Family Closteroviridae Research Articles

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.

MOLECULAR DETECTION OF A CLOSTEROVIRUS ASSOCIATED WITH APRICOT STEM PITTING IN SOUTHERN ITALY

Electrophoretic analysis of cortical and leaf tissue extracts from apricot trees of cv. ‘Tyrinthos’ from Apulia (southern Italy) affected by stem pitting, contained multiple double-stranded RNA (dsRNA) species, the largest with an estimated size of 15 kbp. A segment of 590 nucleotides, showing sequence homology with the HSP70 homologue gene of members of the family Closteroviridae was amplified by RT-PCR from symptomatic trees using degenerated primers designed on the conserved phosphate 1 and 2 motifs of the HSP70 gene sequence. Computer-assisted analysis showed that the 590 nt fragment from Italian apricot trees had 97% sequence homology with Plum bark necrosis stem pittingassociated virus (PBNSPaV), a recently reported closterovirus infecting Black Beaut plum (Prunus salicina) in California. A set of primers that amplified a 290 nt virus-specific fragment was designed and a cRNA probe was generated. This riboprobe and RT-PCR assays with these primers detected PBNSPaV in diseased but not in healthy apricot trees.

Coronary artery ectasia (angiographic and clinical findings of our patients)

For the last 70 years, long before the virus nature of the tristeza disease was recognized, Citrus tristeza virus (CTV), genus Closterovirus, family Closteroviridae, was considered as a serious threat to citrus production. The costs of CTV infections varied geographically and temporally, especially since growers worldwide have recognized the grave consequences of CTV epidemics. A variety of control measures including replacing sensitive rootstocks, prevention of spread with propagation material, and wide scale use of cross-protection for handling stem pitting-related problems are now commonly used in attempts to control the range of CTV diseases.Despite these efforts, the virus which apparently originated in Asia continued its spread and its now present throughout most citrus growing areas of the world, with only a few of the Mediterranean basin countries, and some Western USA areas still free of massive infections. However, even these areas are now under a serious threat due to the recent spread of the most efficient aphid vector to nearby areas. CTV is naturally transmitted in a semipersistent mode by several aphid species. The brown citrus aphid (Toxoptera citricida) is the most efficient vector because of its great abundance on citrus trees and its high efficacy of vectoring highly pathogenic isolates. The symptoms of CTV depend on the nature of the invading isolate and the sensitivity of the citrus host. The most dramatic symptom referred to as ‘quick decline’ results from rapid death of trees grafted onto sour orange rootstocks. Other CTV-related diseases such as ‘stem pitting’ of grapefruit or sweet orange trees are not related to a specific rootstock and do not result in death, but are often associated with stunting and poor productivity. Symptomless CTV isolates are used to provide protection (‘cross-protection’) against stem pitting, but not against the quick decline.The virions of CTV are long flexuous particles (2000 × 12 nm) with an unusual architecture that results from two different coat proteins, the major coat protein (CP), and minor coat protein (CPm) encapsidating approximately 97% and 3%, respectively, of the helically structured particles. Two additional viral proteins, p61 and p64 (a HSP70 homolog), are required for efficient assembly. The 19.3 kbp single-stranded RNA genome is the largest among the plant viruses. Genomic analyses showed a division into 12 open reading frames (ORFs), the organization of which is conserved among isolates. The ORF1a encodes a 349 kDa polyprotein containing two papain-like protease domains plus methyltransferase-like and helicase-like domains. Translation of the polyprotein is thought occasionally to continue through the polymerase-like domain (ORF1b) by a =1 frameshift. Ten 3′ ORFs are expressed by 3′ co-terminal subgenomic messenger RNAs. Infected cells contain a myriad of other subgenomic RNAs. In addition, most CTV isolates contain one or more defective RNAs of different sizes and genomic rearrangements. Among the CTV translation products, at least three function as RNA-silencing repressors, which are correlated with unusual difficulty in conferring pathogen-derived resistance to transgenic citrus plants.

Genetic Diversity and Evolution of Closteroviruses.

The family Closteroviridae comprises more than 30 plant viruses with flexuous, filamentous virions and includes representatives with either mono- or bipartite positive-strand ssRNA genomes. Closteroviruses are transmitted semipersistently by insects from three families of Homoptera, in infected plants are associated with phloem tissue, and demonstrate an astonishing genetic diversity that suggests extensive, on-going evolution. Phylogenetic analyses of their replicative genes as well as the conserved HSP70 demonstrate that closteroviruses co-evolved with their insect vectors, resulting in three major lineages, i.e. aphid-, mealybug-, and whitefly-transmitted viruses. Closteroviruses apparently represent an ancient and diverse virus family that may pose threats to agriculture and needs serious attention.

Severe Yellowing Outbreaks in Tomato in Spain Associated with Infections of Tomato chlorosis virus

Since 1997, yellowing disease outbreaks have occurred in tomato (Lycopersicon esculentum) crops in southern Spain. The outbreaks were associated with high populations of the whitefly Bemisia tabaci. Symptoms consisted mainly of interveinal yellowing that developed initially on lower leaves and then progressed to the upper part of the plant. Affected plants were less vigorous and yielded less due to reduced fruit growth and delayed ripening. During 1998 and 1999, the yellowing disease was widespread and occurred at high incidences in the Málaga province. The disease agent was readily transmissible from tomato to tomato by B. tabaci biotype Q. Samples from symptomatic tomato plants were analyzed and shown to be infected with Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae). This is the first report of ToCV epidemics in Europe.

The 23-kDa Protein Coded by the 3′-Terminal Gene of Citrus Tristeza Virus Is an RNA-Binding Protein

The 23-kDa protein (p23), encoded by the 3′-proximal gene of the RNA of Citrus tristeza virus (CTV), was overexpressed in Escherichia coli fused to the maltose-binding protein and purified by affinity chromatography. Gel retardation and UV crosslinking assays demonstrated that p23 has the ability to cooperatively bind single-stranded RNA in a non-sequence-specific manner. Formation of the p23-RNA complex was dependent on the conformational state of p23 and on the presence of a basic region, but the complex was stable at high salt concentrations, suggesting that interactions other than those between the negatively charged RNA and the basic region of p23 are involved. Competition assays showed that the affinity of p23 for single-stranded and double-stranded RNA was similar but considerably higher than for single-stranded and double-stranded DNA. By use of a series of artificially generated mutants, the RNA-binding domain of p23 was mapped between positions 50–86, a region containing several basic amino acids and a putative zinc-finger domain. Additional p23-derivatives lacking the conserved residues presumably involved in coordinating the zinc ion showed RNA-binding activity, but with an apparent dissociation constant higher than the wild-type protein. These conserved residues might confer binding specificity or increase binding stability in vivo. Within the Closteroviridae family, p23 is the only protein characterized so far showing RNA-binding activity.

Genetic Analysis of the Cell-to-Cell Movement of Beet Yellows Closterovirus

A beet yellows closterovirus (BYV) variant expressing green fluorescent protein and leaves of BYV local lesion host Claytonia perfoliata were used to reveal genetic requirements for BYV cell-to-cell movement in leaf epidermis and mesophyll. A series of mutations targeting genes that are not involved in amplification of the viral positive-strand RNA was analyzed. The products of genes coding for a 6-kDa hydrophobic protein (p6) and a 64-kDa protein (p64), as well as for minor and major capsid proteins, were found to be essential for intercellular translocation of BYV. In a previous work, we have demonstrated that the BYV HSP70-homolog (HSP70h) also plays a critical role in viral movement (V. V. Peremyslov, Y. Hagiwara, and V. V. Dolja, 1999, Proc. Natl. Acad. Sci. USA, 96, 14771–14776). Altogether, a unique protein quintet including three dedicated movement proteins (p6, p64, and HSP70h) and two structural proteins is required to potentiate the cell-to-cell movement of a closterovirus. The corresponding BYV genes are clustered in a block that is conserved among diverse representatives of the family Closteroviridae.

First Report of Cucurbit Yellow Stunting Disorder Virus (Genus Crinivirus) in North America.

In late summer 1999, field- and greenhouse-grown melon plants (Cucumis melo) showing severe stunting and yellowing symptoms were observed near Donna in southern Texas and near the town of Reynosa in northern Mexico. Symptoms were typical of those caused by viruses in the genus Crinivirus, family Closteroviridae. High populations of Bemisia spp. whiteflies were associated with these plantings, with many plants showing heavy infestation. Laboratory analyses showed that melon plants from both locations were infected by the whitefly-transmitted Cucurbit yellow stunting disorder virus (CYSDV). Positive hybridization reactions with digoxigenin-UTP-labeled transcript probes corresponding to the CYSDV heat shock protein 70 (HSP70) homolog coding region (1) were obtained for RNAs extracted from symptomatic plants. Similar probes for the related Lettuce infectious yellows virus (LIYV) and Beet pseudo-yellows virus (BPYV), two whitefly-transmitted viruses previously reported from North America (2), did not hybridize with the RNAs. Definitive confirmation of CYSDV was obtained by performing reverse-transcription polymerase chain reaction (RT-PCR) analyses for two distinct CYSDV coding regions. RT-PCR with primers corresponding to CYSDV, but not LIYV or BPYV HSP70 homolog coding regions, gave specific (≈500 bp) products from corresponding test plants. RNAs from healthy control plants gave no RT-PCR product. Because the HSP70 coding region is highly conserved (2), we also performed RT-PCR with primers designed for the Spanish CYSDV capsid protein gene (GenBank accession AJ243000). Positive RT-PCR products of ≈700 bp were obtained only from the Texas and Mexico melon plants. CYSDV is a widespread and damaging virus of cucurbits in southern Europe and the Middle East (2). This is the first report of this important virus in North America.

PARTIAL MOLECULAR CHARACTERIZATION AND RT-PCR DETECTION OF A PUTATIVE CLOSTEROVIRUS ASSOCIATED WITH OLIVE LEAF YELLOWING

SUMMARY Electrophoretic analysis of extracts from cortical tissues of olive trees of cvs ‘Biancolilla’ and ‘Nostrana’ from Sicily (southern Italy) showing bright chrome yellow discolourations of the leaves, consistently revealed a number of double-stranded RNA (dsRNA) bands, the largest of which had a size (ca 15 kbp) similar to that of the full genomic dsRNA of some species of the genus Closterovirus. A segment of 611 nucleotides, showing sequence homology with the HSP70 homologue gene of Closteroviridae was amplified by RT-PCR from symptomatic trees of both cultivars, using degenerated primers designed on the conserved phosphate 1 and 2 motifs of the HSP70 homologue gene sequence. Computer-assisted phylogenetic analysis showed that the 611 nt HSP70 homologue sequence from olive differed from that of other members of the family Closteroviridae, suggesting that it probably belonged to an undescribed closterovirus, for which the tentative name olive leaf yellowing-associated virus (OLYaV) is proposed. A set of OLYaV-specific primers was designed which amplified a 383 nt fragment of the HSP70 homologue. RT-PCR assays with these primers detected OLYaV sequences in olive trees with leaf yellowing from some Italian regions (Calabria and Latium) and Jerusalem, and in individuals of an unidentified pseudococcid mealybug species and of the psyllid Euphyllura olivina that had fed on symptomatic cv. ‘Biancolilla’ trees. No amplification was obtained with extracts from controls (symptomless olives and apparently healthy olive seedlings) or from olive trees from other Italian regions (Sardinia, Tuscany) that showed somewhat differing yellowing symptoms.

A Closterovirus (Family: Closteroviridae) Isolated from Tobacco Crops in Northern Greece (Macedonia).

In 1997, viruslike symptoms similar to those caused by potato potyvirus Y (PVY) were observed in tobacco (Nicotiana tabacum ) plants of the cv. Basmas in the area of Drama (Macedonia) in northern Greece. Diseased plants showed vein clearing and necrosis of the petioles and main veins, and were severely stunted. Serological tests with polyclonal antibodies prepared against PVY, cucumber mosaic cucumovirus (CMV), alfalfa mosaic alfamovirus (AMV), and tomato spotted wilt tospovirus (TSWV) gave negative results. The virus was mechanically transmitted from infected field tobacco plants to plants of N. tabacum cvs. White Burley, Samsun NN, Xanthi, Christie, and Basmas, and to plants of the species N. benthamiana, N. clevelandii, N. glutinosa, N. roselata, and N. rustica. All tobacco species and cultivars generally reacted with chlorotic local lesions on the inoculated leaves followed by systemic vein clearing, often veinal necrosis, leaf curling, and severe stunting. Similar symptoms were produced on Petunia hybrida. Other plant species such as beet, faba beans, lettuce, tomato, Datura stramonium, and watermelon were not infected by the virus. Aphid transmission studies with Myzus persicae showed that the virus was transmitted in a semi-persistent manner. Electron microscopic observations of leaf squash preparations from infected plants revealed the presence of long, filamentous, virus particles of 1,600 to 1,800 nm in length, typical for the family Closteroviridae. The virus was partially purified in a process in which the extract was treated with Triton for 1 h at 4°C, followed by two cycles of high-speed centifugation on a 20-40-60 % sucrose cushion. The virus was denaturated on CsCl or CsSO 4 gradients. The results show that the virus isolated is the first member in the Closteroviridae reported to naturally infect N. tabacum.