Abstract
Several species of Botryosphaeriaceae and Phaeomoniella chlamydospora are common agents of grapevine decline worldwide. Currently, the use of culture independent PCR based techniques for detection of Botryosphaeriaceae within grapevine tissues has been limited to Botryosphaeria dothidea. In the present study, two Botryosphaeriaceae specific nested PCR assays were developed. One with a narrow target range, to detect Neofusicoccum parvum and the closely related species complex (Neofusicoccum parvum/N. ribis sensu Pavlic et al. Molecular Phylogenetics and Evolution 51:259–268, 2009) and another, with a wider range, to detect all 17 species of Botryosphaeriaceae which have been reported as potential wood pathogens of grapevine. The effectiveness of these assays was validated in vivo on naturally infected wood samples collected from standing vines and dormant grafted rooted cuttings commercialized in Italy by different nurseries in different years. All samples were also screened by means of a previously published nested PCR assay specific for Phaeomoniella chlamydospora. It was found that: 1) propagation material may play an important role as source of primary inoculum, not only of Phaeomoniella chlamydospora, as previously reported, but also for members of the Botryosphaeriaceae, among which Neofusicoccum parvum, Botryosphaeria dothidea and Diplodia seriata are the most common, and 2) multiple infections by different species belonging to Botryosphaeriaceae and/or Phaeomoniella chlamydospora occur frequently both in standing vines and propagation material. This last finding supports the hypothesis that at least some of the non-specific symptoms of grapevine decline may be due to the presence of different pathogens within host tissues.
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Acknowledgements
This research was commissioned from ARSIA-Toscana (Regional Agency for Development and Innovation in Agriculture and Forestry) by fourteen administrative Regions and autonomous provinces and financed with funds provided by the Ministero per le Politiche Agricole e Forestali (Ministry for Agricultural and Forestry Policy) to implement the inter-Regional Project “Grapevine esca: research and experiment in the nursery and in the field for prevention and cure”. Thanks are due to all nurseries and the Stazione Sperimentale per la Viticoltura Sostenibile S.r.l. of Gaiole in Chianti (Siena, Italy) for providing the propagation material that was used in this work, and the WWF of the Burano Lake Conservation Area (Grosseto, Italy) for allowing us to sample V.vinifera subsp. sylvestris plants. A. Phillips was financed by grant number SFRH/BCC/15810/2006 from Fundação para a Ciência e a Tecnologia (FCT) Portugal and project number PPCDT/AGR/56140/2004 from FCT. Prof. Marlene Jaspers, Dr. Jordi Luque, Prof. Sandra Savocchia and Dr. José Ramón Úrbez-Torres provided many of the fungal strains.
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Spagnolo, A., Marchi, G., Peduto, F. et al. Detection of Botryosphaeriaceae species within grapevine woody tissues by nested PCR, with particular emphasis on the Neofusicoccum parvum/N. ribis complex. Eur J Plant Pathol 129, 485–500 (2011). https://doi.org/10.1007/s10658-010-9715-9
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DOI: https://doi.org/10.1007/s10658-010-9715-9