Detection of herbaceous-plant pararetrovirus in lichen herbarium samples.

Cauliflower mosaic virus (CaMV) - a plant pararetrovirus that naturally causes diseases in Brassicaceae and Solanaceae plant hosts worldwide - has been detected by PCR for the first time in herbarium samples of Usnea sp. lichens. The virus's presence in these lichens did not result in any micro- or macromorphological changes, and the herbarium records were classified as representative for the distinct species. Sequence analyses classified all the detected viruses into one lineage of CaMV isolates. We have shown here that herbarium samples could be a good source for virus study, especially where a longer time span is involved.

Reflects the coat protein variability of apple mosaic virus host preference?

Apple mosaic virus (ApMV) is a widespread ssRNA virus which infects diverse species of Rosales. The phylogenetic analysis of complete capsid protein gene of the largest set of ApMV isolates discriminated two main clusters of isolates: one cluster correlates with Maloideae hosts and Trebouxia lichen algae hosts; a second with hop, Prunus, and other woody tree hosts. No correlation was found between clusters and geographic origin of virus isolates, and positive selection hypothesis in distinct hosts was not confirmed: in all virus populations, purifying selection had occurred. GGT→AAT substitution resulted in Gly→Asn change inside the zinc-finger motif in the capsid protein was revealed specific for discrimination of the clusters and we hypothesise that could influence the host preference.

Molecular analysis of gooseberry vein banding associated virus.

UNLABELLED: The intraspecies variability of gooseberry vein banding associated virus (GVBaV) was analyzed by using 5 complete and 9 partial sequences and compared with other badnavirus species. GVBaV was recognized to be a monophyletic and very homogeneous species with up to 94% identities in distinct proteins. Analysis of non-synonymous and synonymous substitution ratios (dNS/dS) revealed higher values for ORF4 in comparison with other genes. This could reflect different evolutionary pressure upon this ORF. A highly variable region with possible diagnostic value has been localized in the intergenic region of the virus. KEYWORDS: badnavirus; complete genome; phylogeny.

New in vitro method for evaluating antiviral activity of acyclic nucleoside phosphonates against plant viruses.

A new method was developed for testing antiviral compounds against plant viruses based on rapidly growing brassicas in vitro on liquid medium. This method enables exchange of media containing tested chemicals in various concentrations and simultaneous evaluation of their phytotoxicity and antiviral activity. While using ribavirin as a standard for comparison, phytotoxicity and ability of the acyclic nucleotide analogues (R)-PMPA, PMEA, PMEDAP, and (S)-HPMPC to eliminate ssRNA Turnip yellow mosaic virus (TYMV) were evaluated by this method. Double antibody sandwich ELISA and real-time PCR were used for relative quantification of viral protein and nucleic acid in plants. Ribavirin had the most powerful antiviral effect against TYMV. On the other hand, (R)-PMPA and PMEA had no antiviral effect and almost no phytotoxicity compared to the control. (S)-HPMPC and PMEDAP showed moderate antiviral effect, accompanied by higher phytotoxicity. The tested compounds can be screened within 6-9 weeks in contrast to the 6 months for traditionally used explants on solid medium. The method enables large-scale screening of potential antivirals for in vitro elimination of viruses from vegetatively propagated crops and ornamentals.

First Report of Blueberry red ringspot virus in Highbush Blueberry in the Czech Republic.

A collection of highbush blueberry (Vaccinium corymbosum L.) cultivars planted in the field for propagation in South Bohemia was surveyed in May and July of 2009 for the occurrence of detrimental viruses. A total of 67 plants of 10 cultivars (Berkeley, Burlington, Blue Crop, Bluetta, Darrow, Duke, Gila, Jersey, Late Blue, and Northland), were observed for typical Blueberry red ringspot virus (BRRV) symptoms that appear as reddish ring spots and blotches on stems and fruits, exclusively on the upper surface of the older leaves but not the underside. Samples of leaves were collected and maintained at -20°C until used for DNA extraction, then assayed for BRRV infection using PCR. Controls originated from the same blueberry cultivars in vitro. DNA was extracted from leaf tissue with a NucleoSpin Plant II kit for isolating genomic DNA according to the manufacturer's instructions (Macherey-Nagel, Düren, Germany). Primer pair BRRV15/16, which amplified fragments of the reverse transcriptase gene (1), was used in PCR for BRRV detection. The program used for PCR amplification was 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 49°C for 30 s, and 70°C for 45 s, followed by a final extension at 70°C for 5 min. The total PCR volume of 25 µl contained 20 ng of DNA, 200 µmol liter-1 dNTPs, 0.5 µl of each primer BRRV15 and BRRV16 (20 pmol µl-1), 75 mM Tris-HCl pH 8.8, 20 mM (NH4)2SO4, 0.01% Tween 20, 2.5 mM MgCl2, 2.5 U of Taq Purple DNA polymerase, and stabilizers (Top-Bio Ltd., Prague, Czech Republic). Amplifications were conducted in an MJ Research (Waltham, MA) thermocycler. Aliquots (4 µl) of each PCR product were analyzed by electrophoresis in tris-acetate-EDTA buffer. No BRRV symptoms were observed on the plants in early spring, yet BRRV was detected in one symptom-free bush of cv. Darrow by PCR. In July, typical symptoms developed on that and another cv. Darrow bush that was also positive by PCR. DNA fragments of the expected sizes were amplified from total nucleic acid samples of both infected blueberry bushes using primers BRRV15/16, while no amplification products were detected in plants without symptoms. The amplicons obtained with primers BRRV15/BRRV16 were sequenced and revealed 97.5%-nt identity to the BRRV putative reverse transcriptase gene (GenBank Accession No. AF404509). The 845 nt of the amplicon has been deposited at GenBank under Accession No. HM107773. The disease was likely introduced in infected planting material, since no highbush blueberry plantations exist in the vicinity and V. corymbosum is not native to the Czech Republic. In conclusion, to our knowledge, this is the first report of Blueberry red ringspot virus (genus Soymovirus, family Caulimoviridae) in V. corymbosum L. in the Czech Republic. Symptom observation and PCR testing for BRRV should therefore, be incorporated into the certification scheme for highbush blueberry in the Czech Republic. Reference: (1) J. J. Polashock et al. Plant Dis. 93:727, 2009.

Occurrence of Potato virus X on hybrid dock in Czech Republic.

Hybrid dock of Uteush (Rumex patientia L. x Rumex tianschanicus A. Los., the family Polygonaceae) is a perspective high productive crop and in the last decade its farming area has continuously grown in Czech Republic. However, the introduction of this non-native perennial crop into a present plant production creates a new potential reservoir for some plant viruses. Also, the hybrid dock could become a host of currently uncommon or insignificant viruses. We screened two dock-farming localities situated in south-west and north-east part of the Czech Republic for the presence of potyviruses, potexviruses, and carlaviruses. In the south-west part of the country, we detected a high incidence of Potato virus X (PVX, the genus Potexvirus). In contrast, in the north-east part of the country we did not detect any dock plants infected with PVX. Next, two other viruses, Turnip yellow mosaic virus (TYMV) and Radish mosaic virus (RaMV) were mechanically inoculated and tested for their survival capacity and multiplication in the hybrid dock. Both viruses were detected 9 months after inoculation in the infected plants.

Investigating the sensitivity of a fluorescence-based microarray for the detection of fruit-tree viruses.

An oligonucleotide microarray for the detection of some fruit-tree viruses was designed and its theoretical detection limit was assessed using Cy3-labelled oligonucleotides. The real sensitivity of the microarray was compared for different kinds of fluorescently labelled targets: (a) cDNA and PCR amplified targets, (b) PCR amplified targets labelled using three different labelling methods. In the first case (a), the number of viral cDNA molecules was below the assessed detection limit of the microarray and only PCR amplified targets were detected. A second comparison (b), done on 3 selected viruses, included indirect labelling, the direct incorporation of labelled-dUTPs, and the use of Cy3-labelled primer. The targets labelled most intensively were produced by the Cy3-primer labelling (2 of 3 viruses) or by the indirect labelling method (1 of 3 viruses), the weakest signal showed targets labelled directly (all 3 viruses). The use of Cy3-primer labelling involved the simplest preparation and the lowest cost, however occasional weak cross-hybridization appeared. The indirect labelling method was of the highest specificity. The probes hybridizing near the 3-end of the targets showed the lowest intensities of fluorescent signal.

First Report of a Phytoplasma Affecting Tomato in Poland.

During 2006, two tomato plants exhibiting dwarfing, twisting of shoots and leaves, and virescence and phyllody of flowers were observed in a greenhouse in western Poland. Total genomic DNA was extracted from approximately 3.5 g of leaf midribs and petioles using the modified cetyltrimethylammoniumbromide (CTAB) buffer method (3). Direct PCR was done with universal phytoplasma primers P1/P7 for amplification of ribosomal 16S rDNA. The PCR product (1.8 kb) was diluted 1:30 with sterile distilled water and used as DNA template for nested PCR with primers R16F2n/R16R2 (1). The final product was an expected 1.2-kb rDNA fragment amplified from infected tomato tissues. The DNA extracted from a Vinca sp. infected with phytoplasma of 16SrI-B subgroup and from a healthy tomato plant were used as positive and negative assay controls, respectively. Restriction fragment length polymorphism analysis of the final PCR product (1.2 kb) using enzymes MseI, KpnI, AluI, HhaI, HpaII, RsaI, TaqI, and TruI (1) indicated that phytoplasma from the tomato belonged to the subgroup 16SrI-C (1,2). The sequence obtained from the final PCR product (746 bp) was deposited in the GenBank database under accession number EF164961. Multiple sequence alignments with sequences of phytoplasma available from GenBank were performed using ClustalW software. The analysis revealed that the Polish isolate was co-identical (100%) to two phytoplasma sequences (GenBank Accession Nos. AY839617 and DQ078304) that belong to 16SrI-C subgroup. To our knowledge, this is the first report of a phytoplasma affecting tomato plant in Poland. References: (1) I.-M. Lee et al. Inst. J. Syst. Bacteriol. 48:1153, 1998. (2) I.-M. Lee et al. Cell-wall free bacteria. Page 283 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et a.l, eds. The American Phytopathology Society, 2001. (3) A. C. Padovan et al. Austral. J. Grape Wine Res. 1:25, 1995.

Daphne mosaic virus (DapMV), a new potyvirus from Daphne mezereum in the Czech Republic.

Daphne shrubs with light green rings and mosaic on leaves contained flexuous filamentous virions (696 x 13 nm) and cylindrical inclusions typical of the subdivision III of Edwardson's classification for inclusions induced by members of the family Potyviridae. Decoration tests using antisera to 67 potyviruses revealed distant serological relations among chilli veinal mottle virus, Colombian datura virus, papaya ringspot virus, tobacco vein mottling virus and yam mosaic virus. The 3' terminal region of the virus genome was amplified by RT-PCR using primers specific for cloned and sequenced members of the family Potyviridae. The most similar sequences in the GenBank were those of isolates of wild potato mosaic virus (WPMV) and yam mild mosaic virus (YMMV), originating from Peru and Guadeloupe, respectively. The new sequence had 63.2% and 61.9% nucleotide identity to WPMV and YMMV in the coat protein gene. The results suggest that the Czech isolate from daphne should be regarded as a new member of the genus Potyvirus. The name daphne mosaic virus (DapMV) is suggested for this virus.

Complete nucleotide sequence of Radish mosaic virus RNA polymerase gene and phylogenetic relationships in the genus Comovirus.

The 3'-terminal part of RNA1 genome segment of Radish mosaic virus (RaMV) including complete RNA polymerase gene was sequenced. The 207 amino acids long polymerase is matured from a polyprotein precursor by cleavage at putative Q/H site by viral protease. The alignment of available amino acid sequences of RNA polymerase genes of comoviruses revealed a closest (55%) identity of RaMV to Red clover mottle virus (RCMV).

First Incidence of Plum Pox Virus on Apricot Trees in China.

Plum pox disease, caused by Plum pox virus (PPV), is the most severe virus disease of plums, apricots, and peaches. The disease causes heavy losses for fruit growers and the international trade of propagation materials and fresh fruits. PPV was first reported in Bulgaria in 1917 (1). It is now widespread in Europe and has been reported from Cyprus, Syria, Egypt, India, Kazakhstan, Chile, the United States, and Canada. Leaves on symptomatic apricot trees (Prunus armeniaca cvs. Hong Mei and Bai Mei and a selected genotype) in the Hunan Province of China showed typical yellow rings and diffused chlorotic spots. Samples from three suspected trees were repeatedly analyzed using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) in the summers of 2001-2003. PPV was detected in leaves, bark, and leaf buds of all three trees using ELISA with polyclonal and monoclonal antibodies provided by M. Navratil, Palacky University, Olomouc, Czech Republic (3). The results were confirmed using RT-PCR amplification of a 243-bp of the coat protein gene with a PPV-specific primer pair (2). BLAST analysis of two RT-PCR product sequences (GenBank Accession Nos. AY750961 and AY795603) showed 100% homology to multiple sequences of the PPV-D strain (GenBank Accession Nos. X81080, AF440743, and AF401295). The third sequence (GenBank Accession No. AY795602) had a C at position 112 rather than the T found in the other sequences. The ELISA, RT-PCR, and sequence results indicate that PPV-D was present in the apricot trees. To our knowledge, this is the first indication of PPV occurrence in China. This sporadic incidence of PPV on apricot trees requires addressing problems with the occurrence and spread of plum pox diseases in China and starting an eradication program. References: (1) D. Atanasoff. Annu. Univ. Sofia Fac. Agron. et Sylvic. 11:49, 1932. (2) T. Candresse et al. Phytopathology. 88:198, 1998. (3) I. Hilgert et al. Hybridoma. 12:215, 1993.

Comparison of genetic variability between Czech and foreign isolates of phytopathogenic bacteria Clavibacter michiganensis subsp. sepedonicus by Rep-PCR technique.

Repetitive-sequence-based polymerase chain reaction (Rep-PCR) method was used for analysis of genetic variability among bacterial populations from different world locations. Collection of 26 Czech and 13 foreign strains of Clavibacter michiganensis subsp. sepedonicus was amplified using BOX primer targeting to repetitive motif occurring in eubacterial genomes. Genetic fingerprints were visually compared and statistically evaluated by cluster analysis. Genetic similarity was estimated to be approximately 80% among all tested strains. Populations of these bacteria seem to be highly homogeneous; potential influence of geographic origin was not confirmed.

Remarkable variability of apple mosaic virus capsid protein gene after nucleotide position 141.

Eight new sequences of European isolates from almond, apple, hop, prune and pear of the Apple mosaic ilarvirus (ApMV) capsid protein gene are presented. A consensus sequence was established as having 654 nucleotides (nt) and two American and two European isolates were identified to have insertions 6 to 15 nucleotides after nt position 141. The insertion resulted in the American isolate A inframeshift repaired with two point insertions 17 and 68 nt downstream. The RNA around the insertion point can potentially form a stable secondary structure with three hairpins. The insertions could stabilise this structure or could be neutral. The predicted folding of the translated protein is not influenced by the insertions or frameshift, and we speculate that the region after nt position 141 is without reasonable selection pressure and represents a hot spot for the accumulation of insertion mutations in ApMV.

Nucleotide sequences of 5'-terminal parts of coat protein genes of various isolates of NTN strain of potato virus Y.

Sequences of the first 300 nucleotides of coat protein (CP) genes of 7 isolates of NTN strain of potato virus Y (PVY, PVY(NTN)) were determined and compared with analogous published sequences of various isolates and strains of PVY. The sequence identity among the sequenced isolates ranged from 96 to 100%. The differences were found at different positions. The nucleotide sequence of this part of CP gene seems to be very conservative among the isolates tested that means that PVY(NTN) is the evolutionary youngest among all PVY strains.

Preparation of recombinant coat protein of Prunus necrotic ringspot virus.

The coat protein (CP) gene of Prunus necrotic ringspot virus (PNRSV) was cloned into pET 16b vector and expressed in Escherichia coli. CP-enriched fractions were prepared from whole cell lysate by differential centrifugation. The fraction sedimenting at 20,000 x g for 30 mins was used for preparation of a rabbit antiserum to CP. This antiserum had a titer of 1:2048 and reacted in a double-antibody sandwich ELISA (DAS-ELISA).

Variability and molecular typing of the woody-tree infecting prunus necrotic ringspot ilarvirus.

The 3'-part of the movement protein gene, the intergenic region and the complete coat protein gene of sixteen isolates of Prunus necrotic ringspot virus (PNRSV) from five different host species from the Czech Republic were sequenced in order to search for the bases of extensive variability of viroses caused by this pathogen. According to phylogenetic analyses all the 46 isolates sequenced to date split into three main groups, which correlated to a certain extend with their geographic origin. Modelled serological properties showed that all the new isolates belong to one serotype.

Potato virus A detection by reverse transcription-polymerase chain reaction.

Simple and reliable procedure for sample preparation and reverse transcription-polymerase chain reaction (RT-PCR) detection of potato virus A (PVA) is described. PVA-specific primers used in the RT-PCR defined a target sequence of 321 bp and did not produce amplification product(s) with potato virus Y.