A virus disease of fig (Ficus carica) known as fig mosaic (FM) is widely spread in Palestine, where its severity varies according to the cultivar and the growing area. At least 10 viruses and three viroids have been detected so far in fig trees. This study reports the results of a preliminary survey carried out in Palestine to secure information on the viruses associated with mosaic-infected figs. Samples were collected from scattered trees, fig orchards and nurseries of different areas of central Palestine, where figs are traditionally grown, and tested for the presence of Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig badnavirus-1 (FBV-1), Fig leaf mottle-associated virus 1 (FLMaV-1) and 2 (FLMaV-2), and Fig cryptic virus (FCV) in addition to Apple dimple fruit viroid (ADFVd). The following viruses. FMV, FBV-1, FLV-1 and FLMaV-2, were detected by RT-PCR. FBV-1 was the most widespread followed by FMV. The genetic diversity of FMV was assessed by sequencing a fragment from the viral p4 protein, revealing a low divergence from the homologous sequences from GenBank.

Mixed viral infections are common in globe artichoke (Cynara cardunculus var. scolymus). Despite lack ofevident symptoms, production losses are relevant. A rapid and accurate detection is advisable for selection and marketingof plant propagation material.Objectives: Our aim was the evaluation of a suspension microbeads array system for simultaneous detection ofArtichoke latent potyvirus (ArLV), Artichoke Italian latent nepovirus (AILV), Artichoke mottled crinkle tombusvirus (AMCV),Tomato spotted wilt tospovirus (TSWV), Turnip mosaic potyvirus (TuMV), Cucumber mosaic cucumovirus (CMV), Potatovirus X (PVX), Tobacco mosaic tobamovirus (TMV) and Pelargonium zonate spot anulavirus (PZSV).Methods: For PCR, oligonucleotides were designed from conserved regions in the viral genomes, using an Illumina´sAssay Design Tool. The actin gene of C. cardunculus was used as control. The microbeads-based procedure started witha first PCR target amplification. A further target-specific primer extension (TSPE), holding a unique 5' motif, was thenperformed. During this step biotinylated dCTPs were incorporated into the extension products. These were then linked tothe holographic microbeads bearing an oligo complementary to the 5' motif. Probe labelling was performed withstreptavidin-Alexa-647, for final scan using an Illumina BeadXpress Reader.Conclusions: Multiplex fluorescent microbeads assays allowed the contemporary detection of several viruses in a singlesample. This highly flexible microarray approach used a suspension of barcoded microbeads bearing specific sequencemotifs. It favoured the tailoring of user defined applications. The method is also suitable for identification of SNPs in viralstrains, within a unique host phenotype.

The complete nucleotide sequence and the genome organization were determined of a putative newmember of the family Tymoviridae, tentatively named Olive latent virus 3 (OLV-3), recovered in southernItaly from a symptomless olive tree. The sequenced ssRNA genome comprises 7148 nucleotides excludingthe poly(A) tail and contains four open reading frames (ORFs). ORF1 encodes a polyprotein of 221.6 kDain size, containing the conserved signatures of the methyltransferase (MTR), papain-like protease (PRO),helicase (HEL) and RNA-dependent RNA polymerase (RdRp) domains of the replication-associated proteinsof positive-strand RNA viruses. ORF2 overlaps completely ORF1 and encodes a putative protein of43.33 kDa showing limited sequence similarity with the putative movement protein of Maize rayado finovirus (MRFV). ORF3 codes for a protein with predicted molecular mass of 28.46 kDa, identified as thecoat protein (CP), whereas ORF4 overlaps ORF3 and encodes a putative protein of 16 kDa with sequencesimilarity to the p16 and p31 proteins of Citrus sudden death-associated virus (CSDaV) and Grapevinefleck virus (GFkV), respectively. Within the family Tymoviridae, OLV-3 genome has the closest identitylevel (49-52%) with members of the genus Marafivirus, from which, however, it differs because of thediverse genome organization and the presence of a single type of CP subunits.

Deep sequencing is a powerful technology that provides information on the infectious agents (viruses and viroids) present in plant tissues and able to explore their intimate relationships with the cellular replication machinery. The technology is being increasingly used to investigate how the plant RNA silencing mechanism attacks viral genomes and, recently, how viruses perturb the antiviral silencing machinery and induce symptoms expression. On the applied side, deep sequencing allowed to investigate diseases of unknown etiology, leading to the identification of new viruses, and to have an overview of the viral and viroids contents of a cultivated parcel (i.e the "virome" of a vineyard). Supported by dedicated bioinformatics tools it is now possible to assemble a complete viral genome thus envisaging the adoption of this unbiased approach for a diagnostic use. We have experienced the Illumina/Solexa system for the analysis of small RNAs populations, in healthy and virus-diseased grapevine plants. The techniques proved efficient for identifying a new virus in a widely diffused grapevine variety, whose detection would otherwise be difficult and time consuming.

Two dsRNA molecules with an estimated lenght of 1.5 Kbp were identified and characterized from leaves of a Japanese persimmon (Diospyros kaki) tree, showing veinlets necrosis on both sides of leaf blades.DOP-PCR recognized two genomic fragments of a bipartite cryptic virus, for which the name of Persimmon cryptic virus (PeCV) is proposed. RLM-RACE leaded to the sequencing of a 1,510 bp contig identified as dsRNA-1 and a 1,491 bp complete segment identified as dsRNA-2. The two genomic fragments resulted both monocistronic and harbored conserved domains related to RNA-dependent RNA polymerase (RdRP) and capsid protein (CP) of species associated to Alphacryptovirus genus. Phylogenetic analysis of RdRP sequence showed highest amino acid identity with Black raspberry cryptic virus (BrCV, 63 %), Pepper cryptic virus 2 (PCV-2, 52 %) and -1 (PCV-1, 46 %). Whereas the CP putatively encoded by dsRNA-2 shared highest identity with Mulberry cryptic virus 1 (MCV-1, 49 %), PCV-2 (39 %) and PCV-1 (32 %). N-J phylogenetic analysis confirmed those relationships and delivered PeCV in a cluster with phyto-cryptoviruses belonging to genera Alpha- and Betacryptovirus, quite far distinguished from myco-cryptoviruses, gathered in genus Partitivirus.Virus-specific primers for RT-PCR were successfully designed inside the CP region to detect PeCV in several symptomless trees found in different orchards of Apulia (Southern Italy), thus proving that infection may be fairly common and presumably latent.Policlonal antiserum (kindly provided by Dr. M. Turina, CNR-IVV, Torino, Italy) specific to the CP of family-related Beet cryptic virus 2 (BCV-2) was profitably used for western blot detection of a 45-50 KDa band, coherent with predicted size of dsRNA-2 product. Furthermore, antibodies were useful for ISEM observation and subsequent decoration of PeCV particles, proved to be isometric, around 30 nm in diameter, with rounded shape lacking in fine structural details, not easily permeable by negative stain.

Nine isolates of Grapevine leafroll-associated virus 7 (GLRaV-7) from diverse geographical regions were sequenced to design more sensitive molecular diagnostic tools. The coat protein (CP) and heat shock protein 70 homologue (HSP70h) genes of these nine isolates were sequenced. Sequences were then used to design more sensitive molecular diagnostic tools. Sequence identity among these isolates ranged between 90 to 100% at the nucleotide and amino acid levels. One RT-PCR and two qRT-PCR assays were used to survey 86 different grapevines from the University of California, Davis Grapevine Virus Collection, the Foundation Plant Services collection and the USDA National Clonal Germplasm Repository, Davis, CA with primers designed in conserved regions of the CP and HSP70h genes. Results revealed that qRT-PCR assays designed in the HSP70h gene was more sensitive (29.07% positives) than that designed in the CP gene (22.09% positives) and both qRT-PCR assays proved to be more sensitive than RT-PCR.

Grapevine rupestris stem pitting-associated virus (GRSPaV, genus Foveavirus, family Betaflexiviridae) is the putative agent of the Rupestris stem pitting (RSP) disease of grapevines. GRSPaV comprises a family of variants whose pathological characteristics are incompletely known. Recently, many of the autochthonous table grape cultivars were tested for the presence of GRSPaV in two major Turkish grape-growing areas, i.e. the Eastern Mediterranean and the Southeast Anatolia regions. Comparative analysis of local GRSPaV isolates from these native cultivars was performed with viral genome sequences from NCBI database. To our knowledge, this is the first report for the presence of GRSPaV in Turkish vineyards.

Through the application of next generation sequencing, in synergy with conventional cloning of DOPPCR fragments, two double-stranded RNA (dsRNA) molecules of about 1.5 kbp in size were isolated from leaf tissue of a Japanese persimmon (accession SSPI) from Apulia (southern Italy) showing veinlets necrosis. Highthroughput sequencing allowed whole genome sequence assembly, yielding a 1,577 and a 1,491 bp contigs identified as dsRNA-1 and dsRNA-2 of a previously undescribed virus, provisionally named as Persimmon cryptic virus (PeCV). In silico analysis showed that both dsRNA fragments were monocistronic and comprised the RNAdependent RNA polymerase (RdRp) and the capsid protein (CP) genes, respectively. Phylogenetic reconstruction revealed a close relationship of these dsRNAs with those of cryptoviruses described in woody and herbaceous hosts, recently gathered in genus Deltapartitivirus . Virus-specific primers for RT-PCR, designed in the CP cistron, detected viral RNAs also in symptomless persimmon trees sampled from the same geographical area of SSPI, thus proving that PeCV infection may be fairly common and presumably latent.

Sharka, caused by Plum Pox Virus (PPV), is by far the most important infectious disease of peach [P persica (L) Batsch] and other Prunus species. The progressive spread of the virus in many important growing areas throughout Europe poses serious issues to the economic sustainability of stone fruit crops, peach in particular. The adoption of internationally agreed upon rules for diagnostic tests, strain-specific monitoring schemes and spatial temporal modeling of virus spread, are all essential for a more effective sharka containment. The EU regulations on nursery activity should be modified based on the zone delimitation of PPV presence, limiting open-field production of propagation materials only to virus free areas. Increasing the efficiency of preventive measures should be augmented by the short-term development of resistant cultivars. Putative sources of resistance/tolerance have been recently identified in peach germplasm, although the majority of novel resistant sources to PPV-M have been found in almond. However, the complexity of introgression from related species imposes the search for alternative strategies. The use of genetic engineering, particularly RNA interference (RNAi)-based approaches, appears as one of the most promising perspectives to introduce a durable resistance to PPV in peach germplasm, notwithstanding the well-known difficulties of in vitro plant regeneration in this species. In this regard, rootstock transformation to induce RNAi-mediated systemic resistance would avoid the transformation of numerous commercial cultivars, and may alleviate consumer resistance to the use of GM plants.

Extensive necrosis of the veinlets were observed in early summer of 2011 on both sides of the leaf blades of a Japanese persimmon (Diospyros kaki) tree growing in a private garden in the vicinity of Bari (Apulia, southern Italy). This tree (accession SSPI) hosted a previously uncharacterized cryptovirus whose presence was apparently unrelated to the vein necrosis condition as it occurred also in a large number of symptomless trees (Morelli et al., 2012). Highthroughput sequencing analysis of the SSPI virome, performed on dsRNA extracts with an Illumina platform, allowed the identification of four contigs which, upon BLAST analysis (Altschul et al., 1997) showed a 97% sequence identity at the nucleotide level with sequences of the putative cytorhabdovirus Persimmon virus A (PeVA, GenBank AB735628), recently described in Japan (Ito et al., 2013). The sequence of the largest contig (983 bp), spanning part of the polymerase gene and the 3'-UTR, was deposited in GenBank under the accession No. KM407515. To confirm deep-sequencing findings, the PCR primer set PeVAfor/Pe- VArev (5'-AGGATCATTACAAAATCCGTGAGG-3'/ 5'- TTCCCGAAAGACAATCTGTCCC-3'), intended to amplify a 250 bp product, was designed on the KM407515 sequence. An amplicon of the expected size was repeatedly obtained from the symptomatic (SSPI) but not from 10 symptomless trees. This product was cloned into pSC-A-amp/kan and custom-sequenced (Macrogen Europe, The Netherlands). BLAST analysis matched previous identification, as cloned sequences shared ca. 98% identity with those of the Japanese PeVA isolate. There was no amplification when RT-PCR assays were extended to the 10 symptomless persimmon trees. Whether or not PeVA is involved in the induction of vein necrosis remains to be ascertained. To our knowledge, this is the first report of PeVA in a country other than Japan.

Surveys were made in the main grape growing region (Southeast Anatolia) of Turkey for the occurrence of Grapevine leafroll-associated virus-5 (GLRaV-5). Plant samples with typical leafroll symptoms and mealybugs, Planococcus ficus (Signoret) were used for assessing the occurrence of GLRaV-5 by RT-PCR. A 272 bp band representing GLRaV-5 infection was successfully detected in plants and mealybugs in some vineyards of the Southeast Anatolia region and the virus is the first time reported in Turkish vineyards. © 2009 Blackwell Verlag GmbH.

Plum pox virus (PPV), agent of Sharka disease, is the most important quarantine pathogen of peach (P. persica L. Batsch). Extensive evaluation of peach germplasm has highlighted the lack of resistant sources, while suggesting the presence of a quantitative disease resistance, expressed as reduction in the intensity of symptoms. Unravelling the genetic architecture of peach response to PPV infection is essential for pyramiding resistant genes and for developing more tolerant varieties. For this purpose, a genome-wide association (GWA) approach was applied in a panel of accessions phenotyped for virus susceptibility and genotyped with the IPSC peach 9 K SNP Array, and coupled with an high-coverage resequencing of the tolerant accession 'Kamarat'.

Early diagnosis of plant virus infections before the disease symptoms appearance may represent a significant benefit in limiting disease spread by a prompt application of appropriate containment steps. We propose a label-free procedure applied on a device structure where the electrical signal transduction is evaluated via impedance spectroscopy techniques. The device consists of a droplet suspension embedding two representative purified plant viruses i.e.,Tomato mosaic virus and Turnip yellow mosaic virus,put in contact with a highly hydrophobic plasma textured silicon surface. Results show a high sensitivity of the system towards the virus particles with an interestingly low detection limit,from tens to hundreds of attomolar corresponding to pg/mL of sap,which refers,in the infection time-scale,to a concentration of virus particles in still-symptomless plants. Such a threshold limit,together with an envisaged engineering of an easily manageable device,compared to more sophisticated apparatuses,may contribute in simplifying the in-field plant virus diagnostics.

A new satellite RNA (satRNA) of grapevine fanleaf virus (GFLV) was identified by high-throughput sequencing of high-definition (HD) adapter libraries from grapevine plants of the cultivar Panse precoce (PPE) affected by enation disease. The complete nucleotide sequence was obtained by automatic sequencing using primers designed based on next-generation sequencing (NGS) data. The full-length sequence, named satGFLV-PPE, consisted of 1119 nucleotides with a single open reading frame from position 15 to 1034. This satRNA showed maximum nucleotide sequence identity of 87 % to satArMV-86 and satGFLV-R6. Symptomatic grapevines were surveyed for the presence of the satRNA, and no correlation was found between detection of the satRNA and enation symptom expression.

Viroids are small (246-401 nt) circular and non coding RNAs infecting higher plants. They are targeted by host Dicer-like enzymes (DCLs) that generate small RNAs of 21-24 nt (sRNAs), which are involved in the host RNA silencing pathways. The accumulation in plant tissues of such viroid-derived small RNAs (vd-sRNAs) is a clear sign of an ongoing viroid infection. In this study, next generation sequencing of a sRNAs library and assembling of the sequenced vd-sRNAs were instrumental for the identification of a viroid resembling apple dimple fruit viroid (ADFVd) in a fig accession. After confirming by molecular methods the presence of this viroid in the fig tree, its population was characterized, showing that the ADFVd master sequence from fig diverges from that of the ADFVd reference variant from apple. Moreover, since this viroid accumulates at a low level in fig, a semi-nested RT-PCR assay was developed for detecting it in other fig accessions. ADFVd seems to have a wider host range than thought before and this poses questions about its epidemiology. A further characterization of ADFVd-sRNAs showed similar accumulation of (+) or (-) vd-sRNAs that mapped on the viroid genome generating hotspot profiles. Moreover, similarly to other nuclear-replicating viroids, vd-sRNAs of 21, 22 and 24 nt in size prevailed in the distribution profiles. Altogether, these data support the involvement of double-stranded RNAs and different DCLs, targeting the same restricted viroid regions, in the genesis of ADFVd-sRNAs. (C) 2014 Elsevier B.V. All rights reserved.

Electron microscope observation of leaf dips from a mulberry (Morus alba) tree from Lebanon showing leaf mottling and vein yellowing disclosed the presence of bacilliform particles ca. 150x30 nm in size, resembling those of members of the genus Badnavirus. BLAST analysis of the sequence of a DOP-PCR clone (810 bp) generated from DNA extracts from partially purified particle preparation disclosed a 67% (nucleotide) and 63% (amino acid) identity with the reverse transcriptase/RNase H gene (ORF3) of the badnavirus Cacao swollen shoot virus (CSSV). In a phylogenetic tree constructed with the amino acid sequence obtained, the putative mulberry badnavirus clustered close to CSSV and Citrus mosaic virus (CiMV) in a branch comprising also Fig badnavirus 1 (FBV-1) and Dioscorea bacilliform virus (DBALV). PCR analyses showed that the virus occurs with high infection rates in Lebanon (7 out of 13 trees examined), Turkey (2 out of 3) and Italy (29 out of 39). However, except for one, all PCR-positive trees were symptomless. Thus, since there is no clear-cut association of the virus in question with a specific disease, we propose for it the provisional name of Mulberry badnavirus 1 (MBV-1).

Vineyards in Turkey were surveyed from July-August of 2006-2007 for the presence of mealybug infestation and the viral agents of leafroll and rugose wood diseases, namely vitiviruses and ampeloviruses, respectively. Plant and insect samples in the main grape-growing regions were collected and processed for nucleic acid isolation. The reverse transcription polymerase chain reaction (RT-PCR) was used to detect the insect-transmitted Grapevine virus A (GVA) and B (GVB) (Vitivirus) and Grapevine leafroll-associated virus-1 (GLRaV-1) and 3 (GLRaV-3) (Ampelovirus). All viruses except GVB were detected in the samples. Planococcus ficus (Signoret) is the only mealybug species found to be widespread in the sampled areas. This is the first epidemiological study of these viruses and their potential vector in Turkey. © 2012 The Canadian Phytopathological Society.

Enation disease of the grapevine is an erratic disorder, whose symptoms recall a teratological condition possibly deriving from hormonal unbalance. Even though graft transmissibility of enations supports a viral aetiology of the disease, its putative agent has not yet been identified.

L'introduzione di nuovi metodi di sequenziamento stà rivoluzionando le potenzialità diagnostiche e di ricerca in campo vegetale. Queste tecnologie di sequenziamento di nuova generazione (NGS), non necessitano di alcuna conoscenza preliminare sul contenuto in acidi nucleici, virali e dell'ospite, dei tessuti sottoposti ad analisi. Il loro utilizzo permette di condurre un'analisi metagenomica sul campione in esame, che può essere costituito da una singola pianta o provenire da più piante. Da un punto di vista fitopatologico, queste nuove tecnologie rivelano la complessità del "viroma" (l'insieme di virus e viroidi) del campione e delle sue interazioni con il genoma dell'ospite, laddove questo è noto. Dal punto di vista diagnostico il metodo velocizza la scoperta e l'identificazione di nuovi o già noti virus, la sequenza del loro genoma e lo sviluppo di nuovi e specifici metodi di diagnosi.

The complete nucleotide sequence of an Albanian isolate of grapevine leafroll-associated virus 7 (GLRaV-7-Alb) was determined. The viral genome consists of 16,404 nucleotides and has nine open reading frames (ORFs) that potentially encode proteins, most of which are typical for members of the family Closteroviridae. Only the 25-kDa (ORF8) and 27-kDa (ORF9) proteins had no apparent similarity to other viral proteins in the sequence databases. The genome structure of GLRaV-7-Alb closely resembles that of little cherry virus 1 and cordyline virus 1. In phylogenetic trees constructed with HSP70h sequences, these three viruses cluster together in a clade next to that comprising members of the genus Crinivirus, to which they are more closely related than to the clostero- and ampeloviruses. The molecular properties of these three viruses differ sufficiently from those of members of the three extant genera of the family Closteroviridae to warrant their classification in a novel genus.

A metagenomic approach was applied to obtain a comprehensive characterization of the B. cinerea virome. dsRNAs segments with sizes ranging from 0.6 to 5.0 kbp were extracted and purified from 12 pooled mycelia from a total of 60 isolates, collected from different host plants and geographical areas, and sequenced. A great abundance and diversity of viruses infecting B. cinerea were found. At least 23 different viruses were detected and identified in one or more of the analysed pools of dsRNA samples.

The consistent identification of Fig badnavirus 1 (FBaV-1) in the totality of samples from mosaic-affected fig trees from New Zealand (Minafra et al., 2012) and Italy (unpublished informa- tion), prompted the examination of additional samples from adult fig trees with mosaic symptoms from Italy (10), France (1), Greece (3), Albania (1), Spain (1), Portugal (1), England (1), Hungary (3), Montenegro (1), Lebanon (19), Syria (11), Tunisia (15), Algeria (2), Turkey (4), South Africa (1), Mexico (1), Cuba (1), and Aus- tralia (1). Additional samples from Italy consisting of volunteer symptomless seedlings (20) and symptomless potted plants de- rived from explants of mosaic-affected trees subjected to heat therapy (4), brought to over 100 the total number of sources ana- lyzed. Total RNA was silica-extracted from cortical scrapings and subjected to RT-PCR using the primers P1s: 5'-GCT GAT CAC AAG AGG CAT GA-3' and P1as: 5'-TCC TTG TTT CCA CGT TCC TT-3' designed on the sequence deposited in GenBank un- der the accession No. JF411989. A product with the expected size (214 bp) was amplified from all samples, regardless of the geo- graphical origin (18 countries) and the type of source (adult trees, seedlings, heat-treated plants). These results confirm what appears to be an amazing and unique association of FBaV-1 with Ficus car- ica. The extent of the association is such (100%) and involves trees from such a wide range of geographical origins, that makes it plausible, as in the case of other pararetroviruses (Staginnus and Richert-Pöggeler, 2006), the suggestion that FBaV-1 may be inte- grated in the fig genome; a likelihood further supported by the virus presence in volunteer fig seedlings.

The RNA2 of seven grapevine fanleaf virus (GFLV) isolates from vines with yellow mosaic (YM) symptoms from different origin were sequenced. These sequences showed a high variability in the homing protein (2AHP ) and, in five of them, a putative recombination with arabis mosaic virus (ArMV) was detected. To investigate recombination frequency, the partial sequences of the 2AHP of 28 additional GFLV isolates from nine different countries, showing either YM or infectious malformations (MF) symptoms, were obtained and compared with those of GFLV isolates from GenBank. The analysis confirmed the high level of sequence variability (up to 41 % at the nucleotide level) among isolates. In phylogenetic trees constructed using different approaches, the sequenced isolates always clustered in four conserved groups, three of which comprised YM strains (groups 1, 2 and 3), and one (group 4) the MF strains. Potential interspecific recombination sites between GFLV and ArMV were predicted in the 2AHP gene of several isolates, all of which were associated with YM symptoms.

Naturally contaminated basil seeds were treated by a surface dielectric barrier discharge driven in the humid air by an amplitude modulated AC high voltage to avoid heat shock. In order to avoid direct contact of seeds with microdischarge filaments, the seeds to be treated were placed at sufficient distance from the surface discharge. After treatment, the seeds were analyzed in comparison with control samples for their microbial contamination as well as for the capability of germination and seedling growth. Moreover, chemical modification of seed surface was observed through the elemental energy dispersive x-ray analysis and wettability tests. We found that treatment applied at 20% duty cycle (effective discharge duration up to 20 s) significantly decreases microbial load without reducing the viability of the seeds. On the other side, seedling growth was considerably accelerated after the treatment, and biometric growth parameters of seedlings (total length, weight, leaf extension) considerably increased compared to the controls. Interestingly, scanning electron microscopy images taken for the different duration of treatment revealed that seed radicle micropylar regions underwent significant morphological changes while the coat was substantially undamaged. Inside the seed, the embryo seemed to be well preserved while the endosperm body was detached from the epithelial tegument. A total of 9 different genera of fungi were recovered from the analyzed seeds. Scanning electron microscopy images revealed that conidia were localized especially in the micropylar region, and after plasma treatment, most of them showed substantial damages. Therefore, the overall effect of the treatment of naturally contaminated seeds by reactive oxygen and nitrogen species produced by plasma and the consequent changes in surface chemistry and microbial load can significantly improve seed vigor.

Heat therapy, meristem tip culture in vitro and a combination of both techniques were used for obtaining fig (Ficus carica) plants free from some viruses associated with fig mosaic, a disease with a worldwide distribution. Source plants were two field-grown adult fig accessions from a germplasm repository of the University of Bari (Italy) that showed severe and mild symptoms of mosaic, respectively, and two symptomless fig seedlings grown under screen. Adult plants were infected by Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1) and Fig badnavirus 1 (FBV-1). Seedlings were infected by FLV-1 and FBV-1. Progeny of explants subjected to meristem tip culture were still infected by FMV (93.8% elimination). This virus, however, was eradicated (100% sanitation) by shoot tip culture combined with heat therapy, or in vitro heat therapy. High sanitation rates from FLV-1 (81 to 100%) were also registered with all sanitation procedures employed, such as in vitro heat therapy alone (two cycles) or combined with tissue culture. By contrast, the DNA virus FBV-1 resisted all attempts of elimination, a behaviour that confirms indirectly its hypothesized integration in the fig genome.

Mulberry is a deciduous tree belonging to the Moraceae family. Although its economical importance and spreading all over the world, due mainly to the domesticated silkworm (Bombyx mori L.) breeding, to date, only few information are available about viral and virus-like diseases affecting this plant. In 2012 a small fragment of a Badnavirus DNA genome was sequenced after a DOP-PCR assay conducted on a mulberry plant, originated from Lebanon, showing symptoms of leaf mottling and vein yellowing. The virus, whose particles were observed with electron microscopy from a partially purified preparation and in tissue thin sections, was provisionally named Mulberry badnavirus-1 (MBV-1). Since different attempts of completing the full-length genome sequence through a conventional approach failed, a small RNA library was constructed for deep sequencing and run according to Illumina protocol ssRNAs analysis allowed the design of a set of primers used in PCR for the achievement of the full length sequence. The complete genome contains all the sequence features and the characteristic functional domains of the genus Badnavirus (highest nucleotide similarity shared with FBV-1 at 54%). By contrast to the badnaviruses, with genomes encoding for 3-4 ORFs, MBV-1 resembles genome organization of Petunia vein clearing virus (PVCV), which bears a single ORF. The study of the distribution of sRNA on the MBV-1 complete genome showed a tidy prevalence of 21- and 22-nt reads, differently from other viruses in the Caulimoviridae family, featuring a nuclear replication and typically supporting an accumulation of the 24-nt sRNAs involved in methylation processes.

Small RNAs were extracted and purified from total nucleic acids in late spring from two pooled samples of cv. Panse Precoce: ridge-like proliferations excised from symptomatic leaves, and symptomless portions. The sRNA libraries were sequenced using High Definition adapters. The resulting short reads were used to investigate the presence of viruses and for the analysis of host responseDifferentially expressed sRNAs between the two samples showed a prevalence of miRNA response. A down regulation of miR166 and miR396 in ena1 was observed. These miRNAs are involved in abaxial/adaxial identity of the leaf lamina and regulation of Growth-Regulating Factors genes implicated in the control of cell proliferation during leaf development

Mulberry badnavirus 1 (MBV1) has been characterized as the aetiological agent of a disease observed on a mulberry tree in Lebanon (accession L34). A small RNA next-generation sequencing library was prepared and analysed from L34 extract, and these data together with genome walking experiments have been used to obtain the full-length virus sequence. Uniquely among badnaviruses, the MBV1 sequence encodes a single ORF containing all the conserved pararetrovirus motifs. Two genome sizes (6 kb and 7 kb) were found to be encapsidated in infected plants, the shortest of which shares 98.95% sequence identity with the full L34 genome. In the less-than-full-length deleted genome, the translational frame for the replication domains was conserved, but the particle morphology, observed under electron microscopy, was somehow altered. Southern blot hybridization confirmed the coexistence of the two genomic forms in the original L34 accession, as well as the absence of cointegration in the plant genome. Both long and deleted genomes were cloned and proved to be infectious in mulberry. Differently from other similar nuclear-replicating viruses or viroids, the characterization of the MBV1-derived small RNAs showed a reduced amount of the 24-mer class size.

Different syndromes of a putative viral origin are comprised under the name ofFig mosaic disease (FMD), an aetiologically ill-defined infectious disease of figtrees, reported from all fig-growing countries in the world. In fact, at least 10different viruses and three viroids have been detected to date in FMD-affectedtrees: five closterovirids (Fig leaf mottle-associated virus 1 and 2, Fig mildmottle-associated virus, Arkansas closterovirus 1 and 2); a Trichovirus (Figlatent virus 1) and several isometric, bacilliform or enveloped viruses (Figcriptic virus, Fig fleck-associated virus, Fig badnavirus 1, Fig mosaic virus). Ofthese, Fig mosaic virus (genus Emaravirus) is the one with the highestassociation with FMD. Fig badnavirus 1, whose DNA is integrated in the figgenome, may not induce a disease even when virus particles are expressed infig seedlings. The development of typical FMD symptoms in seedlings in whichFMV was transmitted by its vector (the eriophyid mite Aceria ficus) support theconclusion that FMV is indeed one, if not the major, agent of FMD. Virtuallynothing is known on the pathogenicity and epidemiology of the other fig infectingviruses. FMD has an extremely high incidence in nature andsymptomless trees are often only apparently healthy. Thus, any campaign forthe improvement of the fig industry should utilize sanitation techniques for theproduction of certified healthy stocks for propagation and trade.

A virus with filamentous articles ca. 700 nm long, denoted Fig latent virus 1 (FLV-1) is widespread in Apulian(southern Italy) fig orchards, in trees showing or not mosaic symptoms and in symptomless seedlings. The virus wastransmitted by sap inoculation to a very restricted range of herbaceous hosts without inducing apparent symptoms andwas transmitted through fig seeds to a very high percentage (80 to 100 %). It was successfully purified from root tissues of infected figs. A virus-specific antiserum raised in rabbits, proved useful for its detection in fig leaf dips byimmunosorbent electron microscopy (ISEM), Western Blot, dot immuno-binding (DIBA), ELISA.The viral genomestructure resembles that of members of the genus Trichovirus in the family Flexiviridae.