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.  

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 (R²=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 0.82–2.62%, respectively, indicating a good reproducibility.  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%). 

A total of 288 grapevine samples of 61 different grapevine cultivars, collected from 22 provinces and regions, were analyzed by reverse transcription-PCR (RT-PCR) for the presence of grapevine fabavirus (GFabV).  PCR detection results showed the incidences of GFabV were 12.8% (30/235) and 48.1% (25/52) in the asymptomatic and symptomatic vines, respectively.  The genetic diversity of GFabV isolates was analyzed based on partial nucleotide and encoded amino acid sequences of the RNA1 and RNA2 polyprotein genes.  Phylogenetic analyses of the RNA1 and RNA2 gene sequences divided the GFabV isolates into five well-defined groups.  Groups 1, 2, and 4 comprised only Chinese isolates.  This article represents the first report for the prevalence and genetic diversity of GFabV in grapevines grown in China.

Apple necrotic mosaic virus (ApNMV) was identified in crabapple trees with mosaic symptoms from Zaozhuang, Shandong Province, China, by reverse transcription polymerase chain reaction (RT-PCR) analysis.  The complete nucleotide sequences of one isolate from crabapple (ApNMV-Hai) and two isolates from apple (ApNMV-Hua and -Qu) were determined.  The sizes of genomic RNA1, 2 and 3 of the three isolates differed from those of the previously reported isolate ApNMV-P126 from Japanese apple, especially RNA3.  Compared with the nucleotide (nt) sequence of RNA3 in isolate P126, those in the Hai and Qu isolates were 7 and 33 nt shorter, respectively, and that of isolate Hua was 7 nt longer.  Alignment analyses showed that these differences in size were mainly due to differences in the lengths of the 5´ untranslated region (UTR) and the UTR region between the ORFs encoding the movement protein and the coat protein.  In the phylogenetic trees constructed using the full genomic sequences of RNA1, 2 and 3, the isolate Hai clustered into a group with the isolate Qu in the RNA1 tree, but formed an individual branch in the RNA2 and 3 trees.  Three recombination events were identified in the nucleotide sequences of RNA1 and 2 among the isolates ApNMV-Hai, -Hua, and -Qu.  This is the first report of the full genome sequence of ApNMV in crabapple.

 

We evaluated the role of pre-culture on survival rate of in vitro apple plants treated by thermotherapy.  Two apple cultivars, Malus×domestica cv. Pink Lady and Huafu, were used in the experiment and both have widely grown in China and infected with Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV).  Results in growth and virus titer of apple plants did not exhibit clear trends during five different periods of pre-culture.  Whilst, pre-culture increased the survival rate of the two cultivars during thermotherapy.  The survival rate of plants pre-cultured for 13 d (P-13d) was 14 and 51% higher than that of P-1d plants for Pink Lady and Huafu, respectively.  Moreover, pre-culture positively influenced regeneration of Huafu plants.  The average survival rate of plants regenerated from P-1d and P-4d was 20% lower than that regenerated from P-7d, P-10d, and P-13d.  The efficiency of virus eradication was determined by reverse-transcription PCR with two primer pairs for each virus, and the detection results showed that pre-culture scarcely affected apple virus elimination.  Despite the fact that the two viruses were hardly detected at 5 d of thermotherapy, no virus-free plants were found in the two cultivars of regenerated apple plantlets after 30-d treatment.