Inter‐laboratory validation of PCR‐based protocol for detection of olive viruses

Abstract

Sanitary selection and certification of olive cultivars require sensitive diagnostic methods and effective sanitation protocols. Although much attention has been paid in the past few years to the development of diagnostic tools for reliable virus identification, the need to define a common and standardized diagnostic protocol led to the implementation of a ring test among nine Italian diagnostic laboratories. A one‐step RT‐PCR protocol and different primer sets, targeting the most common olive viruses covered by phytosanitary rules, were tested in each laboratory, using the same batch of positive and healthy controls as well as the same amplification conditions and reaction components. The one‐step RT‐PCR, performed using several specific primer sets, was able efficiently to detect the target viruses in all laboratories. Furthermore, a one‐step RT‐PCR protocol was used successfully for the first time for detection of Tobacco necrosis virus (TNV) and Olive mild mosaic virus (OMMV). Results showed that all target viruses were not uniformly distributed in the canopy, and that at least two subsets of samples must be collected from each plant. This standardized protocol is now being used to produce nuclear stocks for 70 different Italian olive cultivars, in the framework of the national project OLVIVA, which involves 25 national research institutions.

An integrated approach for understanding the high infection rates of olive viruses in Tunisia

“…This result is extremely important as TNA has been widely used as template for RT-PCR in olive surveys and shows the importance of sequencing. Despite the fact that TNA extracted by using commercial kits show better results (Loconsole et al 2010), the use of dsRNA as template in RT-PCR has previously shown to give more accurate results (Felix et al 2012), because it allows the use of a larger and more representative sample, reducing the constraints of low viral concentration and uneven distribution (Varanda et al 2010(Varanda et al , 2014. In addition it allows the elimination of virus non-related nucleic acids that interfere in the viral genome amplifications (Bertolini et al 2003;Luigi et al 2011) which revealed to be a problem when TNA was used as template in RT-PCR in this study.…”

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

“…The partial genome information available for OLYaV (NC_043417) corresponds to only 4605 nucleotides containing partial ORF1b (RdRp), ORF2 (21kDa), ORF3 (7kDa), ORF4 (HSP70h) and the 5′-end of ORF 5 (HSP90h) [ 2 ], and a subsequent extension of 854 nucleotides in the HSP90h gene towards the 3′-end (AJ844555) [ 3 ]. The development and application of diagnostic methods by RT-PCR of specific regions of the HSP70 gene [ 1 , 4 ] has revealed that OLYaV is one of the most widespread olive viruses with different levels of incidence: California (USA) (93%) [ 5 ], Italy (64–21%) [ 6 , 7 , 8 ], Tunisia (49%) [ 9 ], Lebanon (24%) [ 10 ], Syria (15%) [ 11 ], Greece (5%) [ 12 ] and Albania (2%) [ 13 ]. In addition, studies of OLYaV HSP70 gene diversity revealed a high divergence in this region of samples collected in clonal germplasm repositories in California and Greece [ 5 , 12 ].…”

Phytosanitary Certification

“…Recently, a one step RT-PCR protocol has been set up and validated in an inter-laboratory ring test (Loconsole et al, 2010) for the diagnosis of the eight most important olive viruses. This should be a starting point for anyone wishing to approach the sanitary selection of olive plants.…”