E. Paduch-Cichal

Biological and molecular characterization of Prunus necrotic ringspot virus isolates from three rose cultivars

Prunusnecrotic ringspot virus (PNRSV) is a rose and stone fruit tree pathogen. Three different PNRSV isolates, originating from three rose cultivars were studied. These PNRSV isolates were characterized using molecular techniques. Nearly the complete nucleotide sequence (1,630 nucleotides) of RNA3 of the isolate PNRSV-R1 has been determined (GenBank Acc. No. DQ003584). The sequence of the MP gene of the PNRSV-R1 isolate was determined, the first such results for a rose-derived PNRSV isolate. The reaction of PNRSV infection on test plants was also investigated. Cucumis sativus cv. Wisconsin, Cucurbita maxima cv. Buttercup and Cucurbita pepo cv. Melonowa Żółta appeared to be the most useful test plants for the differentiation of isolate-specific pathogenicity.

Occurrence and phylogenetic analysis of allexiviruses identified on garlic from China, Spain and Poland commercially available on the polish retail market

Garlic plants can be infected by different viruses including eight which belong to the genus Allexivirus, family Alphaflexiviridae. The aim of the research conducted was to detect and identify the allexiviruses GarV-A, GarV-B, GarV-C, GarV-D, GarV-X, GarMbFV and ShVX in garlic (Allium sativum L.) bulbs imported into Poland from China and Spain, and those growing in Poland by ELISA (enzyme-linked immunosorbent assay) as well as reverse transcription polymerase chain reaction (RT-PCR). Bulbs tested were infected with one or more viruses, including species not previously recorded in Poland. Present in various combinations from 146 garlic bulbs were 83 virus isolates representing Garlic virus A, B, D, X and GarMbFV. The most genetically distinct population comprises isolates of GarV-X, while isolates of GarV-B and GarV-D seem to be genetically more uniform. GarMbFV isolates are also genetically uniform, except for isolates from South Korea and Argentina. The high sequence identity of isolates from China, Spain and Poland, detected in this study, probably results from the transmission of the viruses via a vector.

Molecular characterization of polish blueberry red ringspot virus isolate

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.

FIRST REPORT OF GARLIC VIRUS A, B AND C IN GARLIC IN POLAND

Garlic (Allium sativum) can be infected by many viruses, in- cluding members of the genus Allexivirus (Adams et al., 2011). A virus survey was conducted in eleven garlic fields located in dif- ferent regions of Poland. Leaf and bulb samples from 80 plants showing mosaic, deformation and yellow stripes during the 2011- 2012 growing season were tested by DAS-ELISA using antibod- ies to Garlic virus A (GarV-A), Garlic virus B (GarV-B) and Gar- lic virus C (GarV-C) supplied by the Leibniz Institute DSMZ- German Collection of Microorganisms and Cell Cultures (Braun- schweig, Germany). Results indicated that 38 samples (47.5%) were infected with GarV-A, 49 samples (61.3%) with GarV-B and 14 samples (17.5%) with GarV-C. RT-PCR was used to con- firm the occurrence of the three viruses in symptomatic garlic with total RNA extracted from the leaves of 20 DAS-ELISA-posi- tive and five negative samples using the SpectrumTM plant total RNA kit (Sigma, USA), the Titan one tube RT-PCR system (Roche, Switzerland) and primer pairs designed in the conserved region of ORF5 (coat protein) and ORF6 (nucleic acid binding protein) of GarV-A (ACPF/ACPR 5’-ATGTCGAATC- CAACTCAGTCG-3’ and 5’-AGACCATGTTGGTGGCGCG- 3’), GarV-B (BCPF/BCPR 5’-TGACGGGCAAACAGCA- GAATAA-3’ and 5’-ATATAGCTTAGCGGGTCCTTC-3’) and GarV-C (CCPF/CCPR 5’-TTGCTACCACAATGGTTCCTC-3’ and 5’-TACTGGCACGAGTTGGGAAT-3’). Products of the ex- pected size (444 bp for GarV-A, 576 bp for GarV-B and 679 bp for GarV-C) were amplified only from the DAS-ELISA-positive samples. To our knowledge, this is the first report of GarV-A, GarV-B and GarV-C in garlic in Poland.

FIRST REPORT OF LEEK YELLOW STRIPE VIRUS IN FOREIGN AND POLISH GARLIC PLANTS IN CENTRAL POLAND

Leek yellow stripe virus (LYSV), genus Potyvirus, family Potyviridae (King et al., 2011) is the most common and important virus infecting a wide range of Allium worldwide. The aim of this study was to detect and identify LYSV in leek and garlic plants originating in central Poland, and also materials from Belgium, Egypt, and Spain purchased in Polish markets in April 2014. Randomly collected 178 samples were tested by a double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA), according to the manufacturers instructions (DSMZ, Braunschweig, Germany). All leek plants tested negative for LYSV, whereas 31 of 120 garlic bulbs tested positive. The presence of LYSV was confirmed by reverse transcription (RT)-PCR using total RNA extracted with the silica capture method (Boom et al., 1990; Malinowski, 1997) and primers 1-LYSV/2-LYSV (Parrano et al., 2012) designed to amplify a part of the N-terminal domain of the coat protein (CP) gene of the virus (363 bases). A sequence of the partial CP genes of the 12 LYSV isolates was submitted to GenBank (Accession Nos KM032272-KM032283). BLAST analysis of Polish sequences showed 96-99% identity at the nucleotide and amino acid levels. Sequences of Egyptian isolates, first representatives from this locations, showed 92 and 95% nucleotide and amino acid identities, respectively. However Spanish isolates revealed 95% and 97% nucleotide and amino acid identities, respectively. To the best of our knowledge, this is the first report of LYSV in foreign and Polish garlic plants available for purchase in central Poland. The accurate identification of viruses present in garlic plants, especially in imported plant material, will help to use the appropriate strategies to reduce viral incidence in garlic-growing areas.