Real-time RT-PCR (TaqMan^®) assays for the detection of viruses associated with Rugose wood complex of grapevine

Abstract

Real-time TaqMan^® RT-PCR (TaqMan^® RT-PCR) assays were developed to detect the viruses associated with Rugose wood complex of grapevines. The viruses detected were Rupestris stem pitting-associated virus in the genus Foveavirus, Grapevine virus A, Grapevine virus B and Grapevine virus D in the genus Vitivirus. The coat protein was found to be the most conserved gene within the viral species, therefore, the primers and probes for TaqMan^® RT-PCR assays were designed from the multiple alignment of the coat protein sequence of various isolates of each virus. Comparisons were also made between the conventional one step RT-PCR and TaqMan^® RT-PCR for the detection of these viruses using four-fold serial dilutions of both purified RNA and crude extract prepared from grapevine tissue. Results showed that TaqMan^® RT-PCR was more sensitive and could detect viruses at 32- and 256-fold higher dilutions for purified RNA and crude extract, respectively, compared to RT-PCR. The use of an internal control (18S rRNA) allowed comparison of sample preparation protocols and amplification efficiencies between samples.

Introduction

Rugose wood complex is a term used to describe a group of graft-transmissible diseases which affect grapevines (Vitis spp.) worldwide (Graniti and Martelli, 1966, Rosciglione and Gugerli, 1986, Martelli, 1993, Minafra, 2000). Rugose wood is of great economic importance to the grape industry causing severe reduction of growth and yield in affected grapevine plants (Golino et al., 1997, Golino et al., 2000, Goheen, 1988). The Rugose wood complex is classified into different diseases based on symptomatology on specific indicator hosts: Rupestris stem pitting, Kober stem grooving, corky bark and LN 33 stem grooving (Martelli, 1993). To date three different phloem-restricted viruses belonging to two different genera have been associated with the etiology of Rugose wood complex: Rupestris stem pitting-associated virus (RSPaV) in the genus Foveavirus, Grapevine virus A (GVA) and Grapevine virus B (GVB) in the genus Vitivirus (Meng et al., 1998, Zhang et al., 1998, Martelli and Jelkmann, 1998, Martelli et al., 1997). Another vitivirus, i.e. Grapevine virus D (GVD) (Abou-Ghanem et al., 1997, Boscia et al., 2001) has been detected in a vine (V. vinifera) showing corky Rugose wood symptoms, but its role in the Rugose wood complex is unclear. The entire genome of RSPaV has been sequenced and consisted of 8725 nucleotides excluding the poly A tail (Zhang et al., 1998, Meng et al., 1998). GVA is associated with Kober stem grooving disease (Garau et al., 1994) and its complete genome has been sequenced and consisted of 7351 nucleotides (Minafra et al., 1997). GVB is associated with Corky bark disease (Bonavia et al., 1996) and its genome has been completely sequenced and consisted of 7599 nucleotides (Saldarelli et al., 1996). GVD genome is only partially sequenced and the known part of its structure is similar to other vitiviruses (Abou-Ghanem et al., 1997, Choueiri et al., 1997).

Detection of diseases caused by Rugose wood complex is based on bioassays, ELISA and RT-PCR. Bioassays are used widely, but they are time and labor intensive. Detection of viruses associated with Rugose wood complex by ELISA is difficult and sometimes unreliable due to their low concentration in grapevine tissue, seasonal variation in virus titer and uneven distribution in the host plant. In addition, reliable sources of antibodies for all these viruses are not available. Different RT-PCR protocols have been developed for the detection of RSPaV, GVA, and GVB (Minafra and Hadidi, 1994, Minafra et al., 1997, Zhang et al., 1998). Further investigations have shown the existence of large sequence variability among different isolates of RSPaV (Meng et al., 1998, Meng et al., 2005, Rowhani et al., 2000, Lima et al., 2006) and GVB (Shi et al., 2004). Similarly, large sequence variation was found within two serologically distinct groups of GVA (Murolo et al., 2008, De Meyer et al., 2000). Because of this sequence variability among different isolates of a virus, it will be difficult to design efficient and reliable PCR primers for virus detection (Saldarelli et al., 1998). Although the use of degenerate primers allows the simultaneous detection of different viruses, problems such as non-specificity, analysis of PCR product by polyacrylamide gel electrophoresis and silver staining limit their large scale application. In addition, high concentrations of phenolic compounds and polysaccharides that exist in grapevine tissues inhibit the function of enzymes used in RT-PCR and thus prevent virus detection (Nassuth et al., 2000). Real-time PCR is increasingly being used for the detection and quantification of pathogens in plant tissue and soil (Agindotan and Shiel, 2007, Harju et al., 2005, Hren et al., 2007, Ling et al., 2007, López et al., 2006, Lunello et al., 2004, Mumford et al., 2000, Mumford et al., 2004, Osman and Rowhani, 2006, Osman et al., 2007, Roussel et al., 2007, Saponari et al., 2008). The objective of this study was to design sensitive TaqMan^® assays for the detection of RSPaV, GVA, GVB and GVD with the capability to detect different isolates of each virus. The results presented in this paper clearly show that real-time TaqMan^® RT-PCR (TaqMan^® RT-PCR) is a robust and reliable method for the detection of viruses constituting the Rugose wood complex of grapevine whether using purified RNA or crude extract as the starting template.