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

Abstract

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.

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][27][28][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].…”

“…Nevertheless, due to its vegetative propagation through cuttings, it has accumulated several pathogens, including viruses. More than seventeen viruses belonging to different genera or families have been reported to naturally infect olive trees [1][2][3][4][5][6]. Over the last decade, the wide application of high-throughput sequencing (HTS) has allowed the rapid identification and characterization of several novel or new strains of known viruses in various plant species using different technologies [7][8][9][10].…”

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

“…In addition, 94 samples and 14 insect samples of OLYaV candidate vectors were also analyzed. OLYaV has been officially reported in California (USA) [9], Italy [10], Tunisia [11], Lebanon [12], Syria [13], Greece [14], Albania [15], and Brazil [16]. The presence of OLYaV in the USDA National Clonal Germplasm Repository (NCGR) at the University of California from imported plant material suggested its possible presence in Spain, France, Cyprus, Chile, Israel, and Australia [9].…”

Stability investigation of air-dried olive ribo nucleic acids for metavirome studies

“…Air-dried RNA of TRIzol 1 method showed acceptable quality and integrity even for sequencing purposes which proves the capability of out-of-ice transferring method. TRIzol extracted RNAs yielding good quality and purity of fresh RNA extraction were also used for virus detection in Greek olive cultivars showing the samples were free of genomic DNA achieved in less than 3 h without the extra need for purification steps [ 18 ].…”

“…Several limitations such as low antigen titre specifically in phloem restricted viruses [ 11 , 12 ], irregular distribution of viruses in plant tissues [ 13 ], high temperature in sampling season [ 14 ], antiviral secondary metabolites like iridoid glycosides compounds [ 15 ], and cellular components such as oil, polysaccharide and polyphenols influence the accuracy of viral detection in olive [ 16 , 17 ]. Therefore, the extraction and maintenance of high quality and integrity RNA is challenging [ 17 ] and it is basically required for reverse transcription-polymerase chain reaction (RT-PCR) amplification of viral targets [ 18 ]. In woody plants rich in secondary metabolites e.g., glycosides, polyphenols, and polysaccharides, which normally co-purify with nucleic acids, enzymatic reactions in dsRNA extraction are also inhibited [ 16 , 19 – 21 ].…”