Olive Leaf Yellowing-associated Virus Research Articles

Olive Leaf Mottling Virus: A New Member of the Genus Olivavirus.

Studies of the virome of olive trees with symptoms of leaf mottling by high-throughput sequencing (HTS) revealed the presence of a new virus. Full coding genome sequences of two isolates were determined and consisted of a single RNA segment of 16,516 nt and 16,489, respectively. The genomic organization contained 10 open reading frames (ORFs) from 5' to 3': ORF1a, ORF1b (RdRp), ORF2 (p22), ORF3 (p7), ORF4 (HSP70h), ORF5 (HSP90h), ORF6 (CP), ORF7 (p19), ORF8 (p12), ORF9 (p23) and ORF10 (p9). Phylogenetic analyses clustered this virus in the genus Olivavirus, family Closteroviridae, with the closest species being Olivavirus flaviolae, commonly named olive leaf yellowing-associated virus (OLYaV). However, amino acid sequences of all taxonomically relevant proteins showed, in all cases, a divergence higher than 25% between OLYaV and the new virus, indicating that it represents a new species in the genus Olivavirus for which the common name of olive leaf mottling virus (OLMV) is proposed. This study represents an advance in the genus Olivavirus and provides new insights into the olive virome.

Detection by Sensitive Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification of Olive Leaf Yellowing Associated Virus and Its Incidence in Italy and Spain

Olive trees (Olea europea L.) are constantly threatened by many viruses, such as the olive leaf yellowing-associated virus (OLYaV), that belong to the Olivavirus genus, family Closteroviridae. In this work, the OLYaV incidence in different regions of Italy and Spain, which represent the two most important European areas for olive production, was evaluated through the development of a real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for reliable and sensitive OLYaV detection. The specificity and accuracy of the developed real-time RT-LAMP assay were determined; the assay showed that potential cross-reactivity with other viruses belonging to the Closteroviridae family was excluded. The LAMP assay detected OLYaV with a higher sensitivity than conventional end-point RT-PCR, detecting a total of 1.34 × 10−2 genome copies. A total of 80 and 120 plants of different olive cultivars from Spain (Comunitat Valenciana, Andalusia) and Italy (Sicily, Calabria, Apulia, Lazio, and Umbria) regions were tested, respectively. The percentage of infected plants was 46.25% and 30% for Spain and Italy, respectively, while the most susceptible cultivars were “Serrana Espadán” and “Villalonga” from Comunitat Valenciana and Andalusia regions (Spain) and “Ogliarola barese” from Apulia region (Italy). In addition, the survey demonstrated that the real-time RT-LAMP showed good sensitivity for OLYaV-positive sample detection, especially on asymptomatic olive trees. For this reason, the developed assay could be very suitable for phytopathological laboratories as a reliable and efficient method for a rapid and sensitive routine test on olive samples.

PM 4/17 (3) Certification scheme for olive trees and rootstocks

<scp>PM</scp> 4/17 (3) Certification scheme for olive trees and rootstocks

Identification and complete genome sequencing of a divergent olive virus T isolate and an olive leaf yellowing-associated virus isolate naturally infecting olive trees in Greece

Several new full genome sequences of olive viruses came to light recently via high-throughput sequencing (HTS) analysis. In this study, total RNA HTS analysis of two Greek olive trees revealed the presence of an olive virus T (OlVT) isolate and an olive leaf yellowing-associated virus (OLYaV) isolate. The full viral genome of OlVT isolate (50Ch) is composed of 6862 nucleotides encoding for three proteins (replicase, movement protein, and capsid protein) with typical betaflexiviruses’ genomic features. However, both sequence and phylogenetic data analysis exhibited high levels of variability between 50Ch and the previously characterized OlVT isolates. In addition, the almost full genome of the Greek OLYaV isolate (OL2) was obtained, which is composed of 16,693 nucleotides encoding for 11 open reading frames (ORFs) and shares common genomic features with the recently characterized OLYaV isolates from Spain and Brazil. Sequence and phylogenetic analysis revealed high similarity between these three isolates. Due to problems encountered with the detection of both viruses, new nested RT-PCR assays were developed and applied. In addition, recombination events were observed in OlVT isolates (50Ch GR-168), thus highlighting the potential role of this mechanism in the evolution of the virus. This study is adding further knowledge to the limited information available about these recently characterized olive infecting viral pathogens and highlights their widespread distribution in Greece, one of the most important olive producing countries of the world.

Characterization of Spanish Olive Virome by High Throughput Sequencing Opens New Insights and Uncertainties.

The use of high throughput sequencing (HTS) for the analysis of Spanish olive trees showing leaf yellowing discoloration, defoliation, and/or decline has provided new insights into the olive viruses present in Spain and has opened discussions about the pros and cons of these technologies for diagnostic purposes. In this study, we report for the first time in Spanish orchards the presence of olive leaf yellowing-associated virus (OLYaV), for which the second full coding sequence has been determined. This virus has also been detected in a putative vector, the psyllid Euphyllura olivina. In addition, the presence in Spain of Olea europaea geminivirus (OEGV), recently reported in Italy, has been confirmed, and the full-length sequence of two isolates was obtained by HTS and Sanger sequencing. These results, as well as the detection of other viral sequences related to olive latent virus 3 (OLV-3) and olive viral satellite RNA, raises questions on the biological significance of the findings, about the requirement of standardization on the interpretation of HTS results, and the necessity of additional tests to confirm the relevance of the HTS detection of viral sequences.

Detection and phylogeny of viruses in native Albanian olive varieties

Forty samples representing 14 native Albanian and two foreign olive varieties were collected from an olive varietal collection plot in the Valias region (Tirana, Albania). The samples were assayed by RT-PCR for presence of olive-infecting viruses, including arabis mosaic virus (ArMV), cherry leaf roll virus (CLRV), cucumber mosaic virus (CMV), olive latent ringspot virus (OLRSV), olive latent virus 1 (OLV-1), olive leaf yellowing-associated virus (OLYaV), strawberry latent ringspot virus (SLRSV) and by PCR for the bacterium Xylella fastidiosa (Xf). Ninety-eight percent of the samples were infected with at least one virus. OLYaV was the most prevalent (85% of samples), followed by OLV-1 (50%), OLRSV (48%), CMV (28%), SLRSV (3%) and CLRV (5%), whereas ArMV and Xf were absent. Fifty-five percent of the samples were infected with one virus, 13% with two viruses, 20% with three, and 5% with four. Analyses of the nucleotide sequences of the Albanian virus isolates generally showed low genetic variability, and that most were phylogenetically related to Mediterranean isolates, in particular to those from Greece and Italy. Five olive trees, representing three native cultivars (‘Managiel’, ‘Kalinjot’ and ‘Kushan-Preze’) and one foreign (‘Leccino’), were found to be plants of the Conformitas Agraria Communitatis (“CAC”) category i.e. free of ArMV, CLRV, SLRSV and OLYaV. Only one tree of the native cultivar ‘Ulliri i kuq’ was free of all tested viruses, so this is plant material of the “Virus-tested” category. Olives derived from both categories could be used for propagation of standard quality plant materiel in a future certification programme for olive in Albania. This is the first report of CLRV, OLRSV, CMV and OLV-1 in Albania. The study also reveals the precarious health status of native olive varieties in the Valias varietal collection plot. However, the discovery of six plants representing two certifiable categories is a first step in a future olive tree certification program in the country.

Virus Surveys in Olive Orchards in Greece Identify Olive Virus T, a Novel Member of the Genus Tepovirus.

Field surveys were conducted in Greek olive orchards from 2017 to 2020 to collect information on the sanitary status of the trees. Using a high-throughput sequencing approach, viral sequences were identified in total RNA extracts from several trees and assembled to reconstruct the complete genomes of two isolates of a new viral species of the genus Tepovirus (Betaflexiviridae), for which the name olive virus T (OlVT) is proposed. A reverse transcription–polymerase chain reaction assay was developed which detected OlVT in samples collected in olive growing regions in Central and Northern Greece, showing a virus prevalence of 4.4% in the olive trees screened. Sequences of amplified fragments from the movement–coat protein region of OlVT isolates varied from 75.64% to 99.35%. Three olive varieties (Koroneiki, Arbequina and Frantoio) were infected with OlVT via grafting to confirm a graft-transmissible agent, but virus infections remained latent. In addition, cucumber mosaic virus, olive leaf yellowing-associated virus and cherry leaf roll virus were identified.

Molecular Characterization of the Complete Coding Sequence of Olive Leaf Yellowing-Associated Virus

Genome organization and phylogenetic relationships of olive leaf yellowing-associated virus (OLYaV) with other members of the Closteroviridae family were determined. The complete coding sequence of OLYaV was obtained by high throughput sequencing of total RNA from a 35-year-old olive tree (cv. Zarzaleña) from Brazil, showing olive leaf yellowing disease and deformations in the wood. This represents the first report of OLYaV in this country. A genomic sequence of 16,700 nt containing 11 open reading frames (ORFs) was recovered, representing the complete virus coding capacity. The knowledge of the nucleotide sequence of the genome including the gene that codes the coat protein will facilitate the development of diagnostic tests, which are limited so far to PCR-based methods targeting the HSP70h gene. Interestingly, a thaumatin-like protein (ORF2), previously reported in other unassigned viruses in the Closteroviridae family, persimmon virus B and actidinia virus 1, was identified in the OLYaV genome. Phylogenetic analysis of shared proteins (ORF1a, ORF1b, HSP70h, HSP90h and CP) with all members of the Closteroviridae family provides new insight into the taxonomic position of these three closteroviruses and suggests they could represent a new genus in the family.

The Detection of viruses in olive cultivars in Greece, using a rapid and effective RNA extraction method, for certification of virus-tested propagation material

Although Greece is the world’s third largest olive production country, information about the presence of olive viruses is limited. A survey for the presence of virus infections in the ten most important Greek cultivars was conducted in a germplasm collection olive grove located in Chania, Crete. Samples were RT-PCR assayed for the presence of Arabis mosaic virus (ArMV), Cherry leafroll virus (CLRV), Strawberry latent ring spot virus (SLRSV), and Olive leaf yellowing-associated virus (OLYaV), amplifying part of the capsid protein (ArMV), the 3΄UTR (CLSRV, SLRSV) or the HSP70h (OLYaV) gene. Total RNAs were purified using the Trizol method, yielding good quality and purity, thereby confirming application of the method as a rapid economic extraction protocol for detection of olive viruses. SLRSV was the most predominant virus, with an infection rate of 55%, followed by CLRV and OLYaV in 5% of the tested samples. ArMV was detected only in one sample. Mixed virus infections were also commonly detected. The DNA amplicons of the obtained viruses from the infected samples were sequenced. The partial sequences of ArMV, CLRV and SLRSV from olives, which are reported for the first time, showed 74-100% nucleotide similarity with available homologous sequences from other crops, whereas OLYaV isolates showed high sequence variability of 25%. The phylogenetic analysis based on olive-OLYaV HSP70h partial-nucleotide sequences grouped the olive isolate sequences according to the geographical origins of the host germplasm collection. This is the first official report of the occurrence of olive viruses in Greece, emphasizing the need to implement a certification programme for production and distribution of high-quality (virus-free) olive propagation material, in Greece and more generally in the Mediterranean basin.

Establishment of a Sensitive qPCR Methodology for Detection of the Olive-Infecting Viruses in Portuguese and Tunisian Orchards.

Sensitive detection of viruses in olive orchards is actually of main importance since these pathogenic agents cannot be treated, their dissemination is quite easy, and they can have eventual negative effects on olive oil quality. The work presented here describes the development and application of a new SYBR® Green-based real-time quantitative PCR (qPCR) analysis for specific and reliable quantification of highly spread olive tree viruses: Olive latent virus 1 (OLV-1), Tobacco necrosis virus D (TNV-D), Olive mild mosaic virus (OMMV), and Olive leaf yellowing-associated virus (OLYaV). qPCR methodology revealed high specificity and sensitivity, estimated in the range of 0.8–8 copies of the virus genome, for the studied viruses. For validation of the method, total RNA and double strand RNA (dsRNA) from naturally infected trees were used. In a first trial, dsRNAs from trees of cv. “Galega vulgar” from a Portuguese orchard, were subjected to qPCR and from the 30 samples tested, 26 were TNV-D and/or OMMV-positive and 25 were OLV-1 positive. In a second trial, total RNA from trees of different cultivars from Tunisian orchards, were here tested by qPCR and all viruses were detected. From the 33 samples studied, the most prevalent virus detected in Tunisia orchards was OLV-1 (31 samples diagnosed), followed by OLYaV (20 samples diagnosed), and finally the combination in last TNV-D and/or OMMV (12 samples diagnosed). In both trials, qPCR demonstrated to be effective and sensitive, even when using total RNA as template. qPCR through the use of a SYBR® Green methodology enabled, for the first time, a reliable, sensitive, and reproducible estimation of virus accumulation in infected olive trees, in which viruses are usually in low titres, that will allow gaining new insights in virus biology essential for disease control and give an important contribution for establishment of sanitary certification of olive propagative material.

Impact of Olive Leaf Yellowing Associated Virus on Olive (Olea europaea L.) Oil

Among the 15 virus species detected in olive trees, Olive leaf yellowing associated virus (OLYaV) is found with high incidence and frequency in Southern Italy in particular. Effects of OLYaV on virgin olive oil yield and quality of Leccino and Ottobratica cultivars in the Calabria region (Southern Italy) are analyzed. Oil yield, free acidity, number of peroxides, spectrophotometric indices, total content of chlorophylls and carotenoids, total phenol content, composition of the fatty acids, total tocopherols, and total sterols content are determined on oil obtained from olive fruits collected in healthy/virus‐free and OYLaV‐infected trees. Almost all analyzed oil parameters are not statistically different with some exceptions. Oils derived from ‘Ottobratica’ OLYaV‐infected plants have free acidity significantly lower than oil from healthy plants. K232 of oil from virus‐free ‘Leccino’ trees is significantly lower than oil from infected trees. Even though some quality parameter differences between healthy and OLYaV‐infected oils are found, it is important to highlight that all oils can be considered in extra virgin olive oil category, within the UE maximum limit acceptance range. Results suggest a no negative interference by OYLaV in oil yield and quality, except for K232 values, whereas surprisingly suggest a positive effect of virus infection on free acidity parameter.Practical Applications: Based on the evidence that OYLaV does not interfere negatively in oil yield and quality parameters, it seems appropriate that the European Union Council Directives (2014/96/EU, 2014/97/EU, 2014/98/EU) introduced in the last olive certification scheme only ArMV, CLRV, and SLRSV. The application of compulsory EU directive, in Italy as in other countries, will improve the facilities of olive plants commercialization, guaranteeing enough their sanitary status. The findings here reported support the suggestion that the Italian Ministry of Agriculture could be less restrictive in voluntary Italian regulation (D.M. 20/11/2006).Olive leaf yellowing associated virus (OLYaV) is found with high incidence and frequency especially in Southern Italy. Effects of OLYaV on virgin olive oil yield and quality of Leccino and Ottobratica cultivars in the Calabria region (Southern Italy) are analyzed, including free acidity, number of peroxides, spectrophotometric indexes, total content of chlorophylls, carotenoids and phenol, composition of the fatty acids, total tocopherols and total sterols.

Investigations of molecular evolutionary mechanisms in partially sequenced heat shock protein70 homologue-coding gene of Oliveleaf yellowing-associated virus isolates from Tunisia

Abstract Reverse Transcription Polymerase Chain Reaction (RT-PCR) using new designed primers pair for Heat Shock Protein70 homologue (HSP70h) of Olive leaf yellowing-associated virus revealed 667 amplified product of 10 olive accessions collected from various olive-growing regions in Tunisia. Amplicons were cloned and sequenced. The sequences were deposited in the international databases. Pairwise sequence comparisons among 10 Tunisian isolates along with a reference sequence (AJ440010) extracted from GenBank revealed a nucleotide identity of 86.06-99.40 and an amino acid similarity of 91.89-99.55. Sequence multiple alignments were searched for evidence of recombination using three methods, ie. Differences of Sums of Squares (DSS) implemented in TOPALi v2.5 software and Single Breakpoint (SBP) along with GARD, a genetic algorithm, both incorporated in HyPhy package. All used methods pointed out the presence of putative breaking points in partially sequenced HSP70h-coding gene. Since failing to account for recombination can mislead the phylogeny inference and can elevate the false positive error rate in positive selection assessment, the use of GARD resulted in the reconstruction of different phylogenies on the left as well as on the right sides of putative recombination breaking points, and the 11 accessions were distributed into at least three clusters compared to MEGA6 software which delineated only two clades. Nonetheless, by dividing the aligned sequences at breakpoints into separate sequence sets, MEGA6 delineated a clustering pattern different from the former two. As a result, recombination reshuffled the affiliation of the different accessions to the clusters. Analysis of selection pressures exerted on HSP70h encoded protein using different models (SLAC, IFEL, FEL, REL, PARRIS, FUBAR, MEME, GA Branch, and PRIME) taking into account recombination, and implemented in HyPhy package, revealed that it underwent predominantly purifying selection as confirmed by Tajima’s D, Fu and Li’s D and F tests, and SNAP algorithm. However, a few sites were also under positive selection as assessed by various models such as FEL, IFEL, REL, MEME, and PRIME.

FIRST REPORT OF CUCUMBER MOSAIC VIRUS AND OLIVE LATENT RINGSPOT VIRUS ON OLIVE TREES IN LEBANON

Olive (Olea europea L.) is cultivated in Lebanon on a surface of ca.53.600 ha, accounting for 43% of the area given over to perennial crops (Anonymous, 2010). Olive trees are affected by a number of virus and virus-like agents that persist in propagating material, with which they can be transmitted and disseminated (Martelli, 2011). Twelve trees of cv. Airouni, which is one of the most important cultivars grown in the country, were sampled in northern Lebanon and assayed for determining their sanitary status, in view of their possible use by nurseries for propagation. No symptoms of putative viral origin were observed in the surveyed orchards. Leaf samples were tested by RT-PCR to assess the presence of viruses commonly found in olive, i.e. Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Cucumber mosaic virus (CMV), Olive latent ringspot virus (OLRSV), Olive latent virus 1 (OLV-1), Olive latent virus 2 (OLV-2), Olive leaf yellowing-associated virus (OLYaV) and Strawberry latent ringspot virus (SLRSV), using the protocol and the sets of specific primers reported by Grieco et al. (2000). Results showed that CLRV, OLV-1, OLV-2 and OLYaV are present in nine trees, whereas ArMV and SLRV were not found. Two samples were positive for CMV and OLRSV, yielding the expected amplified products of 513 bp and 492 bp, respectively. Both these viruses were mechanically transmitted to herbaceous hosts (Cucumis sativus and Chenopodium quinoa, respectively) and their presence in these hosts was ascertained by RT-PCR. One of the trees proved free from all viruses and may constitute a source of virus-tested material for propagation. To our knowledge, this is the first report of CMV and OLRSV in olive in Lebanon.

An assemblage of divergent variants of a novel putative closterovirus from American persimmon

Deep-sequencing analysis of nucleic acids extracted from leaf tissue of an American persimmon (Diospyros virginiana L.) and subsequent-sequencing analyses uncovered at least four distinct closterovirus-like molecules. Two complete genomes of 18,569 and 18,030 nucleotides (nt) and partial genomes of 4,899 and 9,019 nt were determined. The two complete genomes encoded 11 potential open reading frames and the characteristic organization of closteroviruses. Among the four genomes, the putative heat shock protein 70 homolog (HSP70h), RNA-dependent RNA polymerase, and coat protein showed 82-85, 72-91, and 84-87 % amino acid sequence identities, respectively. These results suggested that the four identified viruses could be divergent variants in a single host plant. The phylogenetic tree based on HSP70h showed that their closest relative, although distant, is Olive leaf yellowing-associated virus, a putative unassigned member of the family Closteroviridae. The name Persimmon virus B was proposed for this new virus, representing another unassigned member of the family.

Detection of viruses in olive trees in Croatian Istria.

Normal 0 14 false false false IT ZH-TW X-NONE MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabella normale; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi;} Following identification of four viruses in a general survey of olive trees throughout Croatia, a detailed survey was conducted in 2009 in the field collection of the Institute of Agriculture and Tourism in Porec (an important reservoir of Istrian native olive germplasm) in order to evaluate the sanitary status of the most important Croatian Istria olive cultivars. Twenty five samples from symptomatic or symptomless trees were collected from five autochthonous and four exotic cultivars. All the samples were tested by RT-PCR for the presence of: Olive leaf yellowing associated virus (OLYaV), Cherry leaf roll virus (CLRV), Strawberry latent ring spot virus (SLRSV), Arabis mosaic virus (ArMV), Olive latent virus -1 (OLV-1), Cucumber mosaic virus (CMV), Olive latent virus -2 (OLV-2) and Tobacco necrosis virus D (TNV-D). Six of the 25 plants were found positive to CLRV; all infected plants showed leaf and fruit deformation and leaf yellowing. Four positive samples were from the native cv. Buža whereas the other two were from two exotic cultivars: Ascolana tenera and Frantoio. The presence of CLRV, either in native or imported plants, highlights the importance of strict phytosanitary regulations to prevent incursion of key virus diseases.

Characterization of latent viral infection of olive trees in the National Clonal Germplasm Repository in California.

The oil- and table olive industry in California is growing rapidly. Propagation material is being requested in ever-larger quantities from the USDA National Clonal Germplasm Repository at the University of California, Davis. This collection is recognized as one of the richest sources of olive material in the U.S. The repository maintains 107 different olive varieties imported from 19 different countries. However, the collection’s viral infection status has never been systematically analyzed. We have now completed the first comprehensive virus testing of the collection using molecular diagnostic tools. A total of 49 trees from 36 different cultivars were sampled. Though these trees were asymptomatic, the samples from 97.9% of them showed dsRNA profiles indicating viral infection. The 93.8% of these trees tested positive for Olive leaf yellowing-associated virus (OLYaV; an unassigned virus in the Closteroviridae) by RT-PCR analysis, while 34.7% were positive for Cucumber mosaic virus (CMV). PCR amplicons of the OLYaV heat shock 70 homologue (HSP70h) gene were cloned and sequenced to analyze diversity of isolates from trees originating from different geographical regions. Sequence analysis showed a maximum of 32% divergence between amplicons obtained from these selections.

Detection of Olive-infecting Viruses in Tunisia

During a virus survey in autumn 2007 and spring 2008 of two Tunisian olive mother blocks, 175 olive samples were collected from 19 different cultivars and tested by RT-PCR for the presence of Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Cucumber mosaic virus (CMV), Olive latent ringspot virus (OLRSV), Olive latent virus 1 (OLV-1), Olive latent virus 2 (OLV-2), Olive leaf yellowing-associated virus (OLYaV) and Strawberry latent ringspot virus (SLRSV), using specific sets of primers. The PCR-negative samples were also subjected to dsRNA and mechanical transmission tests. PCR results indicated that c. 86% of the trees were infected with at least one virus, whereas visible bands were shown by 3 of 24 PCR-negative samples in dsRNA analysis. OLYaV was the most prevalent virus (49.1 %), followed by OLV-1 (34.3%), CMV (25.7%), OLRSV (16.6%), CLRV (13.1%), SLRSV (7.4%) and OLV-2 (6.9%), whereas ArMV was not detected. Very high infection rates were found in the two main oil cvs. Chemlali (84.6%) and Chetoui (86.9%).

FURTHER CHARACTERIZATION OF OLIVE LEAF YELLOWING-ASSOCIATED VIRUS

RACE-PCR using a denatured dsRNA extract from an olive plant infected by Olive leaf yellowing-associated virus (OLYaV), extended a previously sequenced large fragment of the viral genome by 854 nt downstream in the heat shock protein 90 (HSP90). This sequence showed from 45 to 53% identity at the nucleotide level with that of putative HSP90s of different members of the family Closteroviridae. In phylogenetic trees constructed with published HSP90 and RdRp sequences, OLYaV was well separated from other viruses, as was Little cherry virus 1 (LChV-1), another unassigned species in the family Closteroviridae. A polyclonal antiserum was raised to a recombinant protein encoded by the HSP70 gene in fusion with sequence encoding the maltose binding protein. In Western blots of extracts from infected olive tissues, IgGs purified from this antiserum reacted with a polypeptide with a Mr of ca. 65 kDa, which was identified as the putative viral HSP70. Closterovirus-like flexuous particles with distinct cross banding and a maximum length of ca. 2000 nm were seen in extracts from infected roots and leaves of rooted olive cuttings. The cytopathology of infected leaves was similar to that induced by other members of the family Closteroviridae. However, the new information is still insufficient for the ultimate classification of OLYaV.

Preliminary evaluation of the status of olive‐infecting viruses in Syria

Field surveys of 80 commercial groves were made in autumn 2003 in six major Syrian olive‐growing regions. A total of 300 olive samples, representative of the main cultivars grown in the country, were collected. As ascertained by dsRNA analysis, 54 out of 125 samples (about 43%) showed visible bands in polyacrylamide gel electrophoresis. All samples were tested by RT‐PCR for the presence of the following viruses: Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Cucumber mosaic virus (CMV), Olive latent ringspot virus (OLRSV), Olive latent virus 1 (OLV‐1), Olive latent virus 2 (OLV‐2), Olive leaf yellowing‐associated virus (OLYaV) and Strawberry latent ringspot virus (SLRSV). All these viruses, singly or in mixed infection, were detected in about 51% of the samples. CMV was the most prevalent (22.7%), followed by CLRV (15%), OLYaV (14.3%) and OLRSV (11.5%). Less represented were the remaining four viruses. Infection rates ranged from 44% in Dara’a region to 67% in Latakia and Hama.

On the presence and distribution of olive viruses in Lebanon

A survey for viruses was carried out in 2003 in the main olive‐growing areas of Lebanon (South Lebanon, North Lebanon, Mount Lebanon and Bekaa). A total of 300 samples was collected in 31 different locations in 76 different commercial orchards and checked by RT‐PCR for the presence of Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Strawberry latent ringspot virus (SLRV), Olive latent virus 1 (OLV‐1) and Olive leaf yellowing‐associated virus (OLYaV), using virus‐specific primers reported in the literature. About one third (31%) of the trees were infected. In particular, the closterovirus OLYaV was the most widespread, as it was detected in 23.7% of the samples, followed by the necrovirus OLV‐1 (8.3%), the two nepoviruses CLRV (2%) and ArMV (0.3%), and the sadwavirus SLRV (0.3%). A high variability was detected in the HSP70 gene of Lebanese and Italian OLYaV isolates, for at least nine different patterns were obtained when this genomic region was subjected to single‐strand conformation polymorphism (SSCP) analysis.