Artichoke Italian latent virus (AILV), Artichoke latent virus (ArLV), Artichoke mottled crinkle virus (AMCV), Bean yellow mosaic virus (BYMV), Cucumber mosaic virus (CMV), Pelargonium zonate spot virus (PZSV), Tomato infectious chlorosis virus (TICV), Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV) and Turnip mosaic virus (TuMV) are damaging to artichoke. We have developed a protocol enabling the simultaneous detection of these artichoke viruses by non-isotopic dot blot hybridisation with DNA probes. The probe mix detected all viruses with high specificity and identical to that obtained using individual probes. The approach is proposed for the routine assessment of phytosanitary status for certified nursery production of globe artichoke.

An update on viruses infecting greenhouse vegetable crops in Italy Updates on some viruses infecting cucurbits and tomato crops grown in greenhouses are briefly outlined. Evolutionary aspects of WMV and ZYMV concerning the emergence of necrogenic strains and seed transmissibility of the two viruses are discussed as well as the unexpected detection of TYLCV in cucumber, melon, squash and watermelon. Since TYLCV infections in cucurbits are symptomless, the risk of latent infections for tomato crops is highlighted. As for tomato crops, for the first time substantial evidence is provided that reassortants constitute a consistent part of the natural population of TSWV. Finally, eco-epidemiological consequences of seed transmission of PZSV and PepMV are discussed envisaging that phytosanitary restrictions must be applied to seed to limit the spread of harmful plant viruses.

I meccanismi coinvolti nell’interazione pianta-agente fitopatogeno, e in particolare le risposte di difesa dell’ospite e i processi coinvolti nella patogenesi e nella induzione/manifestazione dei sintomi costituiscono una delle aree di ricerca più interessanti della fitovirologia moderna. L’interazione di una pianta ospite con un patogeno, quale può essere un virus, può innescare due possibili tipologie di risposta: resistenza della pianta alla malattia, nel caso di un’interazione geneticamente incompatibile; suscettibilità e quindi insorgenza della malattia, nel caso di un’interazione geneticamente compatibile (gReenbeRg, yao, 2004). i percorsi che si sviluppano nei due tipi di interazione, incompatibile e non, si sovrappongono e seguono alcune tappe fondamentali, come la produzione di specie reattive dell’ossigeno (RoS) e l’attivazione di sistemi di detossificazione intenti a regolare i livelli di tali molecole all’interno della cellula vegetale, l’induzione di cascate di trasmissione del segnale mediate da specifiche chinasi e ormoni vegetali, l’induzione di geni coinvolti nella risposta di difesa, l’innesco di fenomeni di morte cellulare programmata e/o risposta ipersensibile. e’ ampiamente riconosciuto che le RoS siano cruciali sia per lo sviluppo della pianta che per la sua difesa da aggressioni di varia natura (boLweLL, 1999; foyeR, noctoR, 2005): se da un lato le piante utilizzano le RoS come messaggeri in cascate di trasduzione del segnale, che regolano diversi processi come mitosi e tropismi, dall’altro la loro natura chimica le rende potenzialmente pericolose per le cellule vegetali. infatti l’incremento di perossido di idrogeno (H2o2) e anione superossido che si osserva nelle cellule a seguito dell’interazione con un patogeno, oltre una certa soglia di concentrazione, risulta tossico. Le cellule, tuttavia, possiedono sistemi di detossificazione di natura enzimatica e non enzimatica. nel presente lavoro sono stati studiati alcuni enzimi quali ascorbato perossidasi (aPx), catalasi (cat), perossidasi generiche (Pod) e superossido dismutasi (Sod), e metaboliti quali acido ascorbico, aventi un ruolo chiave nei processi di detossificazione da H2o2 e anione superossido (tommaSi et al., 1998; conkLin, baRtH, 2004), utilizzando come modello sperimentale il pomodoro (Solanum lycopersicum cv uc82) e Cucumber mosaic virus (cmV, virus del mosaico del cetriolo). in particolare, in piante di pomodoro inoculate con il ceppo cmV-fny (f), che mostrano sintomi di riduzione di crescita e malformazioni fogliari ma non di morte cellulare, e in piante inoculate con lo stesso ceppo in combinazione con la sua variante di Rna satellite necrogenica (fn), che incorrono in una necrosi letale, sono stati confrontati i valori relativi ad attività enzimatica e livelli di espressione di geni coinvolti nella detossificazione delle RoS, nonché di altri geni coinvolti nelle risposte di difesa ai patogeni. i risultati ottenuti su campioni di Rna ed estratti proteici a 9 giorni dopo l’inoculazione mostrano un aumento dell’espressione genica per aPx, in entrambi i campioni f ed fn, e per cat , solo nei campioni f, rispetto al controllo (piante di pomodoro sane). Si osserva, poi, un incremento dell’attività enzimatica dei loro prodotti in entrambi i campioni rispetto al controllo. inoltre, sia il livello di espressione dei trascritti che l’attività enzimatica risulta minore nei campioni fn rispetto ai campioni f. La misura del contenuto di acido ascorbico mostra un incremento nei campioni f ed fn, (maggiore in f) rispetto al controllo. L’incremento osservato riflette il suo ruolo di molecola antiossidante. il dato ottenuto, oltre a indicare il coinvolgimento dell’acido ascorbico nelle risposte antiossidanti di difesa, conferma l’esistenza di una relazione di proporzionalità diretta tra

The biological and molecular characterization of a Potato virus Y (PVY) isolate, denoted PVY(C)-to, associated with a necrotic phenotype of tomato plants grown in the province of Foggia (Apulia, southern Italy) is reported. The fully sequenced PVY(C)-to genome consists of 9,691 nucleotides and is 94,8% similar to that of PVY LYE84.2, an isolate of the PVY(C) strain group. Using programs in RDP package, a putative recombination breakpoint was identified at approximately nucleotide position 2056 to 2632, corresponding to the HC-Pro/P3 coding region. The event seems to represent an introgression of a PVY strain group sequence and, in particular, of PVY-OZ, which is a non recombinant isolate of the 0 lineage. From this analysis PVY(C)-to appeared to be a recombinant isolate. The virus has a very weak infectivity in pepper and possesses a CP coding region characterised by a PVY(C2) restrictotype. Our results seem to support the hypothesis that PVY(C)-to is a recombinant isolate of the PVY(C2) strain group.

Mixed infection with the SON41 strain of Potato virus Y (PVY-SON41) in tomato increased accumulation of RNAs of strains Fny and LS of Cucumber mosaic virus (CMV-Fny and CMV-LS, respectively) and enhanced disease symptoms. By contrast, replication of PVY-SON41 was downregulated by CMV-Fny and this was due to the CMV-Fny 2b protein. The CMV-FnyΔ2b mutant was unable to systemically invade the tomato plant because its movement was blocked at the bundle sheath of the phloem. The function needed for invading the phloem was complemented by PVY-SON41 in plants grown at 22°C whereas this complementation was not necessary in plants grown at 15°C. Mutations in the 2b protein coding sequence of CMV-Fny as well as inhibition of translation of the 2a/2b overlapping region of the 2a protein lessened both the accumulation of viral RNAs and the severity of symptoms. Both of these functions were complemented by PVY-SON41. Infection of CMV-Fny supporting replication of the Tfn-satellite RNA reduced the accumulation of CMV RNA and suppressed symptom expression also in plants mixed-infected with PVY-SON41. The interaction between CMV and PVYSON41 in tomato exhibited different features from that documented in other hosts. The results of this work are relevant from an ecological and epidemiological perspective due to the frequency of natural mixed infection of CMV and PVY in tomato.

Complete genomic sequences of Artichoke latent virus (ArLV) have been obtained by classical or high-throughput sequencing for an ArLV isolate from Italy (ITBr05) and for two isolates from France (FR37 and FR50). The genome is 8,278 to 8,291 nucleotides long and has a genomic organization comparable with that of Chinese yam necrotic mosaic virus (CYNMV), the only macluravirus fully sequenced to date. The cleavage sites of the viral polyprotein have been tentatively identified by comparison with CYNMV, confirming that macluraviruses are characterized by the absence of a P1 protein, a shorter and N-terminally truncated coat protein (CP). Sequence comparisons firmly place ArLV within the genus Macluravirus, and confirm previous results suggesting that Ranunculus latent virus (RALV), a previously described Macluravirus sp., is very closely related to ArLV. Serological relationships and comparisons of the CP gene and of the partial RaLV sequence available all indicate that RaLV should not be considered as a distinct species but as a strain of ArLV. The results obtained also suggest that the spectrum of currently used ArLV-specific molecular hybridization or polymerase chain reaction detection assays should be improved to cover all isolates and strains in the ArLV species.

Physiological and nutritional alterations in virus-infected plants This update focuses on metabolic, physiological and elemental content alterations in the framework of virus plant interactions. Plant trascriptome analysis reveals that progression of viral infections follows a cyclic pattern, reflecting different steps of virus replication as well as disease symptoms appearance and regression. In the framework of a global cellular remodelling viral replication is hosted in spherules, vesicles, and/or multivesicular bodies, which are denoted as virus factories or viroplasms. Membrane and organelle alterations are due to the action of few multifunctional viral proteins, which act as a scaffold for the assembly of larger complexes including viral RNA and viral and host proteins. Such viroplasms seem to be also involved in cellto- cell virus transport and in confining RNA replication to keep it away from certain host defence mechanisms. Using cucurbits, grapevine and tomato as model plants it is shown that viral infection affects: i) sugar transport and the amounts of the various sugars in the phloem sap; ii) synthesis of carotenoids and iii) total organic acid contents. The virus-induced altered metabolism is usually accompanied by increased respiration and decreased photosynthetic rates and modifies host selection behaviour by its insect vector, according to the “vector manipulation hypothesis”. Finally an estimate is given of viral protein income as infected vegetables enter human diet.

P>The quantification of messenger RNA expression levels by real-time reverse-transcription polymerase chain reaction requires the availability of reference genes that are stably expressed regardless of the experimental conditions under study. We examined the expression variations of a set of eight candidate reference genes in tomato leaf and root tissues subjected to the infection of five taxonomically and molecularly different plant viruses and a viroid, inducing diverse pathogenic effects on inoculated plants. Parallel analyses by three commonly used dedicated algorithms, geNorm, NormFinder and BestKeeper, showed that different viral infections and tissues of origin influenced, to some extent, the expression levels of these genes. However, all algorithms showed high levels of stability for glyceraldehyde 3-phosphate dehydrogenase and ubiquitin, indicated as the most suitable endogenous transcripts for normalization in both tissue types. Actin and uridylate kinase were also stably expressed throughout the infected tissues, whereas cyclophilin showed tissue-specific expression stability only in root samples. By contrast, two widely employed reference genes, 18S ribosomal RNA and elongation factor 1 alpha, demonstrated highly variable expression levels that should discourage their use for normalization. In addition, expression level analysis of ascorbate peroxidase and superoxide dismutase showed the modulation of the two genes in virus-infected tomato leaves and roots. The relative quantification of the two genes varied according to the reference genes selected, thus highlighting the importance of the choice of the correct normalization method in such experiments.

RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.

Nepoviral infections induce recovery in fully expanded leaves but persist in shoot apical meristem (SAM) by a largely unknown mechanism. The dynamics of infection of a grapevine isolate of Artichoke Italian latent virus (AILV-V, genus Nepovirus) in tobacco plants, including colonization of SAM, symptom induction and subsequent recovery of mature leaves from symptoms, were characterized. AILV-V moved from the inoculated leaves systemically and invaded SAM in 7 days postinoculation (dpi), remaining detectable in SAM at least up to 40 dpi. The new top leaves recovered from viral symptoms earliest at 21 dpi. Accumulation of viral RNA to a threshold level was required to trigger the overexpression of RDR6 and DCL4. Consequently, accumulation of viral RNA decreased in the systemically infected leaves, reaching the lowest concentration in the 3rd and 4th leaves at 23 dpi, which was concomitant with recovery of the younger, upper leaves from disease symptoms. No evidence of virus replication was found in the recovered leaves, but they contained infectious virus particles and were protected against re-inoculation with AILV-V. In this study we also showed that AILV-V did not suppress initiation or maintenance of RNA silencing in transgenic plants, but was able to interfere with the cell-to-cell movement of the RNA silencing signal. Our results suggest that AILV-V entrance in SAM and activation of RNA silencing may be distinct processes since the latter is triggered in fully expanded leaves by the accumulation of viral RNA above a threshold level rather than by virus entrance in SAM.

Development of a commercial diagnostic kit for the certification of the phytosanitary status in nursery production of globe artichoke. Artichoke Italian Latent virus (AILV), Artichoke latent virus (ArLV), Artichoke mottled crinkle virus (AMCV), Bean yellow mosaic virus (BYMV), Cucumber mosaic virus (CMV), Pelargonium zonate spot virus (PZSV), Tomato infectious chlorosis virus (TICV), Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV) and Turnip mosaic virus (TuMV) are damaging to artichoke, so new artichoke fields should be planted with virus-free certified germplasm. We have developed a protocol for the simultaneous detection of these artichoke viruses by nonisotopic dot blot hybridisation with DNA probes. The probe mix detected all viruses with identical specificity to that obtained using individual probes. The kit is proposed for the routine assessment of phytosanitary status of certified nursery production of globe artichoke.

Seed transmission of viruses and phytoplasmas: facts, factors and mechanisms. An account is given of the mechanism(s) involved in the seed transmission of plant viruses, most of which have been elucidated only recently and in few instances. Evidence has been provided that seed transmission by direct invasion of the embryo via the ovule depends upon symplastic transport of the virus from infected maternal cells. Such transport pathways have not been identified in higher plants but it has been shown that symplastic connections at the testa-boundary wall as well as porelike structures at the endosperm–suspensor boundary do exist in virus-infected plants. The role of virus determinants like the suppressors of posttranscriptional gene silencing in seed transmission is also discussed. Control measures to limit seed transmission capitalize on the availability of sensitive, reliable and labour saving immunological and nucleic acid-based laboratory techniques however emphasis is placed on the risk of sample contamination in doing these tests. Finally, some preliminary results suggest that also phytoplasmas agents of plant diseases could be transmitted through seeds, posing the needs for a more accurate investigation.

Most of the 25 viruses found in globe artichoke (Cynara scolymus L.) and cardoon (Cynara cardunculus L.) were recorded from Europe and the Mediterranean basin, where they decrease both the productivity and the quality of the crop. Although, sometimes, these viruses are agents of diseases of different severity, most often their infections are symptomless. These conditions have contributed to spread virus-infected material since farmers multiply traditional artichoke types vegetatively with no effective selection of virus-free plants. This review reports the main properties of these viruses and the techniques used for their detection and identification. ELISA kits are commercially available for most of the viruses addressed in this review but have seldom been used for their detection in artichoke. Conversely, nucleic acid-based diagnostic reagents, some of which are commercially available, have successfully been employed to identify some viruses in artichoke sap. Control measures mainly use virus-free stocks for new plantations. A combined procedure of meristem-tip culture and thermotherapy proved useful for producing virus-free regenerants of the reflowering southern Italian cultivar Brindisino, which kept earliness and typical heads shape.

The present invention concerns a process for the synthesis of a molecular polyprobe comprising nucleic acid useful for simultaneous diagnosis of several harmful viruses affecting the quality of artichoke production, in particular in nursery productions, a diagnostic kit comprising said molecular polyprobe and its use for detecting artichoke viruses