Characterization of the first Rubus yellow net virus genome from blackberry.

Rubus yellow net virus (RYNV) is a badnavirus that infects Rubus spp. Mixed infections with black raspberry necrosis virus and raspberry leaf mottle virus cause raspberry mosaic, a disease that leads to significant losses and even plant death. RYNV has been reported in several European countries and the Americas yet there is substantial lack of knowledge, especially when it comes to virus diversity and the evolutionary forces that affect virus fitness outside its primary host, raspberry. Herein, we report the first RYNV episomal genome isolated from blackberry and this is the first report of the virus in Bosnia and Herzegovina. The isolate has five open reading frames (ORFs) and, when compared with other fully sequenced counterparts, showed 82-97% nucleotide pairwise identity. This communication adds to our limited knowledge on RYNV and addresses some of the gaps in RYNV genetics when it comes to the coding capacity of episomal isolates and the probability of the first fully sequenced isolate of the virus being integrated in the raspberry genome.

'Candidatus Phytoplasma convolvuli', a new phytoplasma taxon associated with bindweed yellows in four European countries.

Plants of Convolvulus arvensis exhibiting symptoms of undersized leaves, shoot proliferation and yellowing, collectively defined as bindweed yellows, were sampled in different regions of Europe and assessed for phytoplasma infection by PCR amplification using phytoplasma universal rRNA operon primer pairs. Positive results were obtained for all diseased plants. RFLP analysis of amplicons comprising the16S rRNA gene alone or the16S rRNA gene and 16-23S intergenic spacer region indicated that the detected phytoplasmas were distinguishable from all other previously described rRNA gene sequences. Analysis of 16S rRNA gene sequences derived from seven selected phytoplasma strains (BY-S57/11, BY-S62/11, BY-I1015, BY-I1016, BY-BH1, BY-BH2 and BY-G) showed that they were nearly identical (99.9-100% gene sequence similarity) but shared less than 97.5% similarity with comparable sequences of other phytoplasmas. Thus, BY phytoplasmas represent a new taxon whose closest relatives are stolbur phytoplasma strains and 'Candidatus Phytoplasma fragariae' with which they share 97.2% and 97.1% 16S rRNA gene sequence similarity, respectively. Phylogenetic analysis of 16S rRNA gene sequences confirmed that bindweed yellows phytoplasma strains collectively represent a distinct lineage within the phytoplasma clade and share a common ancestor with previously published or proposed 'Candidatus Phytoplasma' taxa within a major branch including aster yellows and stolbur phytoplasmas. On the basis of unique 16S rRNA gene sequences and biological properties that include a single host plant species and a geographical distribution limited to parts of Europe, the bindweed yellows (BY) phytoplasmas represent a coherent but discrete taxon, 'Candidatus Phytoplasma convolvuli', with strain BY-S57/11 (GenBank accession no. JN833705) as the reference strain.

Real-time quantitative PCR based sensitive detection and genotype discrimination of Pepino mosaic virus.

Over the last decade a new virus disease caused by Pepino mosaic virus (PepMV) has been threatening the tomato industry worldwide. Reliable detection is vitally important to aid disease control. Methods must be both sensitive and capable of detecting the range of distinct genotypes that have been identified. The development of five new reverse transcription real-time quantitative PCR (RT-qPCR) assays is described, which allow the detection of all known PepMV genotypes. The performance of the assays was evaluated on Peruvian, European tomato, Ch2 and US1 PepMV genotypes and optimised for both two- and one-step RT-qPCR detection formats. One-step RT-qPCR detected PepMV European tomato genotype particles at least two orders of magnitude more sensitively than ELISA. The method detected as little as one naturally infected seed among 5000 uninfected seeds. The genotype-specificity of the five assays was compared using PepMV isolates representing all of the different genotypes. The following genotype combinations were all discriminated successfully: European tomato-Peruvian, Ch2, and US1. In addition to its application for diagnostic purposes, the genotype-specificity and the quantitative potential of the method, makes it very useful for epidemiological studies or for studies evaluating resistance of plants to virus infection.