Recent advances in high-throughput sequencing technologies and bioinformatics have generated huge new opportunities for discovering and diagnosing plant viruses and viroids. Plant virology has undoubtedly benefited from these new methodologies, but at the same time, faces now substantial bottlenecks, namely the biological characterization of the newly discovered viruses and the analysis of their impact at the biosecurity, commercial, regulatory, and scientific levels. This paper proposes a scaled and progressive scientific framework for efficient biological characterization and risk assessment when a previously known or a new plant virus is detected by next generation sequencing (NGS) technologies. Four case studies are also presented to illustrate the need for such a framework, and to discuss the scenarios.

Following the introduction and establishment of the plant pathogenic bacterium Xylella fastidiosa (Xf) in the Apulia Region (southern Italy), olive turned to be the main host of the Salentinian bacterial strain and the majorly devastated crop. The mechanism of pathogenicity of Xf is still not completely understood and no means to cure the bacterium in the infected plants are available yet. Nevertheless, the alteration of microbial communities and effects in the expression of symptoms of Xf-infected plants is poorly studied. We are investigating the microbiome of Xf-infected olives by a shotgun metagenomic DNA sequencing approach that avoids the limitations of amplicon sequencing. Data obtained (28,333,924 and 29,096,610 reads from Xf-infected and healthy plants) were analyzed by MetaPhlAn, a metagenomic abundance estimation tool which maps reads to a set of selected marker sequences. Libraries from xylem tissues revealed a complex community in which small symbiotic bacteria of insects, i.e. Candidatus Zinderia insecticola and Candidatus Carsonella ruddii represented the 31% and 22% of the total population. Xf reaches in infected plants the 12% of the total microbial community. Studies are ongoing to characterize the microbial communities in the xylem sap of tolerant and susceptible olive cultivars, to envisage a control strategy based on the manipulation of these resident communities and to identify endosymbiont(s) which may be used to reduce the severity of symptoms. To this end, the evaluation of an endosymbiont bacterium for its potential to colonize Xf-infected olive tissues is underway.

We report here the complete and annotated genome sequence of the plant-pathogenic bacterium Xylella fastidiosa subsp. pauca strain De Donno. This strain was recovered from an olive tree severely affected by olive quick decline syndrome (OQDS), a devastating olive disease associated with X. fastidiosa infections in susceptible olive cultivars.

Virus-derived short interfering RNAs (vsiRNAs) isolated from grapevine V. vinifera Pinot Noir clone ENTAV115 were analyzed by high-throughput sequencing using the Illumina Solexa platform. We identified andcharacterized vsiRNAs derived from grapevine field plants naturally infected with different viruses belongingto the genera Foveavirus, Maculavirus, Marafivirus and Nepovirus. These vsiRNAs were mainly of 21 and 22nucleotides (nt) in size and were discontinuously distributed throughout Grapevine rupestris stem-pittingassociated virus (GRSPaV) and Grapevine fleck virus (GFkV) genomic RNAs. Among the studied viruses, GRSPaVand GFkV vsiRNAs had a 52 terminal nucleotide bias, which differed from that described for experimental viralinfections in Arabidopsis thaliana. VsiRNAs were found to originate from both genomic and antigenomicGRSPaV RNA strands, whereas with the grapevine tymoviruses GFkV and Grapevine Red Globe associatedvirus (GRGV), the large majority derived from the antigenomic viral strand, a feature never observed in otherplant–virus interactions.

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.

The complete nucleotide sequence and genome organization of a new Badnavirus isolated from the autochthonous grapevine variety ''Bombino nero'' from Apulia (Italy) was determined. The genome of this virus consists of 7097 nt and has four open reading frames (ORFs). Analysis of putative proteins encoded by each ORF revealed greatest sequence similarity to Grapevine Roditis leaf discolorationassociated virus w4 (GRLDaV; NC_027131). In a pairwise alignment with GLRDaV w4 genome sequence, the ''Bombino Nero'' sequence was 109 nt longer with a major 57 nt insertion between positions 2405 and 2413. Furthermore, its putative ORF4 is located after the ORF3, while in the GLRDaV w4 sequence, the putative ORF4 completely overlapped ORF3. Nucleotide analysis classifies this new Badnavirus as a GLRDaV strain, which was named GRLDaV-BN. Multi-year field observations showed that the GLRDaV-BN-infected vine was symptomless.

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.

The recent Xylella fastidiosa subsp. pauca (Xfp) outbreak in olive (Olea europaea) groves in southern Italy is causing a destructive disease denoted Olive Quick Decline Syndrome (OQDS). Field observations disclosed that Xfp-infected plants of cv. Leccino show milder symptoms, than the more widely grown and highly susceptible cv. Ogliarola salentina. To ascertain whether these field observations underlie a tolerant condition of cv. Leccino, which could be exploited for lessening the economic impact of the disease on the local olive industry, transcriptional changes occurring in plants of the two cultivars affected by Xfp were investigated. A global transcriptome profiling comparing susceptible (Ogliarola salentina) and tolerant (Leccino) olive cultivars, infected or not by Xfp, was done on messenger RNA (mRNAs) extracted from xylem tissues. The study revealed that 659 and 447 genes were differentially regulated in cvs Leccino and Ogliarola upon Xfp infection, respectively, whereas 512 genes were altered when the transcriptome of both infected cultivars was compared. Analysis of these differentially expressed genes (DEGs) shows that the presence of Xfp is perceived by the plants of both cultivars, in which it triggers a differential response strongly involving the cell wall. Up-regulation of genes encoding receptor-like kinases (RLK) and receptor-like proteins (RLP) is the predominant response of cv. Leccino, which is missing in cv. Ogliarola salentina. Moreover, both cultivars react with a strong re-modelling of cell wall proteins. These data suggest that Xfp elicits a different transcriptome response in the two cultivars, which determines a lower pathogen concentration in cv. Leccino and indicates that this cultivar may harbor genetic constituents and/or regulatory elements which counteract Xfp infection. These findings suggest that cv. Leccino is endowed with an intrinsic tolerance to Xfp, which makes it eligible for further studies aiming at investigating molecular basis and pathways modulating its different defense response.

The draft genome sequence of Xylella fastidiosa CO33 isolate, retrieved from symptomatic leaves of coffee plant intercepted in northern Italy, is reported. The CO33 genome size is 2,681,926 bp with a GC content of 51.7%. Isolate CO33, cultured from a coffee plant intercepted in northern Italy, represents a novel multilocus sequence typing profile, ST72 (G. Loconsole, personal communication). Isolates genetically related to CO33 were found in several coffee plants imported in October 2014 from Costa Rica through the Netherlands (European Food Safety Authority [EFSA] 2015).

We determined the draft genome sequence of the Xylella fastidiosa CoDiRO strain, which has been isolated from olive plants in southern Italy (Apulia). It is associated with olive quick decline syndrome (OQDS) and characterized by extensive scorching and desiccation of leaves and twigs.

The quarantine bacterium Xylella fastidiosa (Xf) is responsible for diseases of a wide range of cultivated and wild plants. Few ef- forts have been made to investigate the potential use of endophytic symbionts on the disease phenotype of Xf-infected plants. The aim of our study was to evaluate if Paraburkholderia phytofirmans PsJN strain, a plant growth-promoting rhizobacterium, whose beneficial effects in the reduction of symptom severity caused by Xf in grape- vine affected by Pierce's Disease have recently been proven, may play a role as biocontrol agent against Xf CoDiRO strain, the agent of a severe disease of olives in Apulia (southern Italy). Greenhouse trials are being conducted to test the ability of P. phytofirmans to colonise xylem vessels of olive, Nicotiana benthamiana and oleander, following inoculation of bacterial suspensions by needle puncture and root dipping. A conventional PCR assay for detection of P. phytofirmans movement in plants has been developed to be used in combination with plate isolation and a qPCR specific assay. Preliminary results showed that needle-inoculated bacterial cells were detectable in the leaf petioles of the three hosts, away from the inoculation site. Root dipping proved successful in infecting in vitro-cultured olive plantlets. Double-infection assays, currently underway, will prove if P. phytofirmans PsJN shows a beneficial interaction with Xf CoDiRO.

The recent findings of the plant pathogenic bacterium Xylella fastidiosa (Xf), infecting several plant species in Italy and France, raised major concerns for its potential impact on the EU and Mediterranean agriculture. In the current EU outbreaks, olive is the predominantly affected crop, in which the bacterium has been consistently associated with a new severe syndrome, denoted Olive Quick Decline". So far, no effective treatments are available to cure infected plants. However, several approaches have been explored, mainly in grapevine and citrus, to reduce bacterial movement and multiplication or directly targeting Xf-cells for lysis.Current knowledge shows that the virulence of the pathogen relies on a fine balance between more motile bacterial forms, able to move and proliferate within xylem vessels, and sticky cells forming a biofilm, which are responsible for vessels blockage and insect acquisition. This different behavior is regulated in a cell density-dependent manner by a diffusible signaling factor (DSF), produced by rpfF-gene, that initiates a transduction cascade resulting in up- or down-regulation of several genes.The aim of our investigation is to explore "pathogen confusion" strategy, by altering DSF level in planta, for reducing the impact of Xf-infections in olives. To this end, a plant viral-based vector, harboring the rpfF-gene, has been engineered to induce transient DSF production. Experiments will verify if, upon DSF accumulation, the bacterium will be less motile and more adhesive to the surface of xylem vessels, thus showing a decreased virulence in infected plants.

Stone fruits rank third among the most important crop species in Chile, after grapevine and apple. Specifically, cherry (Prunus avium L.) cultivation have increased during the last 10 years, making of Chile the most important exporter in the Southern hemisphere. Nineteen cherry samples collected in the spring of 2016 were subjected to high-throughput sequencing (HTS) analyses. Small RNA extracts were obtained following the protocol described by Giampetruzzi et al. (2012). Sequencing libraries were prepared using TruSeq smallRNA library preparation kit (Illumina Inc.) and sequenced on Illumina HiScanSQ platform. Trimming and de novo assembly of sequenced reads using CLC genomics workbench v7.0 were carried out, and the obtained contigs were analyzed with BLAST. One sample presented 131 contigs that showed homology with the Plum bark necrosis stem pitting-associated virus (PBNSPaV) reference sequence with accession no. EF546442. The alignment of nine PBNSPaV complete genome references allowed the design of two primer pairs specific for the RdRp gene (PBN-RdRp-F 5?-CTTATTATTGTGCTGAAGTTGATCT-3?/PBN-RdRp-R 5?-TGGAAAAGTATTGAGTCATCACC-3?) and a partial region of CP gene (PBN-CP-F 5?-GAGGCAATGGATGAGGAA-3?/PBN-CP-R 5?-TCTTCCACCGGACTGATTA-3?) to be used in RT-PCR. The RdRp (KY887573) and CP (KY887574) sequences amplified from isolate 10381 shared 99 and 97% identity with reference isolate WH1 (KJ792852) from China and the PBNSPaV type-strain (EF546442) from the U.S.A., respectively. In addition, the HTS analysis showed that 14 out of 19 cherry samples have several contigs showing homology with Cherry virus A (CVA) reference sequence. Two primer pairs (CVAF1 5?-CAATGTTGTTGACAATTCCCAC-3?/CVAR1 5?-CCTACATGAATTTGACCTAAACAAA-3?; CVAF2 5?-ACTGCAGAGAAAACAACTGCC-3?/CVAR2 5?-AGGCCCCTTCTTATCTCGTT-3?) were designed based on the alignment of CVA complete genomes database with the sequences obtained from Chilean isolates. CVA infection was confirmed via RT-PCR in all 14 cherry trees using both primer pairs. BLASTn analysis of the two amplification products of CVA from isolate 10596 (KY887575, KY887577) showed 99% of identity with the isolate Lambert (KU215410) from Czech Republic and the same amplicons obtained from isolate 10395 (KY887576, KY887578) showed 99% of identity with the isolate ChTA12 from China (KT310083). To our knowledge, this is the first report of CVA and PBNSPaV infecting cherry in Chile and South America. Further analyses are in progress in order to determine the prevalence of these viruses in the main cherry producing areas of Chile.

The role of Grapevine Pinot gris virus (GPGV) in the etiology of grapevine leaf mottling and deformation was investigated by biological and molecular assays. A survey on different cultivars from the Trentino Region in Italy showed a widespread distribution of GPGV, which was associated with symptomatic (79%) but also with symptomless vines (21%). Symptomatic and GPGV-infected Pinot gris vines induced symptoms on grafted vines of healthy Pinot gris or Traminer, whereas GPGV-infected but symptomless vines did not. High-throughput sequencing of small RNA (sRNAs) populations of two infected Pinot gris accessions confirmed the existence of nearly overlapping viromes in vines with or without symptoms but phylogenetic analyses of the genomes of seven GPGV isolates from Italy and the Czech and Slovak Republics clearly differentiated those infecting symptomatic vines. The involvement of Grapevine rupestris vein feathering virus (GRVFV) in the disease, which was only infecting the symptomatic vine, was ruled out by RT-PCR studies. Maximum likelihood and Bayesian phylogenetic analysis of two GPGV genomic regions, encompassing part of the movement protein and coat protein gene sequences (MP/CP) and the RNA dependent RNA polymerase domain (RdRp) of the replicase gene, showed that isolates from symptomatic vines form a lineage distinct from that of symptomless vines. Moreover, the presence or lack of the MP stop codon identified in viral isolates from symptomatic or symptomless vines, respectively, is likely responsible for a six amino acids longer MP in symptomless isolates.

Xylella fastidiosa is a plant-pathogenic bacterium recently introduced in Europe that is causing decline in olive trees in the South of Italy. Genetic studies have consistently shown that the bacterial genotype recovered from infected olive trees belongs to the sequence type ST53 within subspecies pauca. This genotype, ST53, has also been reported to occur in Costa Rica. The ancestry of ST53 was recently clarified, showing it contains alleles that are monophyletic with those of subsp. pauca in South America. To more robustly determine the phylogenetic placement of ST53 within X. fastidiosa, we performed a comparative analysis based on single nucleotide polymorphisms (SNPs) and the study of the pan-genome of the 27 currently public available whole genome sequences of X. fastidiosa. The resulting maximum-parsimony and maximum likelihood trees constructed using the SNPs and the pan-genome analysis are consistent with previously described X. fastidiosa taxonomy, distinguishing the subsp. fastidiosa, multiplex, pauca, sandyi, and morus. Within the subsp. pauca, the Italian and three Costa Rican isolates, all belonging to ST53, formed a compact phylotype in a clade divergent from the South American pauca isolates, also distinct from the recently described coffee isolate CFBP8072 imported into Europe from Ecuador. These findings were also supported by the gene characterization of a conjugative plasmid shared by all the four ST53 isolates. Furthermore, isolates of the ST53 clade possess an exclusive locus encoding a putative ATP-binding protein belonging to the family of histidine kinase-like ATPase gene, which is not present in isolates from the subspecies multiplex, sandyi, and pauca, but was detected in ST21 isolates of the subspecies fastidiosa from Costa Rica. The clustering and distinctiveness of the ST53 isolates supports the hypothesis of their common origin, and the limited genetic diversity among these isolates suggests this is an emerging clade within subsp. pauca.

Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management. © 2013 Maree, Almeida, Bester, Chooi, Cohen, Dolja, Fuchs, Golino, Jooste, Martelli, Naidu, Rowhani, Saldarelli and Burger.

The Italian Ministry of Agriculture funded in 2009 the "ARNADIA" Project, aimed at producing validated reference diagnostic protocols for the control and monitoring of plant pathogens of phytosanitary interest and, among them, grapevine viruses. In this framework, the "Working group ARNADIA-grapevine viruses (WG)", composed of eight universities and research bodies, three accredited private laboratories, one plant health service and one association of grapevine nurseries, was established.

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.

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).

In the attempt to identify the causal agent of Citrus chlorotic dwarf disease (CCDD), a virus-like disorder of citrus, the small RNA fraction and total DNA from symptomatic citrus plants were subjected to high-throughput sequencing. DNA fragments deriving from an apparently new geminivirus-like agent were found and assembled by NGS to re-construct the entire viral genome. The newly identified virus has a circular single-stranded DNA genome comprising five open reading frames (ORFs) with sequence homologies with those encoded by geminiviruses. PCR and qPCR assays were successfully used for determining its presence in the CCDD-affected plants obtained by graft propagation. The larger genome size (3.64 vs. 2.5-3.0 kb) and a number of differences in its structural organization, identified this virus as a highly divergent member of the family Geminiviridae, to which the provisional name of Citrus chlorotic dwarf-associated virus (CCDaV) is assigned.

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.

NGS was welcomed as a "gold rush" at CNR-IPSP for the detection of virus and virus-like diseases of Mediterranean woody and horticultural crops, particularly grapevine, citrus, stone fruits and also minor fruits (quince, mulberry, fig). A major input to the adoption of this techniques relied on the propagation practices of particularly woody species, which multiply infections and spread viruses and viroids. The Institute now possesses skilled personnel and hardware facilities able to perform current NGS protocols in Plant Virology and to respond to a continuously evolving technology. Technologies followed the achievements on RNA silencing for the analysis of small RNA libraries as well as addressed mRNAs- and double stranded RNA-based libraries. Results of the last nine-year activities consisting in the discovery of several new viruses and the use of NGS data in the description of plant-virus/viroids interactions will be presented, coming to the recent application of the technology on commercial grapevine cultivars. The advantages of using NGS technology in the production of certified plant propagation material and the proposal of a future "vine metagenome passport" accompanying mother plants in commercial exchanges will be discussed.

Il Ministero per le Politiche Agricole, Alimentari e Forestali (MiPAAF) nel 2009 ha finanziato il Progetto "ARNADIA" finalizzato alla produzione di protocolli diagnostici, armonizzati e validati, che fossero di riferimento per il controllo e il monitoraggio dei patogeni vegetali di interesse fitosanitario. In questo ambito, è stato costituito il "Gruppo di Lavoro ARNADIA - virus della vite" composto da 8 Università ed Enti di Ricerca, 3 Laboratori privati accreditati, un Laboratorio dei Servizi Fitosanitari e un'Associazione di vivaisti viticoli. L'obiettivo del Gruppo di Lavoro è stato quello di produrre protocolli diagnostici di riferimento, sia sierologici che molecolari, calcolandone i parametri di validazione al fine di ottenere l'armonizzazione della diagnosi di 8 virus vite: Grapevine leafroll-associated virus-1, -2, -3, (GLRaV 1, 2, 3 ) Grapevine virus A (GVA), Grapevine virus B (GVB), Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV) e Grapevine fleck virus (GFkV). La validazione di un protocollo consiste nella valutazione di specifici parametri volti a determinare la loro idoneità per identificare la presenza di un "target" specifico. I parametri che influenzano la capacità di un protocollo di definire accuratamente lo stato sanitario del campione in esame sono: la sensibilità diagnostica (capacità del metodo utilizzato di rilevare la presenza del patogeno nei campioni veramente infetti dal patogeno in questione - veri positivi) e la specificità diagnostica (capacità del metodo utilizzato di non per rilevare la presenza del patogeno in campioni non infetti dal patogeno in questione - veri negativi). Altri parametri che devono essere considerati e che determinano l'efficienza di un protocollo sono: la sensibilità analitica (la più piccola quantità di entità infettiva che può essere identificata dal protocollo), la ripetibilità o accordanza (grado di conformità dei risultati ottenuti nella replicazione del protocollo, in intervalli di tempo brevi, utilizzando gli stessi campioni di riferimenti nelle stesse condizioni di lavoro cioè apparecchiature, operatore, laboratorio) e riproducibilità o concordanza (grado di conformità dei risultati ottenuti con lo stesso metodo con gli stessi campioni di riferimento in diversi laboratori). Quest'ultimo parametro è stato definito con la collaborazione di altri 5 laboratori dei Servizi Fitosanitari Regionali. Nello specifico, 122 campioni di vite (varietà, portainnesti e campioni "pool" composti da 5 piante, di cui solo una infetta) sono stati analizzati mediante ELISA, utilizzando 25 antisieri di tre società commerciali (Agritest, Bioreba e Sediag) e multiplex RT-PCR. Per l'ELISA i test sono stati condotti seguendo le indicazioni fornite dalle Ditte; per la multiplex RT-PCR è stato utilizzato il protocollo descritto da Gambino e Gribaudo, (2006). I test sono stati eseguiti in 18 laboratori utilizzando gli stessi campioni (analizzati in condizioni di anonimato) e reagenti.

I protocolli diagnostici descritti in questo volume sono il prodotto dell'attività effettuata nell'ambito del Progetto Finalizzato 'ARON-ARNADIA', finanziato dal Ministero per le Politiche Agricole, Alimentari e Forestali (DM 19738/7303/08 del 29/12/2008).I protocolli forniscono le linee guida per la diagnosi e l'identificazione di patogeni delle piante (funghi, batteri, fitoplasmi, virus e viroidi) nei laboratori preposti alla diagnosi degli organismi di qualità e/o quarantena, presenti sul territorio italiano. L'uso di protocolli diagnostici armonizzati è alla base di un'efficiente applicazione delle misure fitosanitarie e consente il confronto dei risultati ottenuti da diversi laboratori in diverse condizioni operative.Le metodologie di laboratorio riportate nei protocolli di questo volume sono state selezionate sulla base del valore, ottenuto per ciascuna di esse, dei parametri di sensibilità diagnostica (inclusività per le colture batteriche), specificità diagnostica (esclusività per le colture batteriche), selettività, accuratezza, sensibilità analitica (limite di rilevabilità), specificità analitica, ripetibilità e riproducibilità (ISO/IEC standard 16140:2003; ISO/IEC standard 17025:2005; EPPO standard PM7/98).

Gli istituti di ricerca pugliesi riportano i risultati ottenuti dall'impiego di attivatori delle difese della pianta e dell'N-acetilcisteina per il contenimentodei sintomi del complesso del disseccamento rapido dell'olivo.

Osservazioni e rilievi di campo integrati da saggi diagnostici effettuati su diverse cultivar in un'area fortemente infetta da Xylella fastidiosa confermano i fenomeni di resistenza già osservati in precedenti studi sulla cultivar Leccino e individuano nella selezione FS-17® un'ulteriore e potenziale fonte di resistenza al batterio. Al contrario, si rafforzano le evidenze sull'elevata suscettibilità delle cultivar Cellina di Nardò e Ogliarola salentina.

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

Xylella fastidiosa (Xf) is a xylem-limited bacterium, regulated as a quarantine pest, that is causing a devastating disease on olive crops in the southern area of Apulia (Italy) and whose potential spread in the Mediterranean area poses a severe threat to EU agri- culture and landscape environment. Xf virulence is related to the expression of a cluster of rpf (regulation of pathogenicity factors) genes responsible for a signalling system based on small fatty acid molecules called DSF (Diffusible Signalling Factor). Since DSF regulation is involved in pathogenicity traits of Xf and biofilm for- mation, a "pathogen-confusion" strategy, based on the alteration of DSF levels in planta, has been proposed to contrast bacterial infection. In grapevine, the strategy is based on the transgenic ex- pression of the rpfF gene, which encodes the DSF-synthase. We are attempting to express the rpfF gene of the olive-infecting Xf strain CoDiRO in the heterologous Escherichia coli system. The gene product has been successfully detected by Western blot analysis in cell protein extracts. Chemical characterisation by Gas Chromatog- raphy-Mass Spectrometry analysis of the DSF molecules produced by this expression system, in addition to those naturally produced by Xf CoDiRO, are underway. Concurrently, a TMV-based vector has been engineered to harbour the same rpfF gene and induce its transient expression in planta. Biologically active transcripts of the vector have been inoculated to Nicotiana tabacum and N. ben- thamiana plants, to establish a model system on herbaceous hosts. RpfF expression was successfully proved by Western blot analysis, whereas movement and systemic colonisation of plant tissues were evaluated by RT-PCR assays. The same viral vector harbouring GFP in replacement of rpfF is used as a control. Following inoculation with Xf CoDiRO bacterial cells the system is now being tested to monitor the persistence of DSF expression and its efficacy to lower disease susceptibility or movement of bacterial cells behind the point of inoculation.

The CoDiRO strain of Xylella fastidiosa subsp. pauca (Xfp) is ravaging olive (Olea europaea) groves in southern Italy, causing a destructive disease denoted Olive Quick Decline Syndrome (OQDS). Field observations show that the Xfp-infected plants of the cv. Ogliarola salentina develop more severe symptoms than that of cv. Leccino. A global transcriptome profiling comparing the two olive cultivars, infected or not by Xfp, was performed to ascertain whether a tolerant condition of cv. Leccino exists, which could be exploited for lessening the economic impact of the disease on the local olive industry. The study revealed that 659 and 447 genes were differentially regulated upon Xfp infection, in cvs Leccino and Ogliarola salentina, respectively, whereas 512 genes resulted altered between the two infected cultivars. The analysis showed that plants of both cultivars perceive the presence of Xfp, mainly involving cell wall-associated proteins. The predominant response of cv. Leccino, which is missing in cv. Ogliarola salentina, consists on the up-regulation of genes encoding receptor-like kinases and receptor-like proteins. This different transcriptome response determines a lower pathogen concentration in the cv. Leccino, suggesting that it may harbor genetic constituents and/or regulatory elements which counteract Xfp infection. These findings suggest that cv. Leccino is endowed with an intrinsic tolerance to Xfp, which makes it eligible for further studies aimed at investigating molecular pathways underlying its different defense response.

The recent Xylella fastidiosa subsp. pauca (Xfp) outbreak in olive (Olea europaea) groves in southern Italy is causing a destructive disease denoted Olive Quick Decline Syndrome (OQDS). Field observations disclosed that Xfp-infected plants of cv. Leccino show much milder symptoms, than the more widely grown and highly susceptible cv. Ogliarola salentina. To determine whether these field observations underlie a tolerant condition of cv. Leccino, which could be exploited for lessening the economic impact of the disease on the local olive industry, transcriptional changes occurring in plants of the two cultivars affected by Xfp were investigated. Results: A global quantitative transcriptome profiling comparing susceptible (Ogliarola salentina) and tolerant (Leccino) olive cultivars, infected or not by Xfp, was done on messenger RNA (mRNAs) extracted from xylem tissues. The study revealed that 659 and 447 genes were differentially regulated in cvs Leccino and Ogliarola upon Xfp infection, respectively, whereas 512 genes were altered when the transcriptome of both infected cultivars was compared. Analysis of these differentially expressed genes (DEGs) shows that the presence of Xfp is perceived by the plants of both cultivars, in which it triggers a differential response strongly involving the cell wall. Up-regulation of genes encoding receptor-like kinases (RLK) and receptor-like proteins (RLP) is the predominant response of cv. Leccino, which is missing in cv. Ogliarola salentina. Moreover, both cultivars react with a strong re-modelling of cell wall proteins. These data suggest that Xfp elicits a different transcriptome response in the two cultivars, which determines a lower pathogen concentration in cv. Leccino and indicates that this cultivar may harbor genetic constituents and/or regulatory elements which counteract Xfp infection. Conclusions: Collectively these findings suggest that cv. Leccino is endowed with an intrinsic tolerance to Xfp, which makes it eligible for further studies aiming at investigating molecular basis and pathways modulating its different defense response.

MicroRNAs are post-transcriptional regulators of eukaryotic organisms that function through mRNA degradation or suppression of translation. Artificial microRNA (amiRNA) act in similar way and this technology has recently attracted much attention as a tool against plant virus infections. In this study, a construct assembled on the Vitis vinifera miR166f (vvi-miR166f) pre-miRNA was used to construct an amiRNA cassette by replacing the 21 nucleotides (nt) of the miR166f sequence with a 21 nt sequence from the Grapevine virus A (GVA) genome. Two amiRNAs cassettes were engineered with 21 nt sequences targeting GVA ORF1 and ORF5. Transient expression of these amiRNAs in Nicotiana benthamiana led to various levels of resistance indicating amiRNA technology as a potential tool for GVA control.

Grapevine Pinot gris virus (GPGV) is a new virus reported in Europe and several other grape-growing countries. In an attempt to identify a vector for GPGV, samples of the eriophyid mite Colomerus vitis collected from buds and erinea in GPGV-infected vines were analysed by RT-PCR, using specific primers. Molecular analysis revealed the presence of GPGV in C. vitis. Transmission trials were conducted using C. vitis collected from GPGV-infected vines. Mites were able to transmit GPGV to healthy grapevines, suggesting that C. vitis is a potential vector of this virus.