Distribution and tuf type characterization of ‘bois noir’ in Tuscany by an improved real-time assay or nested-PCR

In agreement with a regional strategy aimed to monitor grapevine yellows (GY) disease, an extensive survey was carried out covering the ten provinces of the Tuscany region. Symptomatic leaf samples were collected from commercial vineyards and nurseries during 2009 and 2010. Laboratory analyses performed by nested PCR (Botti et al., 2005), and by Real-Time PCR (Angelini et al., 2007) both on 16S ribosomal gene, confirmed that ‘bois noir’ (BN) is largely the most important GY in Tuscany with ubiquitous distribution. Molecular characterization of BN phytoplasmas relied on genetic diversity studied through multiple step amplification process of one or more genes followed by RFLP analysis. In recent times the latter technique was flanked by sequencing of amplicons and/or cloned amplicons followed by in silico RFLP analyses. This procedure enable to find in short time a wider BN genetic diversity through the identification of several single nucleotide polymorphism (SNP) lineages. Nevertheless, little is known about the ecology of these newly defined subgroups and their putative role in the epidemiology of BN. Consequently, the model proposed by Langer and Maixner (2004) permitting the distinction of three biologically differentiable sequence variants (tuf-type a, tuf-type b and tuf-type c) is still widely adopted to study BN epidemiology. Recently, a newly TaqMan allelic discrimination assay was developed and proposed for the distinction of tuf-type a and tuf-type b stolbur phytoplasmas (Berger et al., 2009). A real time PCR assay for the rapid and specific detection tuf-type a and tuf- type b was developed and applied in alternative to nested PCR assays carried out on the same gene. For this purpose, two newly designed BHQplus probes (Biosearch Tech, CA, USA) were used in an allelic discrimination test to distinguish between tuf-type a and tuf-type b among selected BN positive samples identified in grapevine in Tuscany. Real-time PCR analysis was performed in duplicates in 12 μl-reactions, containing 6 μl IQ Powermix (Bio-Rad Laboratories, USA), 500 nM of each primer, 100 nM of FAM-VKI probe, 100 nM of CalFluor orange 560-VKII probe, and 1 μl of sample DNA. Assays were carried out on Rotor-Gene Q (Qiagen, Germany). Standard curves traced on serial dilution of synthetic oligonucleotide permitted to calculate the reaction efficiency. The slopes of the linear fits reached -3.329 for the green channel indicating an efficiency of 99.70% (R2= 0.992) for the tuf type-I assay. Similar results were obtained with the tuf type-II probe (Efficiency = 98.64 %, R2= 0.996). When applied to grapevine samples, results were automatically generated through the allelic discrimination option of the Rotor-Gene Q software (version 2.0.2, Qiagen). According to preliminary results, the newly designed assay for the simultaneous detection of tuf-type a and tuf-type b types resulted to be very efficient. A number of BN positive samples are under characterization with both methods real-time or nested-PCR to establish the presence of tuf-type a and tuf-type b and to trace a distribution map in Tuscany.