Jana Fránová

Association of phytoplasmas and viruses with malformed clovers.

Plants ofTrifolium spp. exhibiting two different kinds of symptoms — phyllody associated with yellowing/reddening, and dwarf growth habit without floral abnormalities — were observed in several areas of the Czechia. Nested polymerase chain reaction (PCR) with phytoplasma specific primers, and restriction fragment length polymorphism (RFLP) analyses of 16SrDNA revealed that phyllody ofT. repens was associated with phytoplasmas belonging to the 16SrI-C subgroup. Similar symptoms inT. hybridum andT. pratense plants revealed the presence of phytoplasmas belonging to two subgroups: 16SrI-C and 16SrIII-B. Dwarf disease of cultivatedT. pratense plants was associated with more than one agent: 11 of 20 plants examined by PCR/RFLP analysis revealed the presence of phytoplasmas belonging to four distinct subgroups: 16SrI-B, 16SrI-C, 16SrIII-B and 16SrX-A. Moreover, two kinds of bacilliform virions were observed in ultrathin sections of 15T. pratense plants. Particles occurred mostly in the parenchymatous cells of vascular bundles and were located in the cytoplasm as aggregates within an extended network of membranous cisternae. Phytoplasmas and rhabdoviruses occurred singly, and both together or in co-presence with filamentous virus-like particles.

Genetic diversity of Czech ‘ Candidatus Phytoplasma mali’ strains based on multilocus gene analyses

In several European countries apple trees are affected by apple proliferation disease, which is usually associated with the presence of ‘Candidatus Phytoplasma mali’. During 2010, samples from several apple trees displaying proliferation symptoms were collected throughout the Czech Republic to verify identity of phytoplasmas detected in association with the disease. The majority of the 74 apple trees examined using molecular tools were positive for ‘Ca. P. mali’ presence. The 16S–23S ribosomal genes, the ribosomal protein genes and the nitroreductase and rhodonase like genes were then studied to verify phytoplasma strain variability on multigenic bases. Two RFLP profiles and correspondingly two genetic lineages were found in the PCR-amplified fragments covering the 16S–23S rDNA spacer region. ‘Ca. P. mali’ strains belonging to rpX-A subgroup were identified in the majority of the apple tree sampled, whereas phytoplasmas belonging to the rpX-B subgroup were distributed sporadically. The apple proliferation subtypes AP-15 and AT-2 exhibited nearly equal occurrence; the AT-1 subtype and a mixture of the two or all three of the AP subtypes were infrequently found. The PCR/RFLP results were confirmed by nucleotide sequence analyses of selected ‘Ca. P. mali’ strains.

Identification and characterization of a new member of the genus Luteovirus from cherry

The complete genomic sequence of a new virus from cherry trees was determined. Its genome is 5857 nt long and resembles that of members of the genus Luteovirus in its genomic organization and nucleotide sequence. Based on the species demarcation criteria for luteoviruses, the virus represents a new luteovirus species. Furthermore, a 47-nt-long inverted repeat was found at the 3’ end of its genome. The virus has been provisionally named cherry-associated luteovirus (ChALV) and is the fourth member of the family Luteoviridae reported to naturally infect woody plants.

Phytoplasma associated with witches'-broom disease of Ulmus minor MILL . in the Czech Republic: Electron microscopy and molecular characterization.

Visual inspections of elm trees in south Moravia in 1997–2007 revealed a rare occurrence of plants with smaller and cowl-forming leaves on some twigs, i.e. a feature resembling witches’-broom disease observed on the end of twigs. The presence of phytoplasma-like bodies was observed by transmission electron microscopy of phloem tissue. On the other hand, no phytoplasmas were found in asymptomatic trees. Nucleic acids extracted from these plants were used in nested-PCR assays with primers amplifying 16S rRNA sequences specific for phytoplasmas. Sequence analyses of the 16S–23S ribosomal operon (1852 bp) allowed for the classification of the detected phytoplasmas in the elm yellows group, but its position remained on the boundary of the 16SrV-A and 16SrV-C ribosomal subgroups. Sequence analyses of the ribosomal protein of the rpl22-rps3 and secY genes lead to further classification and revealed the phytoplasmas’ affiliations to the ‘Candidates Phytoplasma ulmi’. Some exceptions in unique oligonucleotide sequences defined for ‘Ca. Phytoplasma ulmi’ were found in the Czech isolate. This is the northernmost confirmed occurrence of phytoplasma on elm trees within Europe.

Leek Proliferation: A New Phytoplasma Disease in the Czech Republic and Italy

During the summer 1996, twelve of twenty-eight leek plants located in a garden near České Budějovice, South Bohemia exhibited symptoms typical of diseases associated with phytoplasmas. In summer 1998 similar symptoms were detected in leek plants in a field used for seed production located in Romagna, North Italy. In both cases the plants were established in the spring of the previous year. Plants showed flower abnormalities: stamen elongation, anther sterility, pistil proliferation, as well as poor, if any, seed production. Phytoplasma-like structures were detected by scanning and transmission electron microscopy in phloem sieve elements in the Czech diseased plants, but not in healthy ones. Nested-PCR amplifications of extracted DNA with phytoplasma-specific oligonucleotide primer pairs confirmed the presence of phytoplasmas in these plants at low concentrations. Restriction fragment length polymorphism analyses of amplified ribosomal sequences allowed the identification of detected phytoplasmas: all the samples from the Czech Republic contained aster yellows related phytoplasmas (16SrI-B) while in the Italian samples aster yellows related phytoplasmas (16SrI-B) together with stolbur related phytoplasmas (16SrXII-A) were identified. This is the first report of detection and identification of a phytoplasma disease of leek in the Czech Republic and Italy.

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

Summary.Daphne shrubs with light green rings and mosaic on leaves contained flexuous filamentous virions (696 × 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 genome sequence of Daphne mosaic virus – a potyvirus from an ornamental shrub related to papaya leaf distortion mosaic virus

Since 1938, when the first observation of a virus-like disease in daphne was recorded [4], about 15 different viruses have been found in different Daphne species. Among them, two potyviruses have been detected: daphne Y virus in New Zealand, with a particle length of 733 nm [7], and a second virus, also named daphne Y virus, in Germany, with particles of 750 nm in length and serologically related to turnip mosaic virus [10]. The filamentous virus found in the Czech Republic has particles of modal length equal to 696 nm and induces the formation of inclusion bodies that are identical to those of the German isolate, but it is not mechanically transmissible to Chenopodium quinoa and C. amaranticolor, in contrast to the German potyvirus. We named the Czech virus Daphne mosaic virus (DapMV), characterised it thoroughly, and based on the nucleotide sequence of the coat protein (CP) gene, proposed it to be a member of a new species of the genus Potyvirus [8]. Here, we report the complete sequence of DapMV and its sequence relationships.

Association of 'Candidatus phytoplasma asteris' with yellowing and phyllody of Plantago lanceolata.

Long plantains (Plantago lanceolata L.) with symptoms resembling those associated with phytoplasma infection were observed repeatedly during the period 2000–2008 in southern Bohemia (Czech Republic). The symptoms of the plants were leaf yellowing, stunted growth, flower phyllody and lack of seed production. Transmission electron microscopy showed phytoplasmas in the sieve cells of affected plants but not in healthy ones. Association of phytoplasmas with the disease was confirmed by polymerase chain reaction using phytoplasma-specific universal ribosomal primers R16F2n/R16R2. An amplification product of the expected size (1.2 kb) was observed in all samples of the symptomatic long plantains. The restriction profiles obtained from digestion of the PCR products with three endonucleases (AluI, HhaI, MseI) showed that the phytoplasmas infecting long plantains in the Czech Republic were indistinguishable from those belonging to the aster yellows group (subgroup 16SrI-B). Sequence analysis of 1748 bp of the ribosomal operon indicated that the closest related phytoplasma was that associated with ‘Rehmannia glutinosa var. purpurea’, originating also in Bohemia. This is the first report of the natural occurrence of ‘Candidatus Phytoplasma asteris’ in plants of P. lanceolata.

Variability Studies of Two Prunus-Infecting Fabaviruses with the Aid of High-Throughput Sequencing

During their lifetime, perennial woody plants are expected to face multiple infection events. Furthermore, multiple genotypes of individual virus species may co-infect the same host. This may eventually lead to a situation where plants harbor complex communities of viral species/strains. Using high-throughput sequencing, we describe co-infection of sweet and sour cherry trees with diverse genomic variants of two closely related viruses, namely prunus virus F (PrVF) and cherry virus F (CVF). Both viruses are most homologous to members of the Fabavirus genus (Secoviridae family). The comparison of CVF and PrVF RNA2 genomic sequences suggests that the two viruses may significantly differ in their expression strategy. Indeed, similar to comoviruses, the smaller genomic segment of PrVF, RNA2, may be translated in two collinear proteins while CVF likely expresses only the shorter of these two proteins. Linked with the observation that identity levels between the coat proteins of these two viruses are significantly below the family species demarcation cut-off, these findings support the idea that CVF and PrVF represent two separate Fabavirus species.

Identification and molecular characterization of a novel varicosa-like virus from red clover

During aetiological study of diseased red clover (Trifolium pratense L.) using high throughput sequencing, a novel virus with a 10 kb genome divided into two segments was discovered. The virus, tentatively named red clover associated varicosavirus (RCaVV), is phylogenetically related to classifiable members of the genus Varicosavirus (family Rhabdoviridae, order Mononegavirales). Analysis of mRNA levels from the individual RCaVV genes suggested possible differences in transcription regulation between rhabdoviruses with divided and undivided genomes.