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Sanitary assessment of olive varieties in a collection plot in Perugia (Umbria, Italy)
Abstract
A survey was carried out to assess the sanitary status of olive trees in a collection plot in Perugia (Umbria, Italy). A total of 95 samples were tested by RT-PCR to check for the presence of the following olive-infecting viruses: Arabis mosaic (ArMV), Cherry leaf roll (CLRV), Cucumber mosaic (CMV), Olive leaf yellowing-associated (OLYaV), Olive latent ringspot (OLRSV), Olive latent -1 (OLV-1), Olive latent -2 (OLV-2), and Strawberry latent ringspot (SLRSV). About 82% of the plants were infected with at least one virus. All the tested viruses were present, with the prevalence of CMV and OLRSV (more than 30%), followed by OLYaV (21.1%) and SLRSV (19%). DsRNA and mechanical transmission assays were also applied to all PCR-negative samples. Eight out of the 17 samples tested showed different dsRNA patterns, thus suggesting that they were infected with viruses different from those previously checked by PCR. This study allowed the identification of 9 "virus-free" and 39 "virus-tested" (i.e. olive plants free from SLRSV, ArMV, CLRV, OLYaV and OLV-1, according to the Italian certification protocols) candidate clones which could potentially be used as mother plants in future Certification programmes.
Strawberry latent ringspot virus (SLRSV) isolated from symptoms and symptomless strawberry plants and identified with a specific antiserum using Double Antibody Sandwich ELISA (DAS-ELISA). Survey was conducted through three successive seasons during 2013-20l6 on commercial strawberry fields in four governorates in Egypt. The percentages of infection which recorded from the survey were 7.5, 6.3, 5.9% (Al-Dair-region) and 9.4, 10.6, 10.5% (Kafer Al-Shapy-region) in El-Qalubia governorate. In El-Behera governorate the percentage was 5.3, 6.0, 5.7% (Badrregion) and 4.0, 4.4, 5.2% (Al-Nobaria-region). El-Monufyia (Quesna-region) recorded 8.8, 8.9, and 9.2 %. Ismailia (fayed-region) was 4.7, 5.0, and 5.5% respectively. SLRSV mechanically transmitted from infected strawberry plants onto 16 host species belonging to seven families. Mottling, local lesion, leaf deformation, systemic and dwarf plants were appeared two weeks' post inoculation on indicator host Chenopodium quinoa and Chenopodium amaranticolor. Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify 497 bp fragment using specific primers for the viral coat protein gene, as a tool for molecular diagnosis. The amplified PCR fragments were cloned, sequenced and compared with other of those sequences available in GenBank. Results observed that, the Egyptian SLRSV isolate was A+T, G+C (251, 246) identical content with Poland and USA isolates. Obtained sequence comparison with the isolates available in the GenBank indicated that SLRSV Egyptian isolate shared 99% identity with Poland isolate followed by 84% USA isolate, 83% UK isolate and 80% Newzeland isolate.
This datasheet on Cherry leaf roll virus covers Identity, Overview, Distribution, Dispersal, Hosts/Species Affected, Diagnosis, Biology & Ecology, Seedborne Aspects, Impacts, Prevention/Control, Further Information.
Taking into consideration that (i) there are no differential woody indicators plants that could help in the diagnosis of olive viruses, (ii) the mechanical transmission of olive viruses to herbaceous hosts is erratic and presents a low intrinsic sensitivity, (iii) the immunoenzymatic assay (ELISA) is unreliable due to the low antigen titre and high amounts of inhibitors in olive tissues, (iv) there is a unique conventional molecular technique (RT-PCR) reliable to detect olive viruses but also known for its tight spectrum for detecting different isolates, all these factors necessitate the development of a sensitive, fast, reliable and a large spectrum diagnostic technique that could be used in the screening of a large number of olive samples.
The real-time RT-PCR seems, in this case, a perfect diagnostic method that could compensate with all these constraints. Accordingly, in this study, real-time- and Multiplex-TaqMan RT-PCR assays were developed to detect single and multiple infections by the five most important olive viruses, i.e. ArMV, CLRV, SLRSV, OLRSV and OLYaV, for which the results obtained are herein reported.
Strawberry latent ringspot virus (SLRSV) isolated from symptoms and symptomless strawberry plants and identified with a specific antiserum using Double Antibody Sandwich ELISA (DAS-ELISA). Survey was conducted through three successive seasons during 2013-20l6 on commercial strawberry fields in four governorates in Egypt. The percentages of infection which recorded from the survey were 7.5, 6.3, 5.9% (Al-Dair-region) and 9.4, 10.6, 10.5% (Kafer Al-Shapy-region) in El-Qalubia governorate. In El-Behera governorate the percentage was 5.3, 6.0, 5.7% (Badrregion) and 4.0, 4.4, 5.2% (Al-Nobaria-region). El-Monufyia (Quesna-region) recorded 8.8, 8.9, and 9.2 %. Ismailia (fayed-region) was 4.7, 5.0, and 5.5% respectively. SLRSV mechanically transmitted from infected strawberry plants onto 16 host species belonging to seven families. Mottling, local lesion, leaf deformation, systemic and dwarf plants were appeared two weeks' post inoculation on indicator host Chenopodium quinoa and Chenopodium amaranticolor. Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify 497 bp fragment using specific primers for the viral coat protein gene, as a tool for molecular diagnosis. The amplified PCR fragments were cloned, sequenced and compared with other of those sequences available in GenBank. Results observed that, the Egyptian SLRSV isolate was A+T, G+C (251, 246) identical content with Poland and USA isolates. Obtained sequence comparison with the isolates available in the GenBank indicated that SLRSV Egyptian isolate shared 99% identity with Poland isolate followed by 84% USA isolate, 83% UK isolate and 80% Newzeland isolate.
We have used a one-step RT-PCR protocol to detect and identify each of the eight viruses most commonly found in olive trees namely: Arabis mosaic virus (Ar-MV), Cherry leaf roll virus (CLRV), Cucumber mosaic virus (CMV), Olive leaf yellowing associated virus (OLYaV), Olive latent ring spot virus (OLRSV), Olive latent virus-1 (OLV-1), Olive latent virus-2 (OLV-2), and Strawberry latent ring spot virus (SLRSV). We have assayed 345 samples collected from olive fields in Italian areas in which national and local olive tree cultivars and selections are grown. The average percentage virus infection of olive trees was 32.8%. Among the eight virus-es assayed, only OLYaV (20.9%), SLRSV (7.8%) and CLRV (4.9%) were detected. The most common virus was OLYaV, which was found at a high percentage in southern Italy. SLRSV was the main virus in central Italy whereas CLRV was detected in five samples from Latium, Umbria and Sicily and in all analyzed trees from Abruzzo.
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.
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 one-step reverse transcription–polymerase chain reaction (RT–PCR) protocol was developed and used for the detection of Strawberry latent ring spot virus (SLRSV) in leaves of olive trees in Italy. The protocol was used to test for infection in samples from 87 trees and to correlate infection with leaf symptoms. SLRSV was found only in olives grown in Central Italy (the Marche and Umbria regions) and was detected in c. 15% of leaf samples. Most infected plants had narrow and twisted leaves, symptoms which seemed to occur in specific cultivars. The one-step RT–PCR protocol is very rapid and sensitive and has the potential to be used on a mass scale for the diagnosis of SLRSV in olive. This virus should be considered an important pathogen in olive trees, even if its distribution in the field seems to be limited to well defined areas.
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.
Olive hosts 13 viruses belonging in seven different genera. Additional non-mechanically transmissible viruses probably infect olive in nature, as suggested by the widespread occurrence of double-stranded RNAs (dsRNAs) in trees from which no viruses can be recovered by manual inoculation. Because sanitary selection appears to be the only measure for restraining virus dissemination through propagating material, detection methods are needed which are more sensitive and reliable than those currently available (biological and serological). The following molecular techniques have therefore been used and their efficiency compared: (1) dsRNA analysis; (2) dot-blot hybridization with digoxigenin-labelled riboprobes in separate reactions or in mixture; and (3) reverse transcription-polymerase chain reaction (RT-PCR). It was found that: (1) dsRNAs were detected in 210 out of 286 olive accessions (73.4%) coming from six different Italian regions; (2) one-step RT-PCR yielded much better results using TNA extracts than crude sap; and (3) dot-blot hybridization of denatured dsRNAs with digoxigenin-labelled virus-specific riboprobes was the most reliable detection method available.
A single-step multiplex RT-PCR was developed for the simultaneous and colourimetric detection of six RNA viruses (Cucumber mosaic virus, Cherry leaf roll virus, strawberry latent ringspot virus, Arabis mosaic virus, Olive latent-1 virus and Olive latent-2 virus) which infect olive trees. Six compatible primer set for one-step RT-PCR amplification in a single closed-tube and 3' digoxigenin labelled probes were designed in optimal, specific and conserved regions. The method has been assessed with 195 Spanish field olive trees, suggesting that approximately 1.5% of the tested material was infected by Cucumber mosaic virus and 0.5% by Cherry leaf roll virus. This method saves time and reagent costs compared with monospecific RT-PCR which needs several reactions for the same number of tests. Using colourimetric detection, it is possible to analyse many samples, it increases sensitivity 10-fold, and whilst facilitating the interpretation of results, it avoids the use of gels and the toxic ethidium bromide. The method could be used routinely for sanitary and certification programmes.
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.
An account is given of diseases of olive (Olea europaea var. sativa) and related pathogens transmitted with propagation material, with special reference to viruses and phytoplasmas. To date, 14 virus and virus-like diseases have been described and 12 different viruses and five phytoplasmas have been identified in symptomatic or symptomless plants. A brief description of the diseases is given, along with information on epidemiology and diagnosis. Possible remedies, such as sanitary selection, sanitation and implementation of certification schemes are discussed.
Highly purified double-stranded (ds) RNA was obtained from cortical scrapings of mature canes of vines infected by grapevine leafroll-associated closterovirus III (GLRaV III) using phenol-chloroform extraction, chromatography on CF-11 cellulose minicolumns and enzymatic digestion. Complementary (c) DNA fragments of various lengths, obtained by random priming denatured dsRNA templates, were cloned into the plasmid pUC-18 at the Smal site in Escherichia coli strain DH5α. Two 32P-labelled cDNA clones denoted p16ds (c. 1100 bp) and p23ds (c. 1500 bp) were successfully used as probes for detecting GLRaV III sequences in grapevine extracts from leaves and petioles, or cortical tissues. Probe p23ds was virus-specific and did not hybridize with total RNA from healthy controls, or from vines infected by grapevine leafroll-associated closterovirus I (GLRaV I), or with genomic RNA from purified grapevine closterovirus A (GVA) and B (GVB). A riboprobe (pGEM23ds) transcribed from p23ds in transcription vector pGEM3zf specifically recognized GLRaV III sequences, but not GLRaV I or GVA sequences, in extracts from differently infected vines. Moreover, in Northern blots, the same probe hybridized also with smaller dsRNA components, which may be replicative forms of subgenomic RNAs.