Isolation of RNA from plants rich in secondary metabolites using commercial kits often results in contaminated preparations which are not suitable for downstream applications. Although many specific protocols appropriate for plants with a high content of phenolics, anthocyanins and polysaccharides have been developed, these are often expensive, time consuming and not applicable to different types of tissues. This study presents a simple and efficient modification of RNA extraction from different types of tissues using two commercial reagent kits. By simple improvement, we routinely obtained high-quality RNA of the following plants: the blackcurrant bush, black chokeberry bush, pear tree, apricot tree, apple tree, hardy kiwi, tangerine tree, highbush blueberry and cranberry plant.

Prune dwarf virus (PDV) isolates have been investigated for genetic diversity. Full-length nucleotide and amino acid sequences of viral coat protein from 23 isolates collected from different stone fruit trees (sour and sweet cherry trees, wild cherry tree, plum tree, almond tree, peach tree) and different countries (Poland, Italy, Germany, USA, Israel) were analysed and compared to 57 others available in GenBank. Comparison of all sequenced virus isolates revealed diversity of 86-100 % at nucleotide level and 79-100 % at amino acid level. The ratio of non-synonymous to synonymous polymorphic sites indicated that purifying selection dominated in case of PDV. However, six codons showed to be under strong positive selection, including the codon located inside the structure involved in RNAbinding activity.

Blueberry scorch virus (BlScV) is a member of the genus Carlavirus and one of the most widespread pathogens of highbush blueberry (Vaccinium corymbosum L.). The virus was first reported in the United States and has been reported in several countries in Europe, including Italy, Germany, the Netherlands, and Poland. Symptoms of scorch disease in highbush blueberry include necrosis of flower blossoms and leaves, shoot blight, and chlorosis. Sometimes BlScV infection is symptomless or limited to single blossoms and shoots, but all highbush blueberry cultivars are susceptible to virus infection. Cranberry (V. macrocarpon L.) and wild black huckleberry (V. membranaceum L.) are known as natural and symptomless hosts of BlScV (1). In June 2012, during the research concerning the occurrence of BlScV in plants outside Vaccinium sp., 15 leaf samples from five elderberry bushes (Sambucus nigra L., family Adoxaceae) were randomly collected from the Lodzkie region in Central Poland and three were positive in double antibody sandwich (DAS)-ELISA using specific antiserum (Agdia Inc., Elkhart, IN). To confirm the presence of the virus, total nucleic acid was extracted from ELISA-positive elderberry samples according to established protocol (T. Malinowski. Proc. 4th Int. EFPP Symposium, 445, 1996) and used in one step reverse transcription PCR. Primers were developed against the published NJ-2, BC-1, and BC-2 sequences of BlScV (GenBank Accession Nos. NC_003499, AY941198, and AY941199, respectively). The forward primer, RDP_1 (5′-ATGGCACTCACATACAGAAGTCC-3′), and the reverse primer, RDP_2 (5′-TGCCTCTTCAATGCACGATGTTC-3′), were used to amplify a 420-bp fragment of the RNA-dependent RNA polymerase gene of the virus. Amplicons of expected size were obtained from three DAS-ELISA-positive samples, while no products were observed for the negative control (DAS-ELISA-negative elderberry tissues). Sequence of one selected PCR product revealed 100, 88, and 87% nucleotide sequence identity and 100, 96, and 96% amino acid sequence identity with BC-2, NJ-2, and BC-1, respectively. BlScV-infected elderberry bushes were asymptomatic. As BlScV is transmitted by aphids in a non-persistent manner, infected elderberry bushes near highbush blueberry plantings may play an important role in virus spread. The potential for BlScV infection of plants outside family Ericaceae should be investigated. To the best of our knowledge, this is the first report of BlScV infecting elderberry. Reference: (1) R. R. Martin et al. Viruses 4:2831, 2012.

In this study, we determined the complete sequence of the genomic DNA of a Polish isolate of Blueberry red ringspot virus (BRRSV24) and compared it with a Czech (Darrow 5), and the US isolates of the virus and those of other Caulimoviridae family. The genomic DNA of BRRSV24 consists of 8,265 nucleotides and encodes eight open reading frames (ORFs). The sequence homologies of the eight ORFs of BRRSV24 were from 95 to 98% in respect of Darrow 5 and from 91 to 98% in respect of the US isolates at the amino acid level. This high level of amino acid sequence identity within the coding regions among the Czech, the US and Polish BRRSV isolates is suggestive of their common origin.Electronic supplementary materialThe online version of this article (doi:10.1007/s11262-011-0679-4) contains supplementary material, which is available to authorized users.

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Blueberry red ringspot virus (BRRSV) isolates have been investigated for genetic diversity. Nucleotide sequences of the coat protein (CP) gene of 19 isolates from Poland, Czech Republic, Slovenia and the United States were analysed. The nucleotide and amino acid sequence identity were 92–100% and 89–100%, respectively. Estimations of the distribution of synonymous and non‐synonymous changes indicated negative selection within the analysed CP gene and confirmed the genetic stability of the virus. At a capsid protein level, our results revealed BRRSV to be distinct from other, recombination‐prone pararetroviruses.

The genetic variability of blueberry scorch virus (BlScV) isolates from New York was determined within a portion of the RNA-dependent RNA polymerase gene and the triple gene block and coat protein (CP) genes. Phylogenetic analysis of 19 New York isolates and other isolates for which sequence information is available in GenBank revealed two distinct clades, regardless of the coding region analyzed, and limited variability within (0.029 ± 0.007) and between (0.183 ± 0.032) phylogroups. Recombination events were identified in the CP gene of three New York isolates, and codons of the five BlScV genes characterized were found to be under neutral or negative selective pressure.