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Hop stunt viroid (HSVd) is the viroid with the broadest host range known so far. In some hosts, such as grapevine and apricot, the infection seems to be latent. In other species, HSVd is found associated with disorders of serious economic importance e.g. hop stunt and dapple fruit in plum and peach, and citrus cachexia. A recent study (Canizares et al., 1998) revealed the presence of HSVd in 81 % of the apricot trees tested in south-eastern Spain. By using a non-isotopic tissue printing technique we found that petiole tissue was a very good source of viroid. We then used transversal sections of infected petiole tissue to follow the presence of HSVd in apricot trees during a whole year. The results showed that viroid titter was higher from May to July, after which the accumulation level of HSVd slightly decreased. Surprisingly, by December, the viroid title had recovered levels similar to those observed in July. The significance of these results is discussed.

The three most economically damaging ilarviruses affecting stone fruit trees on a worldwide scale are the related Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple mosaic virus (ApMV). Nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction (RT-PCR) methodologies were developed that could detect all these viruses simultaneously. The latter technique was advantageous because it was discriminatory. For RT-PCR, a degenerate antisense primer was designed which was used in conjunction with three virus-specific sense primers. The amplification efficiencies for the detection of the three viruses in the multiplex RT-PCR reaction were identical to those obtained in the single RT-PCR reactions for individual viruses. This cocktail of primers was able to amplify sequences from all of the PNRSV, ApMV, and PDV isolates tested in five Prunus spp. hosts (almond, apricot, cherry, peach, and plum) occurring naturally in single or multiple infections. For ApMV isolates, differences in the electrophoretic mobilities of the PCR products were observed. The nucleotide sequence of the amplified products of two representative ApMV isolates was determined, and comparative analysis revealed the existence of a 28-nucleotide deletion in the sequence of isolates showing the faster electrophoretic mobility. To our knowledge, this is the first report on the simultaneous detection of three plant viruses by multiplex RT-PCR in woody hosts. This multiplex RT-PCR could be a useful time and cost saving method for indexing these three ilarviruses, which damage stone fruit tree yields, and for the analysis of mother plants in certification programs.

Viral sequences amplified by polymerase chain reaction from 25 isolates of Prunus necrotic ringspot virus (PNRSV), varying in the symptomatology they cause in six different Prunus spp., were analyzed for restriction fragment polymorphisms. Most of the isolates could be discriminated by using a combination of three different restriction enzymes. The nucleotide sequences of the RNA 4 of 15 of these isolates were determined. Sequence comparisons and phylogenetic analyses of the RNA 4 and coat proteins (CPs) revealed that all of the isolates clustered into three different groups, represented by three previously sequenced PNRSV isolates: PV32, PE5, and PV96. The PE5-type group was characterized by a 5′ untranslated region that was clearly different from that of the other two groups. The PV32-type group was characterized by an extra hexanucleotide consisting of a duplication of the six immediately preceding nucleotides. Although most of the variability was observed in the first third of the CP, the amino acid residues in this region, which were previously thought to be functionally important in the replication cycle of the virus, were strictly conserved. No clear correlation with the type of symptom or host specificity could be observed. The validity of this grouping was confirmed when other isolates recently characterized by other authors were included in these analyses.

Mature canes were collected from vines in the main grapevine-growing areas in Tunisia (Cape Bon, Bizerte, Ben Arous), from commercial vineyards and mother-plant plots, to assess the presence of virus and virus-like diseases. Biological (mechanical transmission onto herbaceous hosts and grafting onto indicator woody plants) and serological detection (ELISA) methods were applied. ELISA showed that 96.4% of 669 vines tested were infected, most of them (88.1%) by at least two viruses. Grapevine leafroll-associated 3 closterovirus (GLRaV-3) was the most widespread virus (87.9%), followed by grapevine A vitiviras (GVA, 69.4%), grapevine fleck virus (GFkV, 51.9%), grapevine leafroll-associated 1 closterovirus (GLRaV-1, 36.8%), grapevine leafroll-associated 2 closterovirus (GLRaV-2, 19.1%), grapevine fan leaf nepovirus (GFLV, 18.2%) and grapevine B vitiviras (GVB, 14.8%). ELISA tests yielded negative results for grapevine leafroll-associated 7 closterovirus (GLRaV-7) and potato X potexvirus (PVX). The highest infections were found in Bizerte and Cape Bon regions (100 and 99.2%), and in vineyards aged over 20 years (98.5%) as compared with the younger ones (81.1%). Rootstocks in mother-plant plots were practically free from all the viruses tested (1 plant infected out of 81), whereas severe infections were found in Vitis vinifera mother plants (67.4% of 341 samples), in particular table grapes (92.6%) compared with wine grapes (47.9%). In these mother-plant plots, the prevailing viruses were GLRaV-3 (41.3%), followed by GFkV (36.7%), GVA (27.9%), GLRaV-1 (17%) and GLRaV-2 (15.2%). GFLV and GVB were far more limited (1.5 and 0.6%, respectively). The presence of vein necrosis and vein mosaic was ascertained by transmission onto 110R and Vitis riparia indicators, whereas only GFLV was mechanically transmitted onto herbaceous hosts (from about 20% of the samples).

Quand les premiers foyers de la sharka ont ete decouverts en Apulia (sud-est de l'Italie) a la fin des annees 1980, les autorites agricoles regionales ont lance un programme pour suivre le Plum pox virus (PPV) et eradiquer la maladie. La souche de virus presente a ete identifiee comme etant le PPV-D. Entre 1989 et 1993 une importante campagne d'eradication a ete menee avec succes; elle concernait 13 vergers de pruniers et 2 d'abricotiers avec differents niveaux d'infection. En 1994-2000, en plus des pruniers, abricotiers et pechers, le suivi a ete etendu aux cerisiers. A cette epoque, les prospections et les analyses n'ont revele aucun nouveau foyer de PPV, mais l'eradication des arbres infectes a continue dans quelques vergers. En 2001-2005, une attention particuliere a ete portee aux pechers, car de graves foyers de PPV-M s'etaient developpes dans d'autres zones du pays. Un nouveau foyer de PPV a ete trouve sur abricotier, provoque par le PPV-Rec, et il a ete rapidement eradique. Les annees suivantes, des prospections dans le verger qui avait ete infecte et les plantations environnantes de pechers n'ont pas detecte de dissemination du virus. Ceci est le plus long programme de suivi et d'eradication en Italie, ses resultats indiquent que l'industrie fruitiere de l'Apulia peut desormais etre consideree comme etant majoritairement indemne du PPV.

Two disorders of almond, know as 'stem pitting’and‘graft union necrosis (black line)', were described years ago from Puglia (southern Italy). The etiology of these diseases was uncertain, the causal agent unknown and reliable diagnostic methods were not available. Investigations were therefore carried out to identify Prunus species and/or cultivars as possible indicators for a quick and reliable diagnosis. In 1990, two different almond sources (cv. Filippo Ceo) affected by black line and stem pitting were budded onto seedlings of P. persica, P. cerasifera, P. amygdalus cv. Don Carlo, GF305, self-rooted cuttings of GF677 (P. persica×P. amygdalus) and onto Don Carlo and GF305 seedlings that had been already grafted with almond cvs Genco, Tuono and Filippo Ceo. At least two inoculated plants were inspected each year for the presence of symptoms on the woody cylinder after removal of the cortex. Stem pitting developed on P. persica and P. cerasifera seedlings during the first year after graft inoculation. On the grafted plants that had been inoculated, pitting and grooving of the wood were much more pronounced in cvs Filippo Ceo and Genco, with a higher incidence when GF305 was used as rootstock. Clear symptoms were observed (from the first year in cvs Tuono and Genco), regardless of the rootstock, all along the junction line and often not associated with pitting of the rootstock and scion.

A monoclonal antibody to an Albanian isolate of plum pox potyvirus (PPV) was obtained (MAbAL), that specifically recognized strain M of this virus. The specificity of MAbAL, assessed by comparative ELISA on 130 PPV isolates of different geographical origin, 22 of which were also tested by comparative IC-PCR, gave consistent and highly reproducible results. MAbAL seems to be elicited by a stable surface determinant that makes it particularly suitable for successful use under a wide range of conditions. MAbAL is an useful addition to the panel of PPV-specific MAbs available to date.

Surveys were carried out in the main stone-fruit growing areas of Albania to assess the phytosanitary status of Prunus in conimercial orchards and varietal collections. The presence of virus and virus-like diseases and their identification was ascertained through field observations, sap transmission to herbaceous hosts, graft transmission to woody indicators, ELISA and IEM tests. The mean infection level was 42%. In particular, infections in apricot and almond were 12 and 16%, respectively, i.e. lower than in plum and cherry (47 and 56%, respectively). The following viruses were identified: plum pox potyvirus (PPV). apple chlorotic leaf spot trichovirus (ACLSV). prunus necrotic ringspot (PNRSV) and prune dwarf (PDV) ilarviruses. PPV infection was very severe in plum, and limited in apricot and peach. Apple mosaic ilarvirus (ApMV), and six nepoviruses tested for (SLRV, TBRV, RRV, CLRV, ArMV and ToRSV) were not encountered in Primus.

Surveys were carried out in the traditional areas of almond and apricot cultivation in Tunisia to assess the phytosanitary status of these species in varietal collections, mother block stands and commercial orchards. The presence of virus and virus-like diseases was ascertained through field surveys, sap transmission to herbaceous hosts, and ELISA and IEM tests. The mean infection level in almond was 34%. compared with only 4.7% in apricot. The following viruses were identified: apple chlorotic leaf spot trichovirus (ACLSV), apple mosaic (ApMV), prune dwarf (PDV) and prunus necrotic ringspot (PNRSV) ilarviruses. Plum pox potyvirus (PPV) was not detected. Likewise, none of the six nepoviruses tested for (SLRV, TBRV, RRV. CLRV. ArMV and ToRSV) was encountered.

Investigations were carried out in the main stone-fruit growing areas of Lebanon to assess the phytosanitary condition of commercial orchards. The presence of virus and virus-like diseases and their identification was ascertained through: (i) field surveys, (ii) sap transmission to herbaceous hosts, (iii) graft transmission to woody indicators; and (iv) ELISA and IEM tests. The mean infection level was 25%. ranging from 5% in apricot to 45% in cherry. The following viruses were identified: apple chlorotic leaf spot trichovirus (ACLSV), prunus necrotic ringspot (PNRSV) and prune dwarf (PDV) ilarviruses. Plum pox potyvirus (PPV), apple mosaic ilarvirus (ApMV), and six nepoviruses tested for (SLRV, TBRV, RRV, CLRV, ArMV and ToRSV) were not encountered.

Grapevine phloem-limited isometric virus (GPLIV) is the name proposed for a non mechanically-transmissible virus found in Italian and Tunisian grapevines. In density gradient centrifugation purified virus preparations sedimented as two components: T, made up of empty protein shells, and B, composed of intact nucleoprotein particles. B particles had a buoyant density of 1.45 g/cm3 at equilibrium in CsCl and contained 35% RNA consisting of a single molecule with an apparent size of 7.4 kb. The coat protein consisted of a single species with a mol.wt of 28,000 daltons. Purified virus preparations did not infect herbaceous hosts by manual inoculation. A specific antiserum with a titre of 1: 64 raised in rabbits, was used for identification of, GPLIV in field-grown Tunisian grapevines and in leafroll-affected Italian vines before and after heat treatment. Although heat treatment eliminated the virus from the majority of the plants, leafroll symptoms persisted in several GPLIV-free vines, indicating that there is no clear-cut relationship between GPLIV and this disease. ZusammenfassungEinige Eigenschaften eJnes im Phloem begrenzten, nicht mechanisch iibertragbaren Weinstockvirus Der Name grapevine phloem-limited isometric virus [GPLIV) wird fur ein nicht mechanisch ubertragbares, in Italien und Tunesien vorkommendes Virus vorgeschlagen, Nach Dichtegradiemenzentnfugation konnten zwei Komponenten aus gereinigten Viruspraparaten festgestellt werden: T, die aus leeren Proteinhullen bestand, und B, die ganze Nucieoproteinpartikein enthielt. Die B-Komponenten hatten eine schwimmende Dichte von 1,45 g/cm3 im Gleichgewicht mit CsCl und emhielten 35 % RNS, die aus eincm einzelnen Molekul mit einer scheitibaren Grose von 7,4 kb bestand. Das Hullprotein setzte sich aus einer Art mit einem Mokkulargewicht von 28 000 Dalton zusammen. Gereinigte Viruspraparaie waren nicht in der Lage, Wirtspflanzen nach einer kiinsthchen Inokulation zu lnfizieren. Ein aus Kaninchen gewonnenes, spezifisches Antiserum, mit einem Titer von 1: 64, wurde bei der Feststellung vom CPLIV in Weinstocken aus Feldern in Tunesien und in Reben in Italien. die geroiite Blatter aufwiesen, vor und nach einer Hitzebehandlung angewandt. Obwohl Nach einer Hitzebehandlung das Virus in fast alien Pflanzen nicht mehr nachzuweisen war, wurden die gerollten Blattersymptome bei einigen GPLIV-freien Reben nicht aufgehoben. Dies deutet darauf hin, das es keinen eindeutigen Zusammenhang zwischen dem GPLIV und dieser Krankheit gibt.

Plum pox virus (PPV) is a major threat to the expanding Mediterranean stone fruit industry. In order to control the plum pox disease it is of utmost importance to detect early PPV foci and to identify the PPV isolates involved. A survey was therefore carried out in Albania, Cyprus, Egypt, Greece, Italy and Turkey by a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with the following monoclonal antibodies (MAbs): 5B (universal), 4DG5 (PPV-D-specific), AL (PPV-M-specific), TUV and AC (PPV-C-specific), and EA24 (PPV-El Amar-specific). A hundred and seventy Mediterranean PPV isolates were tested for strain type. PPV-M was detected in Albania, Cyprus, Greece, Italy, and Turkey; PPV-D was detected in Albania and Italy, whereas samples with natural mixtures of both strains were found in a couple of orchards in Albania. Seven PPV isolates from apricots in two Egyptian localities were recognized only by MAb EA24. In conclusion, DAS-ELISA with a combination of the universal MAb5B and the MAbs specific to the four PPV serotypes currently known (M, D, C and El Amar) is an efficient tool for a simple, sensitive and routine detection of PPV and discrimination of its serotypes.

Four different rootstocks were cultured, namely Primus mahaleb L, Gi-Sel Al (P.fruticosa Pall.X P. avium L.), Damil (P. dawyckensis Sealy) and Tabel (Edabriz). Plants were inspected for symptoms from mid-April through mid-May. Leaf samples from Prunus mahaleb and Gi-Sel Al were collected in the middle of May for ELISA and sap transmission tests. Damil and Tabel were not tested because they had already been certified. Meristems from both axillary and terminal buds were cultured in spring (May), summer (July-August) and autumn (September- October). ELISA and sap transmission tests did not give specific reactions for the viruses. P. mahaleb and Gi-Sel Al were classed as healthy rootstocks. Spring was the best time for culturing meristems. 92 % of meristems of P. mahaleb survived, while Gi-Sel Al explants did not proliferate and showed vitrification and callus induction after the second and third subcultures. Explants of Damil did not even proliferate and most were dead 2-3 weeks after the establishment of culture. In summer, survival of meristems of all plants was considerably less whereas bacterial contamination was very high, ranging from 17 %- 84 %. Meristems of all plants showed the lowest survival in autumn. In general, plants were subcultured every 3 or 4 weeks. During the rooting phase, only P. mahaleb plants were successfully rooted in culture media having 0.5-1.0 mg/1 IBA. 50 % of the explants showed good rooting capacity in 0.5 mg/1 IBA, 75-85 % of them with 1.0 mg/1 IBA. Roots were vigorous in both cases.

Biotinylated cloned DNA probes were employed in dot hybridizations and restriction fragment length polymorphism (RFLP) analyses to compare mycoplasmalike organisms (MLOs) associated with two grapevine yellows diseases (strain FDU of flavescence doree MLO from northern Italy and strain FDB of southern European grapevine yellows MLO from southern Italy) and Italian periwinkle virescence disease (MLO strain G from northern Italy). Results from dot hybridizations using six probes containing cloned DNA of MLO strain FDU, of MLO strain G, or of American aster yellows MLO strain AY1, revealed that FDU and FDB shared some regions of DNA sequence homology with one another as well as with MLO strains G and AY1, but all four MLOs were mutually distinguished [...]

Olive (Olea europaea) is host of 13 different viruses but, it is possible that other viruses, which are either non mechanically transmissible or occur in low concentration in plant tissues, are present in nature. This likelihood is supported by the widespread occurrence of double-stranded RNAs (dsRNAs) in plants negative to biological tests. As very little information is available on the geographical distribution of olive-infecting, investigations were conducted for gathering information on the prevalence and distribution of olive viruses in the Mediterranean basin based on the presence of double stranded RNA (dsRNA). A total of 527 samples were collected throughout the surveys covering 10 countries and 83 locations. Out of 286 tested samples collected from 6 Italian regions, 210 (73.4%) were dsRNA positive, whereas the average of viral infections in the Mediterranean basin was 64.5%. Molecular hybridisation tests, on 25 % of dsRNA-positive samples collected in Apulia, revealed the presence of the three nepoviruses (ArMV, CLRV and SLRSV), OLYaV and OLV-1.