Prevalence of Viruses in Commercial Wine Grape Vineyards in Georgia

Virus diseases are major constraints to Vitis vinifera wine grape production worldwide. Grapevine leafroll-associated virus 3 (GLRaV-3) was first confirmed in Georgia in 2008. The negative impacts of GLRaV-3, such as decreased fruit yield and quality, were confirmed from samples taken in 2009 and 2010. In light of these findings, studies were initiated to determine the prevalence and types of grapevine viruses present in vineyards in Georgia. Five vineyard blocks were visited during August and September of 2011 and 2012. Leaf samples were collected from a total of 50 grapevines showing symptoms of and suspected as being infected by grapevine leafroll disease (GLD). Samples from individual grapevines were extracted and tested separately for 20 grapevine viruses listed in standard virus indexing programs. Reverse transcription polymerase chain reaction (RT-PCR) or PCR, depending on the nature of the virus genome, was used to detect these viruses with species-specific primers. The results showed the presence of GLRaV-1, -2, -3, and -4 (strains 4, 5, and 6), grapevine virus B, grapevine rupestris stem pitting-associated virus, and grapevine red blotch virus. Many vineyards had multiple viruses detected from individual grapevines showing typical GLD or GLD-like symptoms.

Ex Vitro Rooting of American Chestnut Improves Acclimatization Survival and Plantlet Quality

Tissue culture of plants has many applications, from producing genetically identical horticultural varieties, to production of secondary metabolites, to virus indexing, and most relevantly, developing novel traits by genetic transformation. Using Agrobacterium-mediated transformation on somatic embryos, blight-resistant American chestnuts [Castanea dentata (Marsh.) Borkh.] have been developed as shoot cultures in plant tissue culture. Rooting tissue-cultured shoots and acclimatizing the rooted plantlets are key steps in tree production. In this study, in vitro and ex vitro rooting methods were compared. The ex vitro method resulted in a lower initial rooting percentage but an overall higher survival percentage, resulting in higher potted plant production. The higher survival was likely due to partial acclimatization taking place before the plantlets were transplanted into potting mix. After 8 weeks, plantlets rooted via the ex vitro method were taller, and had more, and larger, leaves than the in vitro-rooted plantlets. These trees are currently in high demand for inoculation studies for federal regulatory review and eventually for restoration of this keystone species to its native habitat.

Evaluation of Tissue Culture Seedlings for Their Genetic Fidelity and Virus Indexing in Sugarcane

Plant tissue culture technique is a powerful tool for rapid and large scale multiplication of virus free seed material. Clonal fidelity of the in vitro raised plants was carried out using RAPD. A total of fifteen primers were used for detecting polymorphism by RAPD analysis and the results revealed that all the tissue culture plants were grouped into one cluster and indicated that all the tissue culture developed plantlets are true-to-type. There are no somaclonal variations and are genetically identical with the mother plant. Virus indexing through ELISA indicated that the tissue culture developed seedlings does not contain the protein particles of ScMV and are free from the viral disease. The wells coated with leaf samples with suspected ScMV infection (97 A 85, C3) gave visual confirmation by yellow colour change, with OD 405 values of 0.526. As the reading is 2 times more than the negative control (OD 0.215) we can presume mild infection of mosaic in the test sample with lower titre of ScMV. All the ScMV +ve controls showed significant higher OD values of more than 2.515 confirming the earlier observations. Thus, viral detection using DAS-ELISA method can be used for detection and indexation in large scale sugarcane crop especially for in vitro regenerated plants with relatively cheaper cost and faster for supplying virus free seedlings to farming community in a large scale.

Virus elimination in potato through meristem culture followed by thermotherapy

Virus elimination in potato through meristem culture followed by thermotherapy and virus indexing was studied. Three levels of thermotherapy, viz. 27±1°C (control), 30±1°C and 35±1°C, sixteen combinations of BAP (Benzyl Amino Purine) plus GA3 (gibberellic Acid) concentrations viz. 0.0+0.0 (control), 0.0+0.2, 0.0+0.4, 0.0+0.6, 1.5+0.0, 1.5+0.2, 1.5+0.4, 1.5+0.6, 3.0+0.0, 3.0+0.2, 3.0+0.4, 3.0+0.6 , 4.5+0.0, 4.5+ 0.2, 4.5+0.4 and 4.5+0.6 were used in this study in three potato varieties viz. Diamant, Heera and Lalpakri. Among the thermo therapies, 27±1°C showed the highest (24.55) survival response followed by 30±1°C, 35±1°C, respectively. The poorest (20.47) survival response of meristem derived plantlets was noticed in 35±1°C which gave the highest percentage (43.79) of virus free plantlets followed by 30±1°C. The best (25.85%) survival response was found in Lalpakri and the lowest (19.08%) survivality was recorded in Diamant. The highest (33.27) percentage of PVY (Potato Virus Y) free plantlets was observed in Heera. The combined treatment 3.0 mg L-1 BAP and 0.2 mg L-1 GA3 showed the highest (63.39) percentage of virus free plantlet production followed by 4.5 mg L-1 BAP and 0.2 mg L-1 GA3DOI: http://dx.doi.org/10.3329/sja.v11i1.18376 SAARC J. Agri., 11(1): 71-80 (2013)

First Report of Grapevine leafroll-associated virus 5 in Spain

Grapevine leafroll-associated viruses (GLRaVs) cause significant reductions in yield and quality in the wine industry worldwide. At least nine different GLRaVs have been found in different regions of the world. In the process of virus indexing of candidate grapevine clones for certification, which includes grafting of scions onto rootstocks, we observed strong leafroll symptoms 1 year after grafting with one vine of cv. Estaladina in Castilla y León, Spain and one vine of cv. Tempranillo in La Rioja, Spain, collected in 2008 and 2007, respectively. Both vines tested positive by real-time reverse transcription (RT)-PCR with TaqMan probes specific for Grapevine leafroll-associated virus 5 and double-antibody sandwich (DAS)-ELISA with a mix of monoclonal antibodies that recognizes GLRaV-4, 5, 6, 7, and 9 (Bioreba, Reinach, Switzerland). RNA extracts of both GLRaV-5 positive vines were analyzed by conventional RT-PCR with a pair of consensus degenerated primers derived from GLRaV-5 hsp70 sequences available in GenBank: LR5HYF (5′-TGGGATGAAYAARTTCAATGC-3′) and LR5HYR (5′-TGAAATTCCTCATRTARGAGC-3′) that amplified a 250-bp fragment. Amplicons were cloned and the comparison of the amino acid sequences (Estaladina isolate, Est110: Accession No. HM208622; Tempranillo isolate, Tem020: Accession No. HM208618) showed in the case of the Est110 isolate, 100 and 82.6% identity, respectively, with the homologous genes of one GLRaV-5 isolate from the United States (AF233934 [3]) and Argentina (EU815935 [2]). For isolate Tem020, the hsp70 gene showed 97.1 and 81.2% amino acid identity with the homologous hsp70 genes of the United States and Argentina isolates. The coat protein (cp) genes of both isolates were also amplified and cloned using the specific GLRaV-5 primers, LR53413 (5′-CGTGATACAAGGTAGGACAACCGT-3′) and LR53843 (5′-CTTGCACTATCGCTGCCGTGAAT-3′), designed according to the sequence of AF233934. Fragments were of the expected size (430 bp) and the nucleotide sequences were obtained (Est110: Accession No. HM363522; Tem020: Accession No. HM363523) and used for pairwise nucleotide comparisons. The Est110 isolate showed 96.7 and 97.5% amino acid identity with the isolates from the United States and Argentina, respectively, while the Tem020 isolate showed 94.8 and 95.6% identity, respectively. Amino acid identity of Est110 and Tem020 cp genes was 100% when compared with the homologous genes of isolates AF233934 and EU815935. To our knowledge this is the first report of GRLaV-5 in Spain. Since 2008, we have detected eight additional vines positive for this virus in 200 clones analyzed for certification, suggesting that the incidence of GLRaV-5 in Spain could be widespread. This research indicates that virus indexing for GLRaV should be included in certification schemes for grapevine candidate clones (1) in Spain. References: (1) Anonymous. OEPP/EPPO Bull. 38:422, 2008. (2) S. Gomez Talquenca et al. Virus Genes 38:184, 2009. (3) F. Osman et al. J. Virol. Methods 141:22, 2007.