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Most of the 26 viruses that infect globe artichoke are detrimental to the crop’s production, quality, and nutritional value, hamper the development of a nursery activity in the respect of current EU legislation and pose risks for the new plantings. Worldwide movement of virus-controlled plant propagative material require fast, accurate and feasible methods to guarantee reliable results and detection of multiple viruses in a single assay to reduce time and costs in routine tests. Here we describe the use of a digoxigenin-labelled polyprobe assembled from synthetic 50 nt single-stranded DNA sequences derived from genomes of the most commonly occurring viruses in globe artichoke. These included artichoke Italian latent virus (AILV), artichoke latent virus (ArLV), artichoke mottled crinkle virus (AMCV), turnip mosaic virus (TuMV), tomato infectious chlorosis virus (TICV), bean yellow mosaic virus (BYMV), cucumber mosaic virus (CMV), pelargonium zonate spot virus (PZSV), tomato spotted wilt virus (TSWV) and tobacco mosaic virus (TMV). The polyprobe described herein represent the first cheap and reliable example of a technology for broad-spectrum surveys in globe artichoke.

Fibered Laser Shock Peening has been implemented thanks to a record-breaking fiber injection system allowing the transmission of 63MW in the nanosecond regime. Results obtained on aluminum and titanium are presented.

RNA interference (RNAi) is a sequence identity-dependent RNA degradation mechanism conserved in eukaryotic organisms. One of the roles of RNAi is as a defense system against viral infections, which has been demonstrated in filamentous fungi but not in oomycetes. We investigated the virus-RNAi interplay in the oomycete Phytophthora infestans using a crucifer-infecting strain of the plant virus tobacco mosaic virus (TMVcr) and its derivative TMVcr-Δ122 that is mutated in the sequence of the p122 replicase subunit and thus inhibited in RNA suppression activity. In this study we provide evidence that replication of TMVcr-Δ122 but not of TMVcr was impaired in P. infestans as well as in tobacco plants used as positive control. The interference was associated with induction of high transcription of dicer-like genes Pidcl2 and NtDCL2 and of RNA-dependent-RNA-polymerase Pirdr1 and NtRDR1 in P. infestans and tobacco, respectively. These high transcription levels suggest an RNAi-based response that TMVcr-Δ122 mutant was not able to suppress. Taken altogether, results of this study demonstrated that an antiviral silencing activity operates also in P. infestans and that a plant virus could be a simple and feasible tool for functional studies also in oomycetes.

Grafting is routinely implemented in modern agriculture to manage soilborne pathogens such as fungi, oomycetes, bacteria, and viruses of solanaceous crops in a sustainable and environmentally friendly approach. Some rootstock/scion combinations use specific genetic resistance mechanisms to impact also some foliar and airborne pathogens, including arthropod or contact-transmitted viruses. These approaches resulted in poor efficiency in the management of plant viruses with superior virulence such as the strains of tomato spotted wilt virus breaking the Sw5 resistance, strains of cucumber mosaic virus carrying necrogenic satellite RNAs, and necrogenic strains of potato virus Y. Three different studies from our lab documented that suitable levels of resistance/tolerance can be obtained by grafting commercial tomato varieties onto the tomato ecotype Manduria (Ma) rescued in the framework of an Apulian (southern Italy) regional program on biodiversity. Here we review the main approaches, methods, and results of the three case studies and propose some mechanisms leading to the tolerance/resistance observed in susceptible tomato varieties grafted onto Ma as well as in self-grafted plants. The proposed mechanisms include virus movement in plants, RNA interference, genes involved in graft wound response, resilience, and tolerance to virus infection.

Grafting of commercial tomato varieties and hybrids on the tomato ecotype Manduria resulted in high levels of tolerance to the infection of Sw5 resistance-breaking strains of tomato spotted wilt virus and of severe cucumber mosaic virus strains supporting hypervirulent satellite RNAs that co-determine stunting and necrotic phenotypes in tomato. To decipher the basis of such tolerance, here we used a RNAseq analysis to study the transcriptome profiles of the Manduria ecotype and of the susceptible variety UC82, and of their graft combinations, exposed or not to infection of the potato virus Y recombinant strain PVYC-to. The analysis identified graft- and virus-responsive mRNAs differentially expressed in UC82 and Manduria, which led to an overall suitable level of tolerance to viral infection confirmed by the appearance of a recovery phenotype in Manduria and in all graft combinations. The transcriptome analysis suggested that graft wounding and viral infection had diverging effects on tomato transcriptome and that the Manduria ecotype was less responsive than the UC82 to both graft wounding and potyviral infection. We propose that the differential response to the two types of stress could account for the tolerance to viral infection observed in the Manduria ecotype as well as in the susceptible tomato variety UC82 self-grafted or grafted on the Manduria ecotype.

Viruses are common in fungi and oomycetes and some of these viruses share sequence identities with plant viruses belonging to different families and genera. Replication of the plant virus tobacco mosaic virus has been demonstrated in the phytopathogenic fungi Colletotrichum acutatum, C. clavatum and C. theobromicola and more recently, infection of cucumber mosaic virus has been reported in Rhizoctonia solani isolated from potato plants in the field. Here we show that artichoke mottled crinkle virus, potato virus X, potato virus Y and cucumber mosaic virus plus its satellite RNA can replicate and persist in C. acutatum at least through the first subculture and that artichoke Italian latent virus, artichoke mottled crinkle virus, potato virus X, potato virus Y, tobacco mosaic virus and cucumber mosaic virus plus its satellite RNA can replicate and persist in Phytophthora infestans at least through the first subculture. These observations suggest that, unlike in plants, there may be a different basis for host-virus specificity in plant virus infection in filamentous fungi and oomycetes.

Cucumber mosaic virus (CMV) lists among the most important etiological agents of tomato diseases. Some isolates of CMV function as helper virus for replication, encapsidation and transmission of satellite RNAs (satRNA), which may exacerbate symptoms induced by CMV in certain hosts. Outbreaks of CMV strains supporting hypervirulent variants of satRNAs are recurrent in tomato with devastating effects on crop production and efficient control measures are still unavailable. In this study, we examined the dynamics of infection of the CMV strains tomato top stunting (TTS) and 77 supporting replication of satRNA variants that codetermine top stunting (TTS-satRNA) and necrotic (77-satRNA) phenotypes in two tomato cultivars denoted Solanum lycopersicum Manduria (Sl-Ma) and S. lycopersicum UC82 (Sl-UC). Sl-Ma but not Sl-UC recovered from disease symptoms induced by CMV-TTS while both the cultivars succumbed to the infection of CMV-77 and its necrogenic satRNA. Ability to recover of the Sl-Ma plants was transmitted by grafting to the susceptible genotype Sl-UC. More interestingly, recovery was observed also against the challenge inoculation of CMV plus 77-satRNA in plants grafted on Sl-Ma and in self-grafted plants of both the Sl-Ma and Sl-UC cultivars. Analysis of small RNAs and genes of the defence plant response based on RNA interference (RNAi) suggested that RNAi is involved in the recovery of Sl-Ma against CMV with hypervirulent satRNAs and in scions grafted on this rootstock. The response of Sl-Ma to the inoculation of CMV-77 plus 77-satRNA was compared with that of the transgenic tomato line S. lycopersicum transgenic line UCTC5.9.2 that expresses constitutively the benign variant of the satRNA denoted Tfn-satRNA. Comparative analysis suggested that the response may operate via similar mechanisms, which involve RNAi, the graft and the presence of the satRNA.

Next-generation sequencing (NGS) allowed the assembly of the complete RNA-1 and RNA-2 sequences of a grapevine isolate of artichoke Italian latent virus (AILV). RNA-1 and RNA-2 are 7,338 and 4,630 nucleotides in length excluding the 3' terminal poly(A) tail, and encode two putative polyproteins of 255.8 kDa (p1) and 149.6 kDa (p2), respectively. All conserved motifs and predicted cleavage sites, typical for nepovirus polyproteins, were found in p1 and p2. AILV p1 and p2 share high amino acid identity with their homologues in beet ringspot virus (p1, 81% and p2, 71%), tomato black ring virus (p1, 79% and p2, 63%), grapevine Anatolian ringspot virus (p1, 65% and p2, 63%), and grapevine chrome mosaic virus (p1, 60% and p2, 54%), and to a lesser extent with other grapevine nepoviruses of subgroup A and C. Phylogenetic and sequence analyses, all confirmed the strict relationship of AILV with members classified in subgroup B of genus Nepovirus.

Metagenomic surveys and data from next generation sequencing revealed that mixed infections among plant viruses are probably a rule rather than an exception in natural pathosystems. The documented cases may range from synergism to antagonism, which may depend from the spatiotemporal order of arrival of the viruses on the host and upon the host itself. In synergistic interactions, the measurable differences in replication, phenotypic and cytopathological changes, cellular tropism, within host movement, and transmission rate of one of the two viruses or both are increased. Conversely, a decrease in replication, or inhibition of one or more of the above functions by one virus against the other, leads to an antagonistic interaction. Viruses may interact directly and by transcomplementation of defective functions or indirectly, through responses mediated by the host like the defense mechanism based on RNA silencing. Outcomes of these interactions can be applied to the risk assessment of transgenic crops expressing viral proteins, or cross-protected crops for the identification of potential hazards. Prior to experimental evidence, mathematical models may help in forecasting challenges deriving from the great variety of pathways of synergistic and antagonistic interactions. Actually, it seems that such predictions do not receive sufficient credit in the framework of agriculture.

Schena, L., Finetti Sialer, M., and Gallitelli, D. 2002. Molecular detection of strain L47 of Aureobasidium pullulans, a biocontrol agent of postharvest diseases. Plant Dis. 86:54-60. The strain L47 of Aureobasidium pullulans is an effective biocontrol agent of postharvest diseases. When applied in the field before harvesting it requires a specific monitoring method to evaluate colonization and dispersal in the environment. The randomly amplified polymorphic DNA technique (RAPD) was used for a preliminary screening of A. pullulans genetic variability among 205 isolates. This approach allowed the selection of a 1.3-kb fragment (L4) present solely in isolates L47 and 633. In Southern blots, a digoxigenin (DIG)-labeled L4 amplicon specifically recognized the corresponding fragment present in the polymorphic pattern of L47 and 633. The L4 fragment was cloned, sequenced, and used to design two sequencecharacterized amplification region (SCAR) primers and a 242-bp riboprobe. Both the SCAR primers and the 242-bp DIG-labeled riboprobe were highly specific for L47. In classical polymerase chain reaction (PCR), with a series of 10-fold dilutions of L47 DNA, the limit of detection was 20 pg/µl. The Ap13 primer was also modified to obtain a Scorpion primer for detecting a 150-bp amplicon by fluorescence emitted from a fluorophore through a self-probing PCR assay. This assay specifically recognized the target sequence of L47 strain over a number of other A. pullulans isolates in field-treated grape berry washings. The limit of detection was 105 cells per ml, i.e. 10 times higher than the limit of the CFU method. The method is also proposed as a way to demonstrate the ability of L47 strain to penetrate the epidermis of sweet cherry fruits and to track it in the mesocarp.

International audience; 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.

Despite the evidence that certain viral satellites do not encode any functional product, they can either induce specific disease symptoms or alter (exacerbate or attenuate) symptoms that helper viruses induce in some hosts. Viral satellites that attenuate symptoms have the potential to be used as viral biocontrol agents. Satellite RNAs associated with cucumber mosaic virus (CMV) are well known for viral-symptom modulation properties, and in several instances ameliorative CMV satellites have been exploited to protect plants against hypervirulent CMV strains carrying satellite RNAs molecules that are necrogenic in tomato. Preinoculation and transgenesis of ameliorative CMV satellite variants have been tested under experimental and field conditions with encouraging results, but with a warning for potential risks for the spontaneous change of an ameliorative satRNA to a pathogenic variant and its dissemination by aphids.

The recent demonstration that the plant virus tobacco mosaic virus replicates and expresses in the plant pathogenic fungus Colletotrichum spp. provides opportunities for examining fundamental aspects of the biology of plant pathogenic fungi and of their interaction with the host. The small genome size and the ability in colonizing systemically the host have implemented the use of plant viruses to carry segments of host genes that can then promote the silencing of the RNAs expressed from the corresponding endogenous genes in a process called virus-induced gene silencing (VIGS). This chapter presents support for the view that VIGS with a direct transfection of a plant virus vector in fungal cells can be used for functional genomics also in fungi that activate an antiviral defense based on RNA interference (RNAi). The silencing of genes in filamentous fungi is technically more problematic and labor intensive than in plants, especially if transgenic plants need to be generated first. Compared to current strategies to employ RNAi to investigate the basis of fungal pathogenesis, the VIGS approach described here is more direct, easy to do, and feasible. Future perspectives of both basic and practical aspects of this technology are discussed.

International audience; Manufacturing industries perform mechanical surface treatments like shot peening at the end of the manufacturing chain to protect important working parts. This treatment modifies the near surface of the treated part with the introduction of compressive residual stresses due to the repeated impacts of the shot. Then, the treated part exhibits, not only a longer life, but also a better fretting behavior, an improved resistance to corrosion… The objective of the present paper is first to study the relation between the process parameters and the material state (residual stress and plastic variables…) for a complex geometry. Next, a numerical tool is proposed, able to predict this material state in a time frame that is consistent with industrial constraints. The originality of the proposed approach thus consists in the chaining of the different steps. The first step is to choose the process parameters for the shot peening process considering conventional or ultrasonic shot peening and model the shot dynamics for a complex geometry. Once the impact velocity field is known, the objective is to compute the local incompatible plastic deformation field due to the repeated impacts using analytical methods. Then, a finite element model is used to compute the residual and deformation fields in the considered mechanical part. The complete method has been performed on the model of a gear, a mechanical part that is most often shot peened and exhibits a complex geometry.

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.

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 PVY-SON41 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.

RNA silencing controls endogenous gene expression and drives defensive reactions against invasive nucleic acids like viruses. In plants, it has been demonstrated that RNA silencing can be transmitted through grafting between scions and silenced rootstocks to attenuate virus and viroid accumulation in the scions. This has been obtained mostly using transgenic plants, which may be a drawback in current agriculture. In the present study, we examined the dynamics of infection of a resistance-breaking strain of Tomato spotted wilt virus (RB-TSWV) through the graft between an old Apulian (southern Italy) tomato variety, denoted Sl-Ma, used as a rootstock and commercial tomato varieties used as scions. In tests with non-grafted plants, Sl-Ma showed resistance to the RB-TSWV infection as viral RNA accumulated at low levels and plants recovered from disease symptoms by 21 days post inoculation. The resistance trait was transmitted to the otherwise highly susceptible tomato genotypes grafted onto Sl-Ma. The results from the analysis of small RNAs hallmark genes involved in RNA silencing and virus-induced gene silencing suggest that RNA silencing is involved in the resistance showed by Sl-Ma against RB-TSWV and in scions grafted on this rootstock. The results from self-grafted susceptible tomato varieties suggest also that RNA silencing is enhanced by the graft itself. We can foresee interesting practical implications of the approach described in this paper.

Field surveys were carried out in the main vegetable-growing areas of Western and Central Albania to evaluate the sanitary status of open-field and protected cultivations of capsicum, tomato, potato, watermelon, cucumber, courgette, aubergine, lettuce, cabbage, chicory, leek and celery. The following viruses were detected: Alfalfa mosaic alfamovirus (AMV), Cucumber mosaic cucumovirus (CMV), Potato Y potyvirus (PVY), Tomato spotted wilt tospovirus (TSWV) and Watermelon mosaic potyvirus 2 (WMV-2). The virus found most frequently was CMV and all the isolates identified were of subgroup IA. AMV was also detected in several areas and all isolates were of subgroup II, suggesting a French origin. Finally, at the time of this survey, TSWV infections appeared to be moderately relevant and absent in protected crops. This is the first documented record of AMV, CMV, PVY, TSWV and WMV-2 in Albania. No infection by Tomato yellow leaf curl begomovirus (TYLCV) or Zucchini yellow mosaic potyvirus (ZYMV) was detected during this survey.

Real-time PCR technologies open increasing opportunities to detect and study phytopathogenic and antagonistic fungi. They combine the sensitivity of conventional PCR with the generation of a specific fluorescent signal providing real-time analysis of the reaction kinetics and allowing quantification of specific DNA targets. Four main chemistries are currently used for the application of this technique in plant pathology. These chemistries can be grouped into amplicon sequence non-specific (SYBR Green I) and sequence specific (TaqMan, Molecular beacons, and Scorpion-PCR) methods. Amplicon sequence nonspecific methods are based on the use of a dye that emits fluorescent light when intercalated into doublestranded DNA. Amplicon sequence specific methods are based on the use of oligonucleotide probes labelled with a donor fluorophore and an acceptor dye (quencher). The fluorescent signal eliminates the requirement for post-amplification processing steps, such as gel electrophoresis and ethidium bromide staining. This significantly reduces time and labour required for the analysis and greatly increases the throughput of PCR testing as an automated diagnostic system, making it suitable for large-scale analyses. Furthermore, the use of different fluorescent dyes facilitates the detection of several target microrganisms in a single reaction (multiplex-PCR). Real-time PCR makes possible an accurate, reliable and high throughput quantification of target fungal DNA in various environmental samples, including hosts tissues, soil, water and air, thus opening new research opportunities for the study of diagnosis, inoculum threshold levels, epidemiology and host–pathogen interactions. Moreover, the quantification of specific mRNA transcription by real-time PCR is being increasingly applied to the study of changes in gene expression in response to phytopathogenic and antagonistic fungi.

Previously characterized monoclonal antibodies (MAbs) to Grapevine virus A (GVA) showed a differential reactivity against intact or partially destabilized virus particles [2]. In the present study, this differential reactivity was confirmed and several peptides reacting with a panel of four different antibodies were identified by the PEPSCAN method of epitope mapping. Oligopeptide sequences comprised between coat protein residues 61 (V) and 72 (T) were recognized by all the antibodies tested. One of these peptides (VGPKASK) was also reactive when expressed on recombinant phage particles as a fusion protein with protein pVIII. The specificity of this sequence for antibody binding was also demonstrated by competitive-ELISA using one of the GVA MAbs. The results of this study suggest that GVA particles carry a highly structured epitope centered on a common peptide region of the coat protein sequence.

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.

Mixed infections of Cucumber mosaic virus (CMV), Potato virus Y (PVY), and CMV-satRNA in tomato are frequent in nature and exhibit different disease phenotypes that are the outcomes of synergistic and antagonistic interactions between the causative agents. Such mixtures of interactions make classic strains acquire new pathogenetic skills, which may change their eco-epidemiologic patterns. The emerging picture is very complex, and the present case study puts mixed infections into the framework of creating a more favorable condition for host-to-host aphid-mediated transmission of CMV and PVY. Field cases were reproduced in the tomato UC82 variety by using authentic mild and severe strains of CMV (namely, CMV-LS and CMV-Fny), a deletion mutant of CMV-Fny (namely, CMV-FnyΔ2b), a mild strain of PVY (PVY-SON41), and a benign variant of CMV-satRNA (namely, CMV-Tfn). PVY and either CMV strain acted synergistically in eliciting a disease pattern that was more severe than that produced by each virus in a single infection. The exacerbation of symptoms was particularly evident in the CMV-LS/PVY-SON41 combination; this may be attributable to a cumulative effect of the two silencing suppressors (i.e. 2b and HC-Pro proteins) encoded by the two viruses, respectively. The disease patterns observed with the different combinations of inoculum parallel the accumulation of viral RNAs in infected tissues. At single-cell level, and at early stages of infection, CMV took most of the advantage from the double infection with PVY, as the titer of its RNAs was increased while that of PVY was reduced. The two viruses seemed unable to invade the same cell—a condition that, at a later stage of infection, enabled the two viruses to be more uniformly distributed in tomato plant tissues. PVY-SON41 complemented movement defects of the CMV-FnyΔ2b deletion mutant, while CMV Tfn-satRNA reduced the antagonism of CMV-Fny vs PVY-SON41 by the downregulation of its RNA. The results of this work are relevant from an ecologic and epidemiologic perspective due to the frequency of natural mixed infection of CMV and PVY in tomato.

An account is given of the ecology of Cucumber mosaic virus (CMV) as a pertinent example of how a virus can affect the sustainability of an important crop. It is now generally accepted that the technologies used in modern agriculture should ensure that production systems are operated in such a way that the quality of the produce is maintained year after year without causing degradation of the environment. Recent experiences in countries of the Mediterranean basin demonstrate that the benefits expected from the introduction of new and highly productive plant varieties may be quickly eroded by the concomitant introduction of new virus strains which can greatly change the structure of the resident virus population. Quarantine inspection of plant propagules and genetic engineering are suggested as powerful tools to help achieve sustainability.

Very recently, a novel nucleic acid detection technology based on a primer with a quenched fluorophore and a probe complementary to an internal portion of the amplicon has been developed. The primer probe, named ‘Scorpion’, fluoresces only upon hybridization with its target, i.e. if the specific extension product of the primer is synthesized during the reaction. We have used Scorpion primers in RT-PCR assays to detect Tomato spotted wilt tospovirus and Plum pox potyvirus in naturally infected plants. Amplified products of the expected size were detected either by real-time measurements or by visual estimation of fluorescence emission from tubes placed over an ultraviolet light source at the end of the Scorpion-RT-PCR. In serially diluted plant sap, the visual method allowed detection of 7.5 pg of infected plant tissue and proved at least as sensitive as visual estimation of ethidium bromide-stained amplicons separated by gel electrophoresis.

An account is given of strain-specific diagnostic methods currently adopted in Italy to detect alfalfa mosaic alfamovirus (AMV), cucumber mosaic cucumovirus (CMV) and tomato spotted wilt tospovirus (TSWV). These viruses are highly detrimental to tomato crops as the introduction of new virus strains has deeply changed the population structure of resident viruses. RT-PCR coupled with restriction analysis of amplicons proved suitable for differentiating CMV and AMV strains whereas single-stranded conformational polymorphism (SSCP) was used for the identification of TSWV isolates. Application of strain-specific diagnostic methods to phytosanitary inspection of plant propagules is suggested as a powerful tool for timely identification of new virus strains

Reverse transcription polymerase chain reaction (RT-PCR) amplification was adapted for detection of either single or mixed viral infections in grapevine and artichoke. DNA primers specific for grapevine A closterovirus (GVA), grapevine B trichovirus (GVB), or grapevine leafroll-associated closterovirus III (GLRaV-III) were used for RT-PCR amplification of specific genomic segments from GVA, GVB and GLRaV-III-infected grapevine tissues. The reactions produced discrete fragments of 0.43, 0.45 and 0.34 kb which were representative of GVA, GVB and GLRaV-III, respectively. Immunocapture RT-PCR (IC-RT-PCR) or multiplex RT-PCR (M-RT-PCR) were utilized for the detection of these viruses in extracts of infected grapevine leaves, dormant cuttings and/or viruliferous mealybugs. IC-RT-PCR was more efficient than RT-PCR for the detection of GVA in viruliferous mealybugs, whereas M-RT-PCR was easier and faster than IC-RT-PCR for the detection of GLRaV-III and GVB in infected grapevine tissues. Six specific primers were used for RT-PCR amplification of specific genomic segments of artichoke mottled crinkle tombusvirus (AMCV), artichoke Italian latent nepovirus (AILV) and artichoke latent potyvirus (ALV) in artichoke plant sap. This set of primers was also successfully used for the simultaneous detection of the three viruses in cases of mixed infections, because each primer pair retained its specificity and reverse transcription-amplification reactions ran independently, yielding products of the expected virus-specific size. The limits of simultaneous detection were about 400 fg for AMCV and ALV and 4 pg for AILV, whereas the limits of detection in separate reactions were about 100fg for all three viruses.

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 biological and molecular characterization is reported of a Watermelon mosaic virus isolate, denoted WMV-Le, associated with a necrotic phenotype of watermelon plants grown in the Provinces of Lecce and Taranto (Apulia, southern Italy). The fully sequenced WMV-Le genome consists of 10,045 nucleotides and is 99.1% similar to that of WMV-C05-270, a French isolate from melon of the WMV molecular group 3. Using recombination detection program RDP3, putative recombination breakpoints were identified close to nucleotide positions 42 to 1892, covering the 5′UTR/P1/HC-Pro region. The event represents the insertion of a sequence fragment of an isolate similar to WMV-FBR04-37 in the background of an isolate similar to WMV-FMF00-LL1. The field symptomatology was reproduced in watermelon plants grown in an experimental greenhouse but the virus induced severe symptoms also in Cucumis sativus, C. melo, Cucurbita maxima and C. pepo .

Most of the 24 viruses which infect globe artichoke are detrimental to the crop’s performance and hamper the development of a nursery activity in the respect of current EU legislation. We describe a procedure to sanitize globe artichoke “Brindisino” from Artichoke Italian latent virus (AILV) and Artichoke latent virus (ArLV), while preserving its valuable early flowering trait. ArLV was successfully eliminated by meristem-tip culture, while AILV was removed when two rounds of meristem-tip culture were spaced out with in vitro thermotherapy. In vivo thermotherapy, followed by meristem-tip culture, was also successful in producing virus-free material but was less efficient in terms of the number of plants recovered post treatment. Due to the multi-clonal composition of the populations at present in cultivation, the selected and sanitised clones were fingerprinted by applying microsatellite and AFLP (amplified fragment length polymorphism) markers. One AFLP primer combination produced 28 informative fragments used to evaluate genetic relatedness among the clones in study. Our results demonstrates that AFLP-based molecular fingerprinting enables to verify the true to clone correspondence in nurseries, ensure the effective correspondence between the real and the declared identity of a clone, so that to avoid commercial frauds, and might represents a valuable tool for assessing somaclonal variation occuring during ‘in vitro’ propagation.

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 1α, 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.

The differential response of 29 genotypes of tomato and wild tomato relatives (Solanum section Lycopersicon species) to cucumber mosaic virus strain Fny (CMV-Fny), alone or in combination with three different satellite RNA (satRNA) variants, allowed the identification of four disease phenotype patterns, each including plants that developed very severe symptoms (leaf malformations, top stunting and lethal necrosis) and plants that remained asymptomatic. No resistance or tolerance to CMV-Fny was observed, whilst individual host genotypes displayed latent infection upon inoculation with one (CMV-Fny/Tfn-satRNA, phenotype patterns 1 and 4), two (CMV-Fny/Tfn-satRNA and CMV-Fny/TTS-satRNA, phenotype pattern 2) or all three (the former two plus CMV-Fny/77-satRNA, phenotype pattern 3) CMV/satRNA combinations. RNA gel-blot analyses showed that latent infection generally correlated with a strong downregulation of CMV RNA accumulation levels. Introgression lines derived from a cross between Solanum habrochaites LA1777, which displayed disease phenotype pattern 2, and Solanum lycopersicum were screened for tolerance to the stunting phenotype induced by CMV-Fny/TTS-satRNA, and only one line, carrying an introgression on chromosome 6, was identified as being partially tolerant. Solanum chilense LA1932×S. lycopersicum back-cross introgression lines were screened for tolerance to lethal necrosis induced by CMV-Fny/77-satRNA (phenotype pattern 3); the tolerant phenotype was observed in 33 % of plants of the BC1F2 progeny and 1F3 progeny. Thus, potentially useful sources of tolerance to CMV/satRNA-induced diseases were identified, although the tolerant phenotypes appeared to be controlled by complex quantitative trait loci.

plants showing conspicuous concentric chrome-yellow bands in the leaves, from which itwas described and derived its name [9, 5]. New records from tomato came later from Italy[4], Spain [7] and France [6]. The Italian tomato isolate was characterized by Gallitelli [4].PZSV has a tripartite genome, particles morphologically similar to those of ilarviruses andgeneral properties resembling those of tripartite genome viruses of the family

The complete sequence of the Pelargonium zonate spot virus (PZSV) genome was determined. It comprises 8477 nt, distributed in three positive-strand RNA species encoding four proteins. RNA-1 is 3383 nt long, with an ORF that encodes a polypeptide with a molecular mass of 108 419 Da (denoted protein 1a). This protein contains the conserved sequence motifs I-III of type I methyltransferases and the seven consensus motifs of the helicases of superfamily 1. RNA-2 is 2435 nt long and encodes a major polypeptide with a molecular mass of 78 944 Da (denoted protein 2a), which shows identity to the RNA-dependent RNA polymerases of positive-strand RNA viruses. RNA-3 is 2659 nt long and contains two major ORFs. The first ORF is located in the 5' portion of the genome and sequence comparison of the putative translation product revealed similarities with the 30K superfamily of virus movement proteins. The second ORF is located in the 3' half and encodes the viral coat protein, which is expressed via a subgenomic RNA, RNA-4. The transcription initiation site of RNA-4 maps to the intergenic region of RNA-3. The organization of the PZSV genome, including the primary structure of terminal non-coding regions, strongly suggests that this virus belongs to the family Bromoviridae. The overall biological and genomic characteristics of PZSV indicate affinities in diverging directions with one or other of the virus species in this family, thus enabling it to be considered as a possible representative of a new genus within the family Bromoviridae.

Functional genomics in plants has been facilitated greatly by the use of plant viruses to carry segments of host genes that can then promote the silencing of the RNAs expressed from the corresponding host genes; a process called virus-induced gene silencing (VIGS). The silencing of genes in filamentous fungi is either technically more problematic or labor-intensive, especially if transgenic plants need to be generated first. However, a recent paper from our team demonstrated that a plant virus could infect three related fungal species, as well as express a reporter gene ectopically, and also silence the correspondingly expressed reporter transgene. The gene expression and RNA silencing of the reporter gene was maintained for six passages in culture and also persisted in plants infected by the virus-infected fungus. Here, we consider how the virus can enter and migrate within the fungus, whether the virus can move back and forth between the fungus and the plant and the ramifications of this, the prospects for VIGS being used to silence fungal endogenes and possible biotechnological or therapeutic applications of using plant viruses for expressing foreign proteins in fungi or silencing fungal endogenes.

During winter 2000-2001, an unusual disease of tomato was observed in some greenhouses in Sardinia, Sicily, and Apulia, in southern Italy. Plants were chlorotic and reduced in size, expanded leaves showed interveinal yellowing, and older leaves developed interveinal reddish-bronze necrosis and downward rolling. The symptoms resembled those recently reported from Portugal (1) as induced by Tomato chlorosis virus (ToCV) (family Closteroviridae, genus Crinivirus), a whitefly-transmitted virus new to Europe. Symptomatic leaf tissues were extracted and analyzed by reverse transcription-polymerase chain reaction as described by Louro et al.(1). The 439-bp ToCV-specific DNA fragment was amplified in samples collected from 6 of 14 greenhouses in Sardinia, 2 of 5 greenhouses in Sicily, and 1 of 1 greenhouse in Apulia. The sequence of the fragment obtained from a Sicilian isolate (GenBank Accession No. AY048854) showed more than 99% identity to ToCV isolates (Accession Nos. AF024630 and AF234029) from the United States and Portugal, respectively. Infestations of Trialeurodes vaporariorum and Bemisia tabaci have been reported in autumn. To our knowledge, this is the first report of ToCV in Italy. Although we found the virus in three regions of the country, its distribution is likely to be wider, since the symptoms can be mistaken for those of a physiological disorder or of Tomato infectious chlorosis virus, another crinivirus infecting tomato. Reference: (1) Louro et al. Eur. J. Plant Pathol. 106:589, 2000.

International audience; The nucleotide sequence of the putative coat protein open reading frame of seven previously uncharacterized AMV strains from Italy and France was determined and aligned with comparable sequences of other AMV strains (425 L, 425 M, YSMV, S, VRU, 15/64 and Da). The data set of AMV sequences was used to determine phylogenetic relationships by both a stochastic (stationary Markov model) and a deterministic method (maximum-parsimony) of analysis. The topology of the trees obtained with the two methods was essentially the same showing that all AMV strains clustered in two monophyletic groups. Close clustering of Italian strains in subgroup I and of French strains in subgroup II seems to suggests the effect of geographic distinctiveness of evolutionary dynamics of these AMV strains. This separation did not correlate with differences in host range or symptoms (necrotic or non necrotic) induced in tomato but rather it reflected variations in the amino acid sequence of their CP, which might be related to structural properties of virus particles. A simple and rapid procedure based on the reverse transcriptase-polymerase chain reaction (RT-PCR) followed by ezymatic digestion (RFLP) was developed to identify and classify AMV isolates into the two subgroups. The method applied to a number of other AMV isolates from Italy and France supported their division in two distinct subgroups. This RT-PCR RFLP method may be useful way to investigate the dynamics of AMV populations in nature.