Multiplex RT-PCR for detection and identification of three necroviruses that infect olive trees

Abstract

An optimized multiplex RT-PCR assay was developed to discriminate three necrovirus (Olive latent virus 1 (OLV-1), Tobacco necrosis virus D (TNV-D) and Olive mild mosaic virus (OMMV)) that infect olive trees. An olive orchard consisting of 54 trees of cv. "Galega vulgar" in the south of Portugal was surveyed. dsRNA fraction was used as template and revealed the 3 viruses, singly or in multiple infections, present in 17 out of 54 trees in the orchard. OMMV was the most frequent occurring in 15 trees, followed by OLV-1 in 12 and TNV-D in 4 plants. The results obtained showed that necrovirus-specific dsRNAs do exist in infected tissues in amounts below the resolution permitted by gel electrophoresis analysis and that the developed multiplex PCR based assay is of much higher sensitivity. The design of the specific primers described enabled, for the first time, to discriminate between OMMV and TNV-D by means of RT-PCR assays, an indispensable tool in identification, epidemiology and survey studies. Document Type: ArticleLanguage: English

A degenerate pair of primers for simultaneous detection of four alpha- and betanecroviruses

“…Cloning and sequencing these 17 PCR products revealed the presence of OMMV in 15 olive trees, OLV-1 in 12 trees and TNV-D in 4 trees, and mixed infections in 10 of them. This is in complete agreement with the findings of previous studies, in which single and multiplex RT-PCR assays were carried out using the same trees (Varanda et al, 2010). No TNV-A was detected in any of the sequenced clones.…”

“…DsRNA extracted from the fruits and stems of 54 olive field trees whose sanitary status concerning OLV-1, OMMV and TNV-D had already been determined (Varanda et al, 2010) were used in this study.…”

“…In general, considerable levels of viral infection have been detected in many regions, including Portugal. In this country, extensive field surveys have revealed Olive latent virus 1 (OLV-1), Olive mild mosaic virus (OMMV) and Tobacco necrosis virus D (TNV-D) as predominant, reaching infection levels of 31%, with OMMV the most abundant virus (Saponari et al, 2002;Varanda et al, 2006Varanda et al, , 2010El Air et al, 2011). These viruses have several natural hosts.…”

“…TNV-like viruses, that were able to be assigned to TNV-D or TNV-A, have been detected in herbaceous plants such as tobacco, tulip, bean, cucumber and potato, as well as woody crop plants such as pear and apple, citrus, grape (Kassanis, 1970;Kurstak, 1981) and olive (Félix and Clara, 2002b). OMMV was identified for the first time in olive and genomic sequences of OMMV, published in the Genbank database, were recovered from tulips affected by the Augusta disease, leading to its identification as the causal agent of that disorder (Pham et al, 2009 Mixed infections of these three viruses occur naturally quite frequently (Félix and Clara, 2002a;Varanda et al, 2010). These alphaand betanecroviruses share important similarities.…”

“…OMMV amino acid CP sequence shows 85.1% identity with that of TNV-D (Cardoso et al, 2009) and its RNA-dependent RNA polymerase (RdRp) has 91.2% identity with that of OLV-1 . In fact, differentiation between OMMV and TNV-D isolates is only possible through PCR-based assays using specific primers (Varanda et al, 2010) or genome sequencing.…”