M. S. Szyndel

P0 proteins of European beet-infecting poleroviruses display variable RNA silencing suppression activity.

Post-transcriptional gene silencing (PTGS), or RNA silencing, is one of the key mechanisms of antiviral defence used by plants. To counter this defence response, viruses produce suppressor proteins that are able to inhibit the PTGS pathway or to interfere with some of its function. The aim of this study was to evaluate the RNA silencing suppressor (RSS) activity of P0 proteins from selected European isolates of the beet-infecting poleroviruses beet chlorosis virus (BChV) and beet mild yellowing virus (BMYV) using two different experimental systems: (i) agro-infiltration of Nicotiana benthamiana green fluorescent protein-positive plants and (ii) mechanical inoculation of Chenopodium quinoa using a beet necrotic yellow vein virus (BNYVV, genus Benyvirus) RNA3-based replicon. The results demonstrated that P0 of most BMYV isolates exhibited RSS activity, although at various efficiencies among isolates. Conversely, P0 of BChV isolates displayed no RSS activity in either of the two systems under the experimental conditions used. These results are the first reported evidence that P0 proteins of two closely related beet poleroviruses show strain-specific differences in their effects on RNA silencing.

Aphid transmissibility of different European beet polerovirus isolates

Different field isolates of the ‘beet poleroviruses’ Beet mild yellowing virus (BMYV) and Beet chlorosis virus (BChV) (genus Polerovirus, family Luteoviridae) collected in France and Poland were evaluated for transmissibility from and to sugar beet plants by different aphid species. In general, both BMYV and BChV were efficiently transmitted by Myzus persicae and by a French clone of Macrosiphum euphorbiae. In contrast, transmissibility of the two poleroviruses by an English clone of M. euphorbiae was evidently weaker, although the aphid samples contained the virus as demonstrated by RT-PCR. None of the BMYV or BChV isolates was transmitted by Aphis fabae or Myzus ascalonicus. In attempting to correlate biological properties with molecular variations, the RT proteins were sequenced. Some amino acid point variations, presumably affecting aphid transmissibility, were identified.

Phylogenetic relationships and the occurrence of interspecific recombination between beet chlorosis virus (BChV) and Beet mild yellowing virus (BMYV)

Samples containing two viruses belonging to the genus Polerovirus, beet chlorosis virus (BChV) and beet mild yellowing virus (BMYV), were collected from French and Polish sugar beet fields. The molecular properties of 24 isolates of BChV and BMYV were investigated, and their genetic diversity was examined in the coat protein (CP)- and P0-encoding genes. For the first time, we have demonstrated that beet polerovirus populations include recombinants between BChV and BMYV containing breakpoints within the CP gene. Moreover, a partial correlation between geographic origin and phylogenetic clustering was observed for BMYV isolates.

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 on the natural occurrence of Beet chlorosis virus in Poland.

Yellowing symptoms on sugar beet (Beta vulgaris L.) are caused by several viruses, especially those belonging to the genus Polerovirus of the family Luteoviridae, including Beet mild yellowing virus (BMYV) and Beet western yellows virus (BWYV), and recently, a new species, Beet chlorosis virus (BChV), was reported (2). To identify Polerovirus species occurring in beet crops in Poland and determine their molecular variability, field surveys were performed in the summer and autumn of 2005. Leaves from symptomatic beet plants were collected at 26 localities in the main commercial sugar-beet-growing areas in Poland that included the Bydgoszcz, Kutno, Lublin, Poznań, Olsztyn, and Warszawa regions. Enzyme-linked immunosorbent assay (ELISA) tests (Loewe Biochemica GmbH, Sauerlach, Germany) detected poleroviruses in 23 of 160 samples (approximately 20 samples from each field). Multiplex reverse-transcription polymerase chain reaction (RT-PCR) (1) (GE Healthcare S.A.-Amersham Velizy, France) confirmed the presence of poleroviruses in 13 of 23 samples. Nine of twenty sugar beet plants gave positive reactions with BChV-specific primers and three with primers specific to the BMYV P0 protein. Two isolates reacted only with primer sets CP+/CP, sequences that are highly conserved for all beet poleroviruses. Leaf samples collected from three plants infected with BChV were used as inoculum sources for Myzus persicae in transmission tests to suitable indicator plants including sugar beet, red beet (Beta vulgaris L. var. conditiva Alef.), and Chenopodium capitatum. All C. capitatum and beet plants were successfully infected with BChV after a 48-h acquisition access period and an inoculation access period of 3 days. Transmission was confirmed by the presence of characteristic symptoms and by ELISA. Amino acid sequences obtained from each of four purified (QIAquick PCR Purification kit, Qiagen S.A., Courtaboeuf, France) RT-PCR products (550 and 750 bp for CP and P0, respectively) were 100% identical with the CP region (GenBank Accession No. AAF89621) and 98% identical with the P0 region (GenBank Accession No. NP114360) of the French isolate of BChV. To our knowledge, this is the first report of BChV in Poland. References: (1) S. Hauser et al. J. Virol. Methods 89:11, 2000. (2) M. Stevens et al. Mol. Plant Pathol. 6:1, 2005.

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.