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

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.

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

“…In our study, OLYaV was frequent (23.5%) in the tested samples similarly to reports from Lebanon, Italy, Spain, and Tunisia [ 12 , 26 – 29 ]. On the contrary, in previous surveys conducted in Greece, both in olive orchards [ 5 ] and in a germplasm collection of the Institute of Olive Tree, Subtropical Crops and Viticulture (IOSV) in Chania [ 4 ], its incidence was very low (1.2 and 5%, respectively) and similar to that reported in Albania (2%) from Luigi et al [ 30 ].…”

“…An explanation provided in the previous studies, regarding the low infection rate of OLYaV in Greece, was related to the absence or low populations of the putative psyllid vector ( Euphyllura olivinae ) in Greek olive orchards [ 4 , 5 ]. However, from our study, we conclude that high genetic variability and/or low virus titer, both frequently encountered in OLYaV and in other closteroviruses, could also explain the differences observed [ 12 , 28 , 29 ].…”

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

“…Although this virus may be associated with a bright yellow discoloration in olive plants [ 2 , 26 , 30 ], changes in vegetative growth [ 31 ], or woody deformations [ 29 ], no such symptoms were observed. OLYaV appears to be one of the most widespread viruses in olives, with infection rates of up to 63% in Tunisia [ 2 , 24 , 26 , 32 ]. High OLYaV infection rates were also recorded in the germplasm repository of the USDA (National Clonal Germplasm Repository, Davis, California) where 93.8% of the tested varieties, including three Greek varieties, were found to be infected [ 9 ].…”

A novel study on bean common mosaic virus accumulation shows disease resistance at the initial stage of infection in Phaseolus vulgaris

“… Jiang et al (2013) measured the viral load of HTNV (Hantaan virus) by using an SYBR Green I-based one-step qRT-PCR technique and observed low viral titres in samples with high Ct values. Campos et al (2019) reported that qRT-PCR based on SYBR Green methodology produced high Ct values in low titres of olive viruses. Similarly, de Freitas-Vanzo et al (2021) used the qRT-PCR to quantify BGMV titre in host plants and found variations in symptom expression and BGMV titre between cultivars and lineages at 10 and 25 pi and the bean cultivar “Tangará” as reported in YLV-14 in this research, had a higher Ct value but showed less symptom severity than the other cultivars.…”