Redescription of Cardiosporidium cionae (Van Gaver and Stephan, 1907) (Apicomplexa: Piroplasmida), a plasmodial parasite of ascidian haemocytes.

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 66bp 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.

Response of tomato and its wild relatives in the genus Solanum to cucumber mosaic virus and satellite RNA combinations.

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 LA1932xS. 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 BC(1)F(2) progeny and <1 % of plants of the BC(1)F(3) 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.

Isolate-Specific Detection of Grapevine fanleaf virus from Xiphinema index Through DNA-Based Molecular Probes.

ABSTRACT 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 x 10(6) 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.

Complete nucleotide sequence of Pelargonium zonate spot virus and its relationship with the family Bromoviridae.

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.

First Report of Tomato chlorosis virus in Italy.

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.

Evidence for two distinct subgroups of alfalfa mosaic virus (AMV) from france and italy and their relationships with other AMV strains Brief report.

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.

Formalin-induced infidelity in PCR-amplified DNA fragments.

A 643-nucleotide-long fragment of rDNA gene was amplified by PCR in the nematode worm Caenorhabditis elegans. When the experiments were performed by using samples fixed in formalin, artefacts were detected. While the size of the amplified fragment resulted unaffected, very striking differences were seen in the nucleotide sequences of the amplified fragments. Furthermore, in many cases, the PCR reaction failed completely. The results obtained might warn of potential problems, especially when the amount of DNA to be amplified is scarce.