be supposed that mainly the exchange of virus infected propagation materials caused the dissemination of GLRaV isolates.展开更多
Nursery plant propagation by grafting has been widely used in modern viticulture to minimize the damage caused by biotic and abiotic stresses.In grapevine(Vitis spp.),an effective way to control disease damage is to p...Nursery plant propagation by grafting has been widely used in modern viticulture to minimize the damage caused by biotic and abiotic stresses.In grapevine(Vitis spp.),an effective way to control disease damage is to provide producers and growers with pathogen-free stock plants.In this study,five grapevine rootstock varieties,‘SO4’,‘101-14’,‘5BB’,‘110R’and‘1103P’,were selected as explants to establish an in vitro culture protocol,and three species of grapevine viruses were tested by real-time RT-PCR.The results showed that MS medium supplemented with 0.2 mg/L IBA,1.0 mg/L 6-BA,0.5 mg/L KT,4.0 mg/L adenine for culture initiation,and WPM supplemented with 0.2 mg/L IBA for subculture were suitable for all five rootstock varieties,with multiplication coefficients ranging from 1.6 to 4.4.Virus testing showed that single RT-PCR was more effective for detecting the three viruses compared to double or triple RT-PCR.Only plantlets free from the aforementioned viruses were retained for subculture.Plantlets were hardened at room temperature under natural lighting in Hoagland solution for 2 weeks and transplanted to pots filled with mixed media in a greenhouse.This protocol is applicable for rapid propagation of the five grapevine rootstock varieties and can be used for commercial production of virus-free grapevine stocks.展开更多
基金This research was supported by the earmarked fund for China Agriculture Research System(CARS-29-bC-1).
文摘To develop a rapid and high-sensitivity method for detection of grapevine virus E(GVE),a SYBR Green based real-time fluorescence quantitative RT-PCR method(RT-qPCR)was established.This method could be used to detect GVE specifically,and the sensitivity was about 100 times greater than conventional RT-PCR.An excellent linear correlation(R=0.997)and a high amplification efficiency(E=97.5%)were obtained from the standard curve of this method.Reproducibility tests revealed that the coefficients of variation in the intra-and inter-assay results were 0.31-1.03%and 0.82--262%,respectively,indicating a good reproduiblity.The RT-qPCR method could be used to detect GVE in a wide range of grapevine sample types.The detection rates of RT-qPCR for nearly all sample types from different positions and seasons were higher than conventional RT-PCR.The detection rates in spring,summer,autumn and winter increased gradually.Samples in autumn and winter were best for detection,and the detection rates of most samples were 80-100%,which were 10 to 40%higher than conventional RT-PCR.In general,old petioles and branches were the best tissues for GVE detection.The detection rates of these samples in each season were all 100%,which were 20 to 40%higher than conventional RT-PCR.The second highest rates were in the old leaf,with detection rates for RT-qPCR of 80-100%in all seasons,which were 20 to 40%higher than conventional RT-PCR.GVE could be difficultly detected in young leaves by conventional RT-PCR,and the detection rates were only 0-50%,while by RT-qPCR the rates could increase to 0--80%.A total of 33 out of 363 samples(belonging to 68 cultivars)from 20 regions in China were detected to be positive by RT-qPCR(9.1%),which was more than twice the rate of the conventional RT-PCR(3.9%).
基金supported by the National Key R&D Program of China (2019YFD1001800)the China Agricultural Research System of MOF and MARA (CARS-29)。
文摘Vitis vinifera 87-1 plants infected by grapevine fleck virus(GFkV) and grapevine rupestris stem pitting-associated virus(GRSPaV) were used as the plant materials for virus elimination treatment. This study evaluated the effects of ribavirin at different concentrations(15 and 25 μg mL^(–1);R15 and R25, respectively), thermotherapy(37°C;T), and the combination of ribavirin and thermotherapy(R15+T and R25+T) on eliminating viruses from grapevine plants in vitro. Both R15 and R25 had phytotoxic effects and weakened plant growth. Thermotherapy positively affected the growth of grapevine plants. Plant height was significantly greater in T, R15+T, and R25+T than in CK, R15 and R25. The proportion of dead plants after T, R15+T, and R25+T was 51.4, 11.4, and 8.6%, respectively. The survival rates of regenerated plants after all treatments were >68.0%. Ribavirin concentration and treatment time were related to the regeneration of shoot tips and elimination efficiencies of the two viruses. The survival rates of plants after R15+T for 30, 40, and 50 days were 97.3, 90.7, and 74.4%, respectively. The elimination rates of GRSPaV from plants in the three time quantum were 55.6, 84.6, and 93.8%, respectively. The elimination rate of GFkV was 23.9% higher in R25(35/44) than in R15(25/45), and that of GRSPaV was 7.0% higher in R25 than in R15. The combination of thermotherapy and chemotherapy was found to have a positive effect on the eradication of GFkV and GRSPaV, and R25+T for 50 days was able to completely eliminate the two viruses from in vitro grapevines.
文摘Hungarian isolates of Grapevine leafroll associated virus 1 and 3 (GLRaV-1, GLRaV-3) were tested using serological (DAS-ELISA) and molecular (RT-PCR) methods. Five hundred bp long PCR products of the part of HSP70 gene of one serologically positive GLRaV-1 and four GLRaV-3 isolates were sequenced. These sequences were applied for phylogenetic analysis and compared to foreign virus isolates of NCBI GenBank. Phylogenetic analysis of GLRaV-1 HSP70 gene supported the earlier results that it could be divided into two clusters: E and A. The Hungarian isolate 6.4.1 belonged to the group E. This isolate showed the highest homology with the AY754914 isolate from the Czech Republic. GLRaV-3 sequence data could cluster five groups. Hungarian 2.2;3.5 and 4.2 isolates were estimated belonging to the group II. The 1.4 isolate from the same vineyard as 2.2 varied in sequence data so it belonged to the other, IV. variant group with two South African, two Austrian and a Syrah isolate. According to the phylogenetic analysis, two variant groups occurred in Hungary. These isolates related with each other, but showed higher similarity of foreign counties. In some cases, they were similar to isolates of the neighbour countries such as Slovakia and Austria. It could be supposed that mainly the exchange of virus infected propagation materials caused the dissemination of GLRaV isolates.
文摘Nursery plant propagation by grafting has been widely used in modern viticulture to minimize the damage caused by biotic and abiotic stresses.In grapevine(Vitis spp.),an effective way to control disease damage is to provide producers and growers with pathogen-free stock plants.In this study,five grapevine rootstock varieties,‘SO4’,‘101-14’,‘5BB’,‘110R’and‘1103P’,were selected as explants to establish an in vitro culture protocol,and three species of grapevine viruses were tested by real-time RT-PCR.The results showed that MS medium supplemented with 0.2 mg/L IBA,1.0 mg/L 6-BA,0.5 mg/L KT,4.0 mg/L adenine for culture initiation,and WPM supplemented with 0.2 mg/L IBA for subculture were suitable for all five rootstock varieties,with multiplication coefficients ranging from 1.6 to 4.4.Virus testing showed that single RT-PCR was more effective for detecting the three viruses compared to double or triple RT-PCR.Only plantlets free from the aforementioned viruses were retained for subculture.Plantlets were hardened at room temperature under natural lighting in Hoagland solution for 2 weeks and transplanted to pots filled with mixed media in a greenhouse.This protocol is applicable for rapid propagation of the five grapevine rootstock varieties and can be used for commercial production of virus-free grapevine stocks.
