D. Alioto

Identification and molecular properties of a 306 nucleotide viroid associated with apple dimple fruit disease

A new viroid associated with an apple fruit disorder similar to, but more severe than, the dapple apple disease induced in some varieties by apple scar skin viroid (ASSVd) has been found. The new viroid, tentatively termed apple dimple fruit viroid (ADFVd), is a circular RNA of 306 nucleotides which adopts a quasi-rod-like conformation of minimum free energy. It contains the core nucleotides of the central conserved region (CCR) of the ASSVd group as well as the terminal conserved region (TCR) present in all members of the ASSVd and potato spindle tuber viroid (PSTVd) monophyletic groups. ADFVd has the highest sequence similarity with ASSVd and the 294 nucleotide citrus viroid CVd-IIIb sharing with the latter an almost identical left terminal domain. The right- and left-hand termini of ADFVd are formed by almost perfect duplications of sequences found in the CCR upper and lower strands, respectively, of PSTVd and closely related viroids.

Variability Among Turnip Mosaic Potyvirus Isolates

ABSTRACT Eight turnip mosaic potyvirus (TuMV) isolates from the Campania region of Italy were characterized. Experiments based on host range and symptomatology indicated that the isolates were biologically different. In addition, the isolates, with the exception of ITA1 and ITA3, were distinguished from each other by using a combination of monoclonal antibodies recognizing the coat protein. Single-strand conformation polymorphism (SSCP) analysis of the coat protein gene revealed that each isolate produced a specific SSCP profile, except for isolates ITA1 and ITA3. This study indicates that (i) even in a small geographical region, there is a great deal of variation in TuMV isolates; (ii) the use of a set of four differential hosts does not always specify the same pathotype in different environments; (iii) the TuMV isolates with the same pathotype on Brassica napus test lines can still differ in host range, symptoms, serology, and SSCP; and (iv) there was perfect correlation between the panel of antibodies and SSCP in differentiating among the isolates; ITA1 and ITA3 were indistinguishable by either assay.

Characterization by Deep Sequencing of Prunus virus T, a Novel Tepovirus Infecting Prunus Species

Double-stranded RNAs purified from a cherry tree collected in Italy and a plum tree collected in Azerbaijan were submitted to deep sequencing. Contigs showing weak but significant identity with various members of the family Betaflexiviridae were reconstructed. Sequence comparisons led to the conclusion that the viral isolates identified in the analyzed Prunus plants belong to the same viral species. Their genome organization is similar to that of some members of the family Betaflexiviridae, with three overlapping open reading frames (RNA polymerase, movement protein, and capsid protein). Phylogenetic analyses of the deduced encoded proteins showed a clustering with the sole member of the genus Tepovirus, Potato virus T (PVT). Given these results, the name Prunus virus T (PrVT) is proposed for the new virus. It should be considered as a new member of the genus Tepovirus, even if the level of nucleotide identity with PVT is borderline with the genus demarcation criteria for the family Betaflexiviridae. A reverse-transcription polymerase chain reaction detection assay was developed and allowed the identification of two other PrVT isolates and an estimate of 1% prevalence in the large Prunus collection screened. Due to the mixed infection status of all hosts identified to date, it was not possible to correlate the presence of PrVT with specific symptoms.

Genetic diversity among potato species as revealed by phenotypic resistances and SSR markers

The evolutionary diversity of wild potato species makes them excellent materials for improving the narrow genetic basis of the cultivated potato Solanum tuberosum. Understanding their genetic diversity is important not only to choose the best parents for breeding, but also to design proper crossing schemes and selection strategies. The objectives of this study were to determine the resistance response to Ralstonia solanacearum, Potato virus Y and low temperatures of 21 clones of 12 potato species, and to determine their genetic diversity through simple sequence repeat (SSR) markers. Sources of resistance have been found for all the investigated traits, with high resistance variability not only between but also within species. Combined resistances were also identified, with positive implications for efficient breeding. SSR analysis allowed the detection of 12 loci and 46 alleles across all genotypes, with an average value of 3.8 alleles per locus. Both unique and rare alleles useful for marker-assisted selection were found. SSR-based cluster analysis revealed that resistant genotypes were distributed among all clusters, suggesting that genetically different resistant genotypes were identified. The information obtained in this study is discussed from a breeding perspective.

Apple dimple fruit viroid: Fulfillment of Koch's Postulates and Symptom Characteristics

The previously reported properties of a small circular RNA isolated from plants of cultivar Starking Delicious with symptoms similar to those of the dapple apple disease induced by Apple scar skin viroid (ASSVd), strongly suggested that it is a distinct viroid species of genus Apscaviroid to which ASSVd also belongs. Nevertheless, the autonomous replication of this novel RNA, termed Apple dimple fruit viroid (ADFVd), and the nature of the symptoms incited when inoculated free of other pathogens that might be present in the original source, were not determined. We addressed these questions by mechanically inoculating purified ADFVd into young apple seedlings (Golden). Dot blot hybridization of leaf extracts collected ten months after inoculation showed the presence of ADFVd in most of the inoculated seedlings, thus proving the ability of this RNA to replicate independently. Material from these ADFVd-infected seedlings grafted onto the ASSVd indicator Starkrimson incited the appearance on the fruit skin of discolored spots more or less depressed which were particularly frequent around the calyx cavity. Apple cultivars such as Gala, Pink Lady, and Braeburn reacted to ADFVd with similar symptoms, occasionally accompanied by scar skin in Braeburn, whereas ADFVd replicated without eliciting any visible symptoms in other apple cultivars such as Golden, or in the pear indicator Fieud 37. No interference in symptom expression or viroid accumulation were observed in plants inoculated first with ASSVd and then with ADFVd.

The first phlebo‐like virus infecting plants: a case study on the adaptation of negative‐stranded RNA viruses to new hosts

A novel negative-stranded (ns) RNA virus associated with a severe citrus disease reported more than 80 years ago has been identified. Transmission electron microscopy showed that this novel virus, tentatively named citrus concave gum-associated virus, is flexuous and non-enveloped. Notwithstanding, its two genomic RNAs share structural features with members of the genus Phlebovirus, which are enveloped arthropod-transmitted viruses infecting mammals, and with a group of still unclassified phlebo-like viruses mainly infecting arthropods. CCGaV genomic RNAs code for an RNA-dependent RNA polymerase, a nucleocapsid protein and a putative movement protein showing structural and phylogenetic relationships with phlebo-like viruses, phleboviruses and the unrelated ophioviruses, respectively, thus providing intriguing evidence of a modular genome evolution. Phylogenetic reconstructions identified an invertebrate-restricted virus as the most likely ancestor of this virus, revealing that its adaptation to plants was independent from and possibly predated that of the other nsRNA plant viruses. These data are consistent with an evolutionary scenario in which trans-kingdom adaptation occurred several times during the history of nsRNA viruses and followed different evolutionary pathways, in which genomic RNA segments were gained or lost. The need to create a new genus for this bipartite nsRNA virus and the impact of the rapid and specific detection methods developed here on citrus sanitation and certification are also discussed.

First Report and High Prevalence in Noncherry Host of Cherry virus A in Italy

Cherry virus A (CVA) has been reported to naturally infect sweet and sour cherry, apricot, peach, and plum but has not been associated with any symptoms. In the spring and summer of 2005, samples were collected in Prunus spp. germplasm collections in Campania (Italy) and analyzed by a polyvalent reverse transcription-polymerase chain reaction (RT-PCR) assay (1). Amplicons were sequenced, revealing CVA infection in seven apricot cultivars (Prunus armeniaca L.), one plum (Prunus domestica L.), and one cherry (Prunus avium L.). CVA sequences (GenBank Accession Nos. DQ445275 to DQ445292) compared among themselves and with databank sequences showed pairwise nucleotide sequence identity levels of 80.3 to 99.6% (86.8 to 100% for amino acid sequences), significantly extending the known variability range of CVA. The presence of CVA was confirmed by hybridization with a CVA-specific probe (P39) (2), targeting region different from that amplified in the polymerase chain reaction assay. All samples containing CVA were found to be in mixed infections with Apple chlorotic leaf spot virus (ACLSV) and Apricot pseudo-chlorotic leaf spot virus (plum, apricot), or ACLSV plus Cherry green ring mottle virus (cherry) so that CVA symptomatology could not be evaluated. To our knowledge, these results constitute the first report of the occurrence of CVA in Italy. They confirm the ability of CVA to naturally infect noncherry Prunus spp. hosts with surprisingly high prevalence levels (6 of 14 and 1 of 6 tested apricot and plum cultivars, respectively). References: (1) X. Foissac et al. Phytopathology 95:617, 2005. (2) W. Jelkmann J. Gen. Virol. 76:2015, 1995.

Sphaceloma coryli: A Reemerging Pathogen Causing Heavy Losses on Hazelnut in Southern Italy

In 2013 to 2014, a disease causing severe crop losses was observed in several growing areas of the Campania region in southern Italy. Disease symptoms consisted of necrotic spots on leaves, bracts, and shells along with vein necrosis. Nuts were necrotic or aborted and symptomatic fruit dropped prematurely. A fungus was consistently isolated from symptomatic tissues and morphologically identified as Sphaceloma coryli. The fungus was first reported in France and Italy (Campania) over 30 years ago and was not subsequently documented until 2006 in the Latium region of Italy. When artificially inoculated on healthy hazelnut plants, disease symptoms were reproduced. The teleomorph of S. coryli was recorded for the first time on overwintering tissues of hazelnut trees. It was also induced in vitro. The morphology of asci and ascospores together with phylogenetic analysis based on internal transcribed spacer sequences indicated that the teleomorph is an undescribed species within the genus Elsinoë for which the name of Elsinoë coryli is proposed. Optimum temperatures for ascospore and conidium germination and mycelium growth were determined in vitro. The inhibition effect of some commercial fungicides on S. coryli was also evaluated in vitro.

NECROTIC POTATO TUBERS INFECTED BY EGGPLANT MOTTLED DWARF VIRUS IN ITALY

Potato (Solanum tuberosum subsp. tuberosum) alterations characterized by severe necrosis of vascular rings and adjacent flesh were observed in potato cv. Vivaldi in Emilia Romagna (northen Italy). The presence of Eggplant mottled dwarf virus (EMDV) was demonstrated in diseased plants by bioassays, electron microscopy, serology and RT-PCR. Symptoms similar to those observed in cv. Vivaldi were observed in other potato cultivars and areas of central and southern Italy. This is the first report of natural occurrence of EMDV in potato in Italy.

Characterization of a putative novel nepovirus from Aeonium sp.

A virus was isolated from potted plants of an unidentified species of Aeonium, a succulent ornamental very common in Southern Italy, showing chlorotic spots and rings on both leaf surfaces. It was successfully transmitted by sap inoculation to a limited range of hosts, including Nicotiana benthamiana which was used for ultrastructural observations and virus purification. Virus particles are isometric, ca. 30nm in diameter, have a single type of coat protein (CP) subunits 54kDa in size, that encapsidate single-stranded positive-sense RNA species of 7549 (RNA1) and 4010 (RNA2) nucleotides. A third RNA molecule 3472 nts in size entirely derived from RNA2 was also found. The structural organization of both genomic RNAs and the cytopathological features were comparable to those of nepoviruses. In addition, amino acid sequence comparisons of CP and the Pro-Pol region (a sequence containing parts of the proteinase and polymerase) with those of other nepoviruses showed that the Aeonium virus belongs to the subgroup A of the genus Nepovirus and is phylogenetically close to, but serologically distinct from tobacco ringspot virus (TRSV). Based on the species demarcation criteria for the family Secoviridae, the virus under study appears to be a novel member of the genus Nepovirus for which the name of Aeonium ringspot virus (AeRSV) is proposed.