A novel mitovirus isolated from the phytopathogenic fungus Botryosphaeria dothidea.

In this study, we isolated and determined the complete genome sequence of a novel mitovirus, "Botryosphaeria dothidea mitovirus 2" (BdMV2), from the phytopathogenic fungus Botryosphaeria dothidea isolate DT-5. BdMV2 has a genome 2,482 nt in length with an A+U content of 67%. The genome of BdMV2 contains a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) of 717 amino acids (aa) with a molecular mass of 81.86 kDa. A BLASTp comparison of the RdRp sequence showed the highest identity (66.67%) with that of Alternaria arborescens mitovirus 1 (AbMV1). Sequence comparisons and phylogenetic analysis revealed that BdMV2 is a new member of the genus Mitovirus of the family Mitoviridae.

Transmission studies of the newly described apple chlorotic fruit spot viroid using a combined RT-qPCR and droplet digital PCR approach.

The transmission of the apscaviroid tentatively named apple chlorotic fruit spot viroid (ACFSVd) was investigated using a one-step reverse-transcription (RT) droplet digital PCR assay for absolute quantification of the viroid, followed by quantification of relative standard curves by RT-qPCR. Our results indicate that ACFSVd is effectively transmitted by grafting, budding and seeds. No transmission has yet been observed to the viroid-inoculated pome fruit species Pyrus sp. and Cydonia sp. ACFSVd was detected in viruliferous aphids (Myzus persicae, Dysaphis plantaginea) and in codling moths (Cydia pomonella). The viroid was also detected systemically in the infected hemiparasitic plant Viscum album subsp. album (mistletoe).

Identification and characterization of an isolate of apple green crinkle associated virus involved in a severe disease of quince (Cydonia oblonga, Mill.).

A quince tree showing severe symptoms of a previously undescribed viral disease occurring in northern Apulia (Italy) was analysed using high-throughput sequencing of small RNA libraries, leading to the identification of a new strain of apple green crinkle associated virus (isolate AGCaV-CYD) showing peculiar traits. RT-PCR with specific primers detected AGCaV-CYD in consistent association with symptoms in the surveyed orchards. Molecular characterization of the reconstructed genome, together with phylogenetic analysis, showed it to be closely related to an AGCaV strain causing green crinkle disease in apple (AGCaV-AUR) and divergent from the type strain of apple stem pitting virus (ASPV-PA66).

VIRION MORPHOLOGY AND STRUCTURAL ORGANIZATION OF POLYVALENT BACTERIOPHAGES TT10-27 AND KEY.

Fine ultrastructure of polyvalent bacteriophages TT10-27 and KEY isolated from affected with fire blight disease plant tissues, was studied using electron microscopy. Phages have isometric heads connected to short complex tail (TT10-27, C1-morphotype) or long non-contractile tail (KEY B-1 morphotype). Maximum diameter of TT10-27 head, measured as the distance between opposite vertices, is 71.3 nm; tail tube of 22 nm in diameter and 9.0 nm in width is framed with 12 appendages that form flabellate structure of 47.0-58.6 nm in diameter. KEY features capsid of 78.6 nm in diameter and flexible non-contractile tail of 172.5 nm long, which ends with a conical tip. Due to a number of features phage TT10-27 was assigned to a group of N4-like phages of Podoviridae family. KEY is a representative of family Siphoviridae, the least freaquent group of Erwinia amylovora phages.

Control of pome and stone fruit virus diseases.

Many different systemic pathogens, including viruses, affect pome and stone fruits causing diseases with adverse effects in orchards worldwide. The significance of diseases caused by these pathogens on tree health and fruit shape and quality has resulted in the imposition of control measures both nationally and internationally. Control measures depend on the identification of diseases and their etiological agents. Diagnosis is the most important aspect of controlling fruit plant viruses. Early detection of viruses in fruit trees or in the propagative material is a prerequisite for their control and to guarantee a sustainable agriculture. Many quarantine programs are in place to reduce spread of viruses among countries during international exchange of germplasm. All these phytosanitary measures are overseen by governments based on agreements produced by international organizations. Also certification schemes applied to fruit trees allow the production of planting material of known variety and plant health status for local growers by controlling the propagation of pathogen-tested mother plants. They ensure to obtain propagative material not only free of "quarantine" organisms under the national legislation but also of important "nonquarantine" pathogens. The control of insect vectors plays an important role in the systemic diseases management, but it must be used together with other control measures as eradication of infected plants and use of certified propagation material. Apart from the control of the virus vector and the use of virus-free material, the development of virus-resistant cultivars appears to be the most effective approach to achieve control of plant viruses, especially for perennial crops that are more exposed to infection during their long life span. The use of resistant or tolerant cultivars and/or rootstocks could be potentially the most important aspect of virus disease management, especially in areas in which virus infections are endemic. The conventional breeding for virus-tolerant or resistant fruit tree cultivars using available germplasm is a long-term strategy, and the development and production of these cultivars may take decades, if successful. Genetic engineering allows the introduction of specific DNA sequences offering the opportunity to obtain existing fruit tree cultivars improved for the desired resistance trait. Unfortunately, genetic transformation of pome and stone fruits is still limited to few commercial genotypes. Research carried out and the new emerging biotechnological approaches to obtain fruit tree plants resistant or tolerant to viruses are discussed.

One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys.

A raspberry bushy dwarf virus isolate from Ecuadorean Rubus glaucus contains an additional RNA that is a rearrangement of RNA-2.

Sequencing of the complete genome of a raspberry bushy dwarf virus isolate from Rubus glaucus in Ecuador revealed that its RNA-1 and RNA-2 were 5449 and 2231 nucleotides (nt) long, respectively, and phylogenetically closest to isolates from Sweden and Slovenia. In dsRNA analysis of infected plants, an additional band of 3 kbp was observed. Sequencing of this band revealed that it was 3279 nt long. BLAST searches revealed that this band contained a modified version of RNA-2, which consisted of RNA-2 (2231 nt) plus an additional 1048-nt fragment that was concatenated in a reverse-complement fashion to its 5' terminus.

Complete genomic sequence of a Rubus yellow net virus isolate and detection of genome-wide pararetrovirus-derived small RNAs.

Rubus yellow net virus (RYNV) was cloned and sequenced from a red raspberry (Rubus idaeus L.) plant exhibiting symptoms of mosaic and mottling in the leaves. Its genomic sequence indicates that it is a distinct member of the genus Badnavirus, with 7932bp and seven ORFs, the first three corresponding in size and location to the ORFs found in the type member Commelina yellow mottle virus. Bioinformatic analysis of the genomic sequence detected several features including nucleic acid binding motifs, multiple zinc finger-like sequences and domains associated with cellular signaling. Subsequent sequencing of the small RNAs (sRNAs) from RYNV-infected R. idaeus leaf tissue was used to determine any RYNV sequences targeted by RNA silencing and identified abundant virus-derived small RNAs (vsRNAs). The majority of the vsRNAs were 22-nt in length. We observed a highly uneven genome-wide distribution of vsRNAs with strong clustering to small defined regions distributed over both strands of the RYNV genome. Together, our data show that sequences of the aphid-transmitted pararetrovirus RYNV are targeted in red raspberry by the interfering RNA pathway, a predominant antiviral defense mechanism in plants.

Molecular characterization and population structure of blackberry vein banding associated virus, new Ampelovirus associated with yellow vein disease.

Blackberry yellow vein disease is the most important viral disease of blackberry in the United States. Experiments were conducted to characterize a new virus identified in symptomatic plants. Molecular analysis revealed a genome organization resembling Grapevine leafroll-associated virus 3, the type species of the genus Ampelovirus in the family Closteroviridae. The genome of the virus, provisionally named blackberry vein banding associated virus (BVBaV), consists of 18,643 nucleotides and contains 10 open reading frames (ORFs). These ORFs encode closterovirid signature replication-associated and quintuple gene block proteins, as well as four additional proteins of unknown function. Phylogenetic analyses of taxonomically relevant products consistently placed BVBaV in the same cluster with GLRaV-3 and other members of the subgroup I of the genus Ampelovirus. The virus population structure in the U.S. was studied using the replication associated polyprotein 1a, heat shock 70 homolog and minor coat proteins of 25 isolates. This study revealed significant intra-species variation without any clustering among isolates based on their geographic origin. Further analyses indicated that these proteins are under stringent purifying selections. High genetic variability and incongruent clustering of isolates suggested the possible involvement of recombination in the evolution of BVBaV.

[Polyvalence of bacteriophages isolated from fruit trees, affected by bacterial fire blight].

Phage populations appearing as a result of a pathogenic process caused by Erwinia amylovora have been discovered and described. They accompany bacterial fire blight development in the process of quince, pear and apple trees vegetation in Zakarpattya region of Ukraine. Phage isolates of the affected pear and quince include polyvalent virulent phages able to develop on bacterial strains associated with plants--E. amylovora. E. "horticola" and Pantoea agglomerans. E. amylovora isolated from the plant tissues affected by the fire blight and detected at the same time as phages proved to be resistant to the viral infection. It is hard to explain now this characteristic however it was noticed that resistance to phages can change drastically in case of dissociation, lysogenization and mutagenesis of erwinia in laboratory conditions. Phage population study shows that they are heterogeneous and can obviously include not only polyvalent but also specific viruses. Further studies of biology and molecular genetics of pure lines of isolated phages will help to get closer to understanding the place and role of bacteriophages in the complicated network of relations between bacterial pathogens and plants.

Experimental and bioinformatic evidence that raspberry leaf blotch emaravirus P4 is a movement protein of the 30K superfamily.

Emaravirus is a recently described genus of negative-strand RNA plant viruses. Emaravirus P4 protein localizes to plasmodesmata, suggesting that it could be a viral movement protein (MP). In the current study, we showed that the P4 protein of raspberry leaf blotch emaravirus (RLBV) rescued the cell-to-cell movement of a defective potato virus X (PVX) that had a deletion mutation in the triple gene block 1 movement-associated protein. This demonstrated that RLBV P4 is a functional MP. Sequence analyses revealed that P4 is a distant member of the 30K superfamily of MPs. All MPs of this family contain two highly conserved regions predicted to form ß-strands, namely ß1 and ß2. We explored by alanine mutagenesis the role of two residues of P4 (Ile106 and Asp127) located in each of these strands. We also made the equivalent substitutions in the 29K MP of tobacco rattle virus, another member of the 30K superfamily. All substitutions abolished the ability to complement PVX movement, except for the I106A substitution in the ß1 region of P4. This region has been shown to mediate membrane association of 30K MPs; our results show that it is possible to make non-conservative substitutions of a well-conserved aliphatic residue within ß1 without preventing the membrane association or movement function of P4.

Rubus canadensis virus 1, a novel betaflexivirus identified in blackberry.

Flexuous filaments, resembling flexivirus virions, were observed in partially purified blackberry preparations showing mild virus-like symptoms. Further tests revealed the presence of a novel betaflexivirus that is phylogenetically related to foveaviruses. The putative virus-encoded proteins shared limited similarity with orthologs of known members of the genus, indicating that the virus, provisionally named Rubus canadensis virus 1 (RuCV-1), represents a novel member of the taxon. Results of a survey in several U.S. states suggest that RuCV-1 is not widespread in the blackberry germplasm.

Population structure of blackberry chlorotic ringspot virus in the United States.

Blackberry chlorotic ringspot virus is a subgroup 1 ilarvirus, detected in several rosaceous hosts exhibiting disease symptoms in Europe and the United States. The population structure of the virus was studied using isolates collected from wild and cultivated plants from six states in the United States. The results suggest a homogeneous virus population in the United States, similar to what observed within single orchards for other ilarviruses. Given the lack of evidence for host or geography-driven adaptation, it is hypothesized that the virus was recently introduced into the New World.

[Electron microscopy and restriction analysis of bacteriophages isolated from quince and pear with symptoms of fire blight].

Phage populations of isolates from quince and pear affected with fire blight disease were studied using electron microscopy, restriction analysis and both agarose gel electrophoresis of particles and host range scoping method. The isolate from quince (pMA1) comprises at least three phage populations and two phage variants that can be detected on different bacterial indicators. After titration of this isolate on Erwinia amylovora the bacteriophage KEY of B1 morphotype with the genome size of 82.4 kb was identified. The isolate pMA1 also includes a unique phage population 4*, which can be identified on the test bacteria Pantoea agglomerans (Pag) g150. Two analogous populations being also present in the isolate pMA1 that appeared to be close phage variants with almost identical Hpal-restriction patterns can be identified using Pag g157 and 9/7-1. The situation is similar in the case of phage isolates from pear, pMG. Three phage populations identified in it using three different indicators represent the same phage of C1 morphotype (TT10-27) with a genome size of 71.4 kb. At least two other phage populations were also detected in the same isolate using P. agglomerans 9/7-2 as an indicator. A model system allowing the most efficient analysis of the isolates for the presence of different phage populations and phage variants in plants infected by fire blight disease has been developed. It provides for using three indicator enterobacterial species closely associated with the plants: E. amylovora, Erwinia "horticola" and Pagglomerans and ignoring of the phage cloning procedure.

Population structure of Blackberry yellow vein associated virus, an emerging crinivirus.

Blackberry yellow vein disease (BYVD), a disorder caused by virus complexes, has become a major threat to fresh market blackberry production in the United States. Blackberry yellow vein associated virus (BYVaV) is the most prevalent virus in the BYVD complexes; detected in about 50% of samples exhibiting typical disease symptoms. Thirty-four virus isolates infecting wild and cultivated blackberries were collected from several areas with high BYVD incidence. Sequence variability and virus evolution predictions were calculated for four genomic regions coding for six proteins and accounting for about 30% of the virus genome. Nucleotide diversity ranged between 7 and 12%, and all proteins studied were under negative selection. Several isolates were identified as potential recombinants suggesting that recombination might be a driving force behind BYVaV evolution.

Evaluation of four virus recovery methods for detecting noroviruses on fresh lettuce, sliced ham, and frozen raspberries.

Although noroviruses play a significant role in causing foodborne illness in developed countries, no standardised method for detecting noroviruses in foodstuffs is currently available. This study compared four virus recovery methods based on ultrafiltration, immunomagnetic separation, ultracentrifugation and PEG precipitation techniques using identical real-time RT-PCR protocols for detection of RNA in eluates from lettuce, sliced ham and raspberries inoculated artificially with genogroup II norovirus. Noroviruses in all the food source matrices were successfully detected by all four methods. Ultracentrifugation yielded the highest recovery efficiencies in lettuce and ham, whereas PEG precipitation recovered the highest yield of noroviruses from raspberries. The repeatability of the results and the applicability of the methods to all food matrices were best with PEG precipitation, which had average virus recoveries of 19%, 47% and 28% for lettuce, ham and raspberries (viral RNA in dilution 1:10), respectively. In each case, a tenfold dilution of the extracted RNA clearly reduced the level of PCR inhibitors, which were released from raspberries in particular. The results of this study show that the detection of noroviruses in food is challenging, and more efforts to develop sensitive methods are still needed to detect noroviruses in food containing viruses in low numbers.

Transmission biology of Raspberry latent virus, the first aphid-borne reovirus.

Raspberry latent virus (RpLV) is a newly characterized reovirus found in commercial raspberry fields in the Pacific Northwest (PNW). Thus far, all members of the plant reoviruses are transmitted in a replicative, persistent manner by several species of leafhoppers or planthoppers. After several failed attempts to transmit RpLV using leafhoppers, the large raspberry aphid, commonly found in the PNW, was tested as a vector of the virus. The virus was transmitted to new, healthy raspberry plants when inoculated with groups of at least 50 viruliferous aphids, suggesting that aphids are vectors of RpLV, albeit inefficient ones. Using absolute and relative quantification methods, it was shown that the virus titer in aphids continued to increase after the acquisition period even when aphids were serially transferred onto fresh, healthy plants on a daily basis. Transmission experiments determined that RpLV has a 6-day latent period in the aphid before it becomes transmissible; however, it was not transmitted transovarially to the next generation. To our knowledge, this is the first report of a plant reovirus transmitted by an aphid. Phylogenetic analyses showed that RpLV is related most closely to but distinct from Rice ragged stunt virus (RRSV), the type member of the genus Oryzavirus. Moreover, the conserved nucleotide termini of the genomic segments of RpLV did not match those of RRSV or other plant reoviruses, allowing us to suggest that RpLV is probably the type member of a new genus in the Reoviridae comprising aphid-transmitted reoviruses.

Cydonia japonica, Pyrus calleryana and P. amygdaliformis: three new ornamental or wild hosts of Apple stem pitting virus.

Japanese quince, ornamental and wild pear symptomless samples were infected with Apple stem pitting virus (ASPV). Identification of ASPV was achieved by different PCR assays that amplified either the RNA polymerase or coat protein gene regions. For further confirmation, 312 bp amplicons within the polymerase gene were sequenced and compared with previously published ASPV sequences and additional sequences of isolates from ancient Italian cultivars. Comparison of the partial sequences isolated from wild/ornamental hosts and from cultivated species revealed significant divergence levels. Among the wild/ornamental isolates, the PCT88 isolate from Pyrus calleryana was the most divergent, having an amino acid deletion and incorporating a unique stretch of amino acids not present in any other isolate. Further to this preliminary partial sequence data, statistical analysis demonstrated that the isolates from wild or ornamental hosts were not more closely related to each other than to isolates from cultivated hosts. These results represent the first report of natural ASPV infection in these novel ornamental and wild Rosaceae hosts.

Multiple norovirus outbreaks linked to imported frozen raspberries.

In 2009, the number of foodborne norovirus outbreaks in Finland seemed markedly high, and many outbreaks seemed to be linked to imported frozen raspberries. We reviewed the data regarding all notified foodborne outbreaks in 2009 in Finland in order to assess the magnitude of the problem and to summarize the information on raspberry-linked outbreaks. Between March and August, 13 norovirus outbreaks affecting about 900 people could be linked to imported frozen raspberries. Two raspberry samples corresponding to two batches of raspberries were positive for norovirus. These two batches proved to have been the likely source in six of the 13 outbreaks. Analytical studies had not been conducted for six outbreaks, and virological test results were inconclusive in two. However, combining epidemiological and microbiological methods often enabled finding the source, as exemplified in investigation of a large school outbreak. To ensure prompt control measures in similar situations in the future, both aspects of outbreak investigations should be strengthened.

Raspberry leaf blotch virus, a putative new member of the genus Emaravirus, encodes a novel genomic RNA.

A new, segmented, negative-strand RNA virus with morphological and sequence similarities to other viruses in the genus Emaravirus was discovered in raspberry plants exhibiting symptoms of leaf blotch disorder, a disease previously attributed to the eriophyid raspberry leaf and bud mite (Phyllocoptes gracilis). The virus, tentatively named raspberry leaf blotch virus (RLBV), has five RNAs that each potentially encode a single protein on the complementary strand. RNAs 1, 2 and 3 encode, respectively, a putative RNA-dependent RNA polymerase, a glycoprotein precursor and the nucleocapsid. RNA4 encodes a protein with sequence similarity to proteins of unknown function that are encoded by the genomes of other emaraviruses. When expressed transiently in plants fused to green or red fluorescent protein, the RLBV P4 protein localized to the peripheral cell membrane and to punctate spots in the cell wall. These spots co-localized with GFP-tagged tobacco mosaic virus 30K cell-to-cell movement protein, which is itself known to associate with plasmodesmata. These results suggest that the P4 protein may be a movement protein for RLBV. The fifth RLBV RNA, encoding the P5 protein, is unique among the sequenced emaraviruses. The amino acid sequence of the P5 protein does not suggest any potential function; however, when expressed as a GFP fusion, it localized as small aggregates in the cytoplasm near to the periphery of the cell.