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Plants rely on RNA silencing (RS) as a major layer of innate defense to neutralize invading viruses. In an opposite way, plant viruses evolved a survival mechanism, based on viral suppressors of RS (RSS), able to hinder the host RS activation system. In the present study, the sequences of the barley yellow striate mosaic virus (BYSMV) proteins P3, P6, P9 and of Phosphoprotein (P), from Chahar Mahall (Iran) isolate, were determined. Furthermore, we carried out agroinfiltration assays in leaves of Nicotiana benthamiana (16c line) to explore the RS suppression mechanism of these proteins. Analyses showed that P and P3 act as weak local and limited short-term systemic inducers of RSS. A reduction of RNA-dependent RNA polymerase 6 (RDR6) gene expression was observed in presence of BYSMV P and P3 proteins, which imply their putative role in interfering with systemic RS, in which RDR6 is involved. In addition, P and P3 RSS activity were reduced when co-expressed with other BYSMV accessory proteins, indicating that, in the system used, only free P or P3 proteins lead to RSS. The BYSMV P protein lacked any detectable Glycine-Tryptophan (WG/GW) or F-box motifs, known to interfere with the AGO1 slicing activity. On the opposite, two WG/GW were identified in P3 sequence.

Potato virus Y (PVY) isolate PVYC-to induces growth reduction and foliar symptoms in tomato, but new vegetation displays symptom recovery at a later stage. In order to investigate the role of micro(mi)RNA and secondary small(s)RNA-regulated mechanisms in tomato defenses against PVY, we performed sRNA sequencing from healthy and PVYC-to infected tomato plants at 21 and 30 days post-inoculation (dpi). A total of 792 miRNA sequences were obtained, among which were 123 canonical miRNA sequences, many isomiR variants, and 30 novel miRNAs. MiRNAs were mostly overexpressed in infected vs. healthy plants, whereas only a few miRNAs were underexpressed. Increased accumulation of isomiRs was correlated with viral infection. Among miRNA targets, enriched functional categories included resistance (R) gene families, transcription and hormone factors, and RNA silencing genes. Several 22-nt miRNAs were shown to target R genes and trigger the production of 21-nt phased sRNAs (phasiRNAs). Next, 500 phasiRNA-generating loci were identified, and were shown to be mostly active in PVY-infected tissues and at 21 dpi. These data demonstrate that sRNA-regulated host responses, encompassing miRNA alteration, diversification within miRNA families, and phasiRNA accumulation, regulate R and disease-responsive genes. The dynamic regulation of miRNAs and secondary sRNAs over time suggests a functional role of sRNA-mediated defenses in the recovery phenotype.

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.

Two rapid detection methods based on dot blot hybridization with a nonradioactive DNA probe and molecular beaconPCR were developed for the specific detection of Pseudomonas syringae pv . tomato , the causal agent of bacterial speck of tomato. A 1378 bp DNA fragment (Acc. No. AM039892), obtained from the extension of a 255 bp fragment generated by a RAPD protocol, was used to find a suitable combination of primers specific for the tomato pathovar. A 138 bp fragment from the genome of P. syringae pv. tomato DC 3000 was used as DNA probe. In dot blots of DNA extracted from either pure cultures or artificially contaminated seeds washes, the probe recognized specifically the tomato pathovar. A molecular beacon was designed from the same region for the specific detection and quantification of P. syringae pv . tomato by real-time PCR. A highly significant correlation was observed between the amount of target DNA and the cycle threshold (Ct). Using a fast protocol for DNA extraction, from pure cultures and from washes of artificially contaminated seeds, the limit of detection was about 1 × 10 2 CFU. The diagnostic tools developed proved highly specific for P. syringae pv. tomato and simple to use. They can therefore be applied to large-scale testing of tomato seeds and seedlings for the assessment of their phytosanitary condition in nurseries.

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.

Strain L47 of Aureobasidium pullulans is an effective biocontrol agent for several post-harvest fruit diseases, even when applied in the field before harvesting. This strategy requires a specific method of monitoring in order to optimize formulations and to evaluate the level of colonization and dispersal in the environment. We have detected strain L47 among other morphologically indistinguishable isolates of A. pullulans using Scorpion PCR, an automated real-time fluorescence detection system that targets a 145-bp fragment of the L47 genome. We propose this approach as a specific, rapid and reliable method of monitoring A. pullulans, strain L47.

Motivations. RNA silencing, or post-transcriptional gene silencing (PTGS), is a conserved mechanism in a broad range of eukaryotes. In plants, PTGS acts as an antiviral system and a successful virus infection requires suppression or evasion of the induced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected with RNA and DNA viruses and provide specificity to this RNA-mediated immune system. High-throughput sequencing has contributed to expanding our previously knowledge of siRNA populations better describing their abundance, complexity and diversity in infected tissues. It is now known that siRNAs from virus-infected plants are extraordinarily abundant and diverse, and are widespread in near saturation at any region of either positive and negative genomic RNAs. However, certain regions of viral genomes ("hot spots") are usually more represented than others in sequenced siRNA populations. A gene involved in chlorophyll biosynthesis has been shown targeted by a siRNA derived from viral satellite RNA, revealing PTGS mechanism at basis of the symptom. Potato virus Y (PVY) is the type species of Potyvirus, a genus of agricultural importance belonging to the largest plant virus family, Potyviridae. The potyviral genome is a single-stranded, positive-sense RNA of ca. 10 kilobases (kb). PVYc-to and PVY-SON41 are two isolates of PVY that infect solanaceous hosts. While PVYc-to induces severe leaf distortion in different cultivars of tomato (Solanum lycopersicum), PVY-SON41 produces in the same host only a mild mosaic, followed by recovery. In order to elucidate the molecular mechanism underlying the different symptoms induced by PVYc-to and PVY-SON41 infections on tomato, we set up an in silico approach, mining genomic regions of PVY isolates and looking at possible RNA-based mechanisms where siRNA putatively generated from the PVY genome could target and suppress accumulation of host messenger RNA (mRNA), leading to dysfunctional biological processes that could explain different disease phenotypes. Methods. A Perl script was used to extract the complete datasets of 21-mers from isolates PVYc-to and PVY-SON41 complete genomes, leading the scanning of the complete sequence, shifted by one base at the time. MySQL was used for identification of the 21-mers shared and unique between the two viral genomes, highlighting sequence differences that could be at the base of diverse induced symptoms. The data obtained in the previous step were used to build the genomic map of identical regions, by fancyGene. A BLAST analysis was conducted with the identified 21-mer dataset, considering only the dissimilar sequences between the two isolates (blastn-short identity > 94%, max 2 mismatch or gap, alignment length 21 bp). The 21-mer were used as query and the tomato mRNA database (Solgenomics, release ITAG2.3) was used as target. The results were further used as input in a gene ontology analysis, through Blast2GO. Results. Despite the high identity (> 91%) shared by the two viral genomes, the 21-mer dataset produced a high degree of variation, as the analyses showed 1750 identical and 9671 and 9680 dissimilar sets of 21-mer, for PVYc-to and PVY-SON41, respectively. Data showed that 315 and 381 tomato mRNA were complementary to, and thus possible targets of, the in silico-generated siRNAs deduced from of PVYc-to and PVY-SON41 sequences, respectively. The GO analysis showed different putative gene targets, involved in several metabolic pathways indicating specific targets for each isolate. Of particular interest, for instance, are putative target mRNAs of transcription factors with a known role concerning leaf development and symmetry. These genes, if differentially modulated during the infections of the two different PVY isolates, could cause the leaf malformations observed as major differential symptoms between the two PVY isolates. To validate the bioinformatic approach, target genes with highly significant complementarity have been selected from the in silico data for experimental validation in tomato plants mechanically infected by virus isolates.

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 .

Expression profiles were identified in the fungus Pochonia chlamydosporia, a biological control agent of plant parasitic nematodes, through a cDNA-amplified fragment length polymorphism approach. Two isolates with different host ranges, IMI 380407 and IMI 331547, were assayed in conditions of saprotrophic-to-parasitic transition, through in vitro assays. Gene expression profiles from three different nutritional conditions and four sampling times were generated, with eggs of host nematodes Globodera pallida and Meloidogyne incognita. Expression of transcripts changed in RNA fingerprints obtained under different nutritional stresses (starvation in presence/absence of eggs, or rich growth media). Transcript derived fragments (TDFs) obtained from the expression profiles corresponded to 6,800 products. A subset was sequenced and their expression profile confirmed through RT PCR. A total of 57 TDFs were selected for further analysis, based on similarities to translated or annotated sequences. Genes expressed during egg parasitism for both IMI 380407 and IMI 331547 were involved in metabolic functions, cellular signal regulation, cellular transport, regulation of gene expression, DNA repair, and other unknown functions. Multivariate analysis of TDF expression showed three groups for IMI 380407 and one for IMI 331547, each characterized by expression of genes related to eggs parasitism. Common amplification profiles among TDF clusters from both isolates also reflected a pool of constitutive genes, not affected by the nutritional conditions and nematode associations, related to general metabolic functions. The differential expression of parasitism related genes suggest a network of induced/repressed products, playing a role in fungal signaling and infection, with partial overlaps in host infection and parasitism traits.

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.

Cardiosporidium cionae (Apicomplexa), from the ascidian Ciona intestinalis L., is redescribed with novel ultrastructural, phylogenetic and prevalence data. Ultrastructural analysis of specimens of C. intestinalis collected from the Gulf of Naples showed sporonts and plasmodia of C. cionae within the host pericardial body. Several merogonic stages and free merozoites were found in the pericardial body, together with sexual stages. All stages showed typical apicomplexan cell organelles, i.e. apicoplasts, rhoptries and subpellicular microtubules. Merogonic stages of C. cionae were also produced inside haemocytes. A fragment of the rSSU gene of C. cionae was amplified by PCR using DNA extracted from the pericardial bodies. The amplified product showed closest affinity with other apicomplexan representatives and a 66 bp unique insertion, specific for C. cionae, at position 1644. Neighbour-joining phylogenetic analysis placed C. cionae in a clade with other piroplasm genera, including Cytauxzoon, Babesia and Theileria spp. The parasite was found in different populations of C. intestinalis with highest prevalence in October–November. Ultrastructural and DNA data showed that the organism, described in 1907 from the same host but not illustrated in detail, is a member of a novel marine apicomplexan radiation of tunicate parasites.

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.

Tests with a real-time reverse transcription-polymerase chain reaction (RT-PCR) were performed on specimens of Xiphinema index collected from the rhizosphere of Grapevine fanleaf virus (GFLV)-infected grapevines at Palagiano, Italy. A 1,157-bp fragment of the GFLV RNA-2 coat protein (CP) gene was amplified and sequenced. A fluorescent Scorpion probe was designed to detect a highly conserved CP region. A second region with isolate-specific multiple nucleotide polymorphisms was used to detect GFLV isolates using molecular beacons (MB). The Scorpion probe allowed quantitative estimation of GFLV RNA-2 in single nematodes, using a dilution series of a 692-nucleotide transcript of the CP gene. The assay allowed detection of GFLV RNA-2 in individual X. index, with a minimum template threshold of 800 fg or 2.8 × 106 RNA-2 molecules per nematode. The CP fragment used for GFLV detection with the Scorpion probe appeared highly conserved among isolates. The probes were tested against other GFLV isolates, which were recognized by the species-specific Scorpion probe and by the corresponding MB specific to the particular isolate. Both tests appeared useful as diagnostic tools or for studies on GFLV in acquisition, retention, and transmission experiments.

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.

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.

Transgenic tomato (Lycopersicon esculentum Mill. cv. UC82) plants expressing a benign variant of Cucumber mosaic virus satellite RNA (CMV Tfn-satRNA) were generated. The transformed plants did not produce symptoms when challenged with a satRNA-free strain of CMV (CMV-FL). The same plant lines initially were susceptible to necrosis elicited by a CMV strain supporting a necrogenic variant of satRNA (CMV-77), but a phenotype of total recovery from the necrosis was observed in the newly developing leaves. The features of the observed resistance were analyzed and are consistent with two different mechanisms of resistance. In transgenic plants inoculated with CMV-FL strain, the symptomless phenotype was correlated to the down-regulation of CMV by Tfn-satRNA, amplified from the transgene transcripts, as the first resistance mechanism. On the other hand, the delayed resistance to CMV-77 in transgenic tomato lines was mediated by a degradation process that targets satRNAs in a sequence-specific manner. Evidence is provided for a correlation between a reduced accumulation level of transgenic messenger Tfn-satRNA, the accumulation of small (approximately 23 nucleotides) RNAs with sequence homology to satRNAs, the progressively reduced accumulation of 77-satRNA in infected tissues, and the transition in infected plants from diseased to healthy. Thus, events leading to the degradation of satRNA sequences indicate a role for RNA silencing as the second mechanism determining resistance of transgenic tomato lines.

Background MicroRNAs (miRNAs) are small RNAs (21-24 bp) providing an RNA-based system of gene regulation highly conserved in plants and animals. In plants, miRNAs control mRNA degradation or restrain translation, affecting development and responses to stresses. Plant miRNAs show imperfect but extensive complementarity to mRNA targets, making their computational prediction possible, useful when data mining is applied on different species. In this study we used a comparative approach to identify both miRNAs and their targets, in artichoke and safflower. Results Two complete expressed sequence tags (ESTs) datasets from artichoke (3.6·104 entries) and safflower (4.2·104), were analysed with a bioinformatic pipeline and in vitro experiments, identifying 17 potential miRNAs. For each EST, using RNAhybrid program and 953 non redundant miRNA mature sequences, available in mirBase as reference, we searched matching putative targets. 8730 out of 42011 ESTs from safflower and 7145 of 36323 ESTs from artichoke showed at least one predicted miRNA target. BLAST analysis showed that 75% of all ESTs shared at least a common homologous region (E-value < 10-4) and about 50% of these displayed 400 bp or longer aligned sequences as conserved homologous/orthologous (COS) regions. 960 and 890 ESTs of safflower and artichoke organized in COS shared 79 different miRNA targets, considered functionally conserved, and statistically significant when compared with random sequences (signal to noise ratio > 2 and specificity ≥ 0.85). Four highly significant miRNAs selected from in silico data were experimentally validated in globe artichoke leaves. Conclusions Mature miRNAs and targets were predicted within EST sequences of safflower and artichoke. Most of the miRNA targets appeared highly/moderately conserved, highlighting an important and conserved function. In this study we introduce a stringent parameter for the comparative sequence analysis, represented by the identification of the same target in the COS region. After statistical analysis 79 targets, found on the COS regions and belonging to 60 miRNA families, have a signal to noise ratio > 2, with ≥ 0.85 specificity. The putative miRNAs identified belong to 55 dicotyledon plants and to 24 families only in monocotyledon.