Spread of ‘bois noir’ in organic vineyards in Tuscany: spatial pattern analysis and identification of the phytoplasma in weeds

In this study the spread of ‘bois noir’ (BN) was monitored from 2005 to 2010 in three organic vineyards of cv. Sangiovese established in 2001 and 2002 in the province of Florence, Tuscany (central Italy). Maps with locations of all symptomatic and non-symptomatic vines, a complete census of binary data, were made for each plot and year of assessment. Three indexes (annual incidence, infection and recovery rates) were calculated from the original data sets to describe changes in the epidemic with time. Moreover with the purpose of determining the spatial pattern of the disease (random vs. non random or clustered), for each year of assessment, field data on annual incidence were analyzed at three spatial levels (hierarchies). The values of annual incidence, infection and recovery rates in each year were different at each study site but with a similar trend over time. In general symptomatic plants showed the phenomenon of recovery in a relatively short time meanwhile the infection rate decreased rapidly after the second year of assessments. Results of spatial pattern analysis were consistent at all levels considered and suggested that the arrangement of diseased vines was random across all disease assessment dates in two out of three study sites. On the other hand in the third vineyard the distribution of the disease was always non random although an intensification of clustering over time never became evident. Since the epidemics were different in different environments, a survey on the herbaceous vineyard weeds was carried out in 2010 to study how organic vineyard management practices may influence disease spread. Symptoms of yellowing or reddening were recorded on some species meanwhile others showed symptoms typical for those associated with phytoplasma infections like dwarfing, virescence, and phyllody. Total DNA was isolated from root and or leaf tissues of about 200 samples and nested PCR was carried out using P1/P7 followed by R16F2/R2 primers. RFLP analyses carried out with TruI restriction enzyme allow to identify the following species infected by 16SrXII phytoplasmas (stolbur): Pichiris hieracioides, Daucus carota, Lactuca saligna, Lactuca sp., Bupleurum tenuissimum, Cichorium intibus, Convolvolus spp., Cuscuta spp., Mercurialis annua, Linaria vulgaris, and Medicago lupulina. In some of these species further polymorphisms in R16F2/R2 amplicons were detected after MboII restriction, and in one case also with Hpy188I; the same samples digested with AluI, did not show any polymorphism. The different profiles observed could be referred to some of those identified in grapevine from diverse geographical areas including Tuscany (Contaldo et al., 2011). Further studies to verify the relevance of these polymorphisms in the BN epidemiology are in progress.