Abstract
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.
Highlights
Olive trees are susceptible to several pathogens that may affect the yield and quality of their products with important economic impact, such as diseases causes by fungi (i.e., Colletotrichum spp. and Spilocaea oleagina) (Salman, 2017; Materatski et al, 2018), and more recently the emergence of Xylella fastidiosa, first noticed in 2013, and responsible for a severe outbreak of Olive quick decline syndrome (Saponari et al, 2013)
Standard curves of all studied olive viruses were constructed based on Cq values obtained from a 10-fold dilution series of the target plasmid DNA obtained from each reference isolate, in the dynamic range that was chosen
Similar standard curves were obtained when Olive mild mosaic virus (OMMV) and Tobacco necrosis virus D (TNV-D) were used as targets, once a single set of primers for amplification of both viruses was used
Summary
Olive trees are susceptible to several pathogens that may affect the yield and quality of their products with important economic impact, such as diseases causes by fungi (i.e., Colletotrichum spp. and Spilocaea oleagina) (Salman, 2017; Materatski et al, 2018), and more recently the emergence of Xylella fastidiosa, first noticed in 2013, and responsible for a severe outbreak of Olive quick decline syndrome (Saponari et al, 2013). Viral symptoms on olive trees include bumpy fruits, leaf yellowing, vein banding, and vein clearing, (Martelli, 1999; Albanese et al, 2012). These symptoms are non-specific and difficult to recognize, and often occur in virus-infected olive trees without apparent symptoms, which makes viral diagnosis in the field impossible to perform (Martelli et al, 2002; Alabdullah et al, 2010; Martelli, 2011; Zellama et al, 2018). The Olive leaf yellowing associated virus (OLYaV) was not found to have a negative interference in oil yield and quality (Fontana et al, 2019)
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