Profiling and accurate quantification of trans-resveratrol, trans-piceid, trans-pterostilbene and 11 viniferins induced by Plasmopara viticola in partially resistant grapevine leaves U. VRHOVSEK 1 , G. MALACARNE 1 , D. MASUERO 1 , L. ZULINI 1 , G. GUELLA 2 , M. STEFANINI 1 , R. VELASCO 1 and F. MATTIVI 1 1 IASMA Research and Innovation Centre, Edmund Mach Foundation,Via E. Mach 1, 38010 San Michele all’Adige, Italy 2 Department of Physics, University of Trento, via Sommarive 14, 38100 Trento, Italy Corresponding author: Dr Urska Vrhovsek, fax: +39 0461 615 200, email: urska.vrhovsek@iasma.it Abstract Background and Aims: In the Vitaceae, stilbenoid viniferins constitute a relatively restricted group of molecules based on a trans-resveratrol structure and represent the main known phytoalexins. The aim of this work was the development of a new method for the chromatographic separation and accurate quantification of viniferins found in grapevine leaves following infection with Plasmopara viticola. Methods and Results: An interspecific F1 population was used, deriving from the cross between Merzling and Teroldego, respectively, partially resistant and susceptible to P. viticola. Analysis was carried on a liquid chromatography-mass spectrometry (LC-MS) system, using a reverse phase column. A method was validated for detailed metabolite profiling, with accurate identification and quantification of trans-resveratrol, trans-piceid, trans- pterostilbene, a condensation product between (+)-catechin and caffeic acid, and the whole class of stilbenoid viniferins in P. viticola-infected grapevine leaves, using their respective standards. Synthesis was ongoing, and 6 days after infection, it was possible to see the highest concentration of stilbenoids for all genotypes. On the other hand, the intensity of stilbene induction did not show a clear and homogeneous correlation with the position of the leaves in the young shoot. Conclusions: The method described in this paper provides the basis for performing in a single run a targeted analysis of resveratrol, piceid, pterostilbene and the whole class of stilbenoids in P. viticola-infected grapevine genotypes. Significance of the Study: To our knowledge, this is the first paper that has reported on detailed metabolite profiling with accurate identification and quantification of the main viniferins in grape leaves following infection with P. viticola, using the respective standards. Keywords: HPLC-DAD-MS, Plasmopara viticola, stilbenoid, viniferin, Vitaceae Introduction Stilbenoids are phenols deriving from the phenylpropanoid and acetate–malonate pathway expressed in many plant families. Within the Vitaceae, stilbenoids constitute a relatively restricted group of molecules based on a trans-resveratrol structure and represent the main known phytoalexins. The synthesis of stilbenes in grapes may be constitutive (Pezet and Pont 1988, Korhammer et al. 1995, Mattivi et al. 1995) or induced in response to biotic and abiotic stress (Langcake and Pryce 1976, Langcake and Pryce 1977, Adrian et al. 1996, Sarig et al. 1997, Douillet-Breuil et al. 1999, Cantos et al. 2001) or elicitors (Bru et al. 2006; Zamboni et al. 2009). Stilbenes have aroused intense interest because of their anti- fungal properties (Hoos and Blaich 1990, Adrian et al. 1997). In the Vitis species, Langcake and Pryce (1977) characterised a range of phytoalexins with antifungal activity. Specifically, the presence of resveratrol has been shown to be closely related to disease resistance in Vitis spp. (Dercks and Creasy 1989, Adrian et al. 1997). In addition to resveratrol, other stilbenoids deriving from oxidative dimerisation have also been found to act biologi- cally against various fungal pathogens in the grapevine (Jeandet 2002). Variability in stilbene production can also be influenced by the genotype and the grapevine’s stage of development (Barlass et al. 1987, Dercks and Creasy 1989, Sbaghi et al. 1995, Gatto et al. 2008) or by leaf age (Stein et al. 1985). In particular, very old leaves and very young leaves have been reported to synthe- sise fewer stilbenes as compared with others, because of the closing and early stages of development of the stomata limiting pathogen infection and indirectly stilbene synthesis (Stein et al. 1985). The toxicity of these compounds was found to be related to their chemical structure. In particular, it was found that d-viniferin, an oxidative resveratrol dimer, and pterostilbene, the 3,5-dimethoxy analogue of resveratrol, are the most toxic stilbenes in terms of mobility and disease development of the Plasmopara viticola oomycete (Pezet et al. 2004a,b, Schmidlin et al. 2008). Different analytical methods have been reported for analysis of viniferins, the most frequently used being the high presure liquid chromatography (HPLC) separation system, coupled with different types of detectors such as diode array detector (DAD) (Jeandet et al. 1997, Vitrac et al. 2005), fluorescence (Jeandet et al. 1997) and more recently mass spectrometers (Pezet et al. 2003, Jean-Denis et al. 2006, Jerkovic et al. 2007, Godard et al. Vrhovsek et al. Stilbenoid viniferins in infected grapevine leaves 11 doi: 10.1111/j.1755-0238.2011.00163.x © 2011 Australian Society of Viticulture and Oenology Inc.