UNIVERSITÀ DEGLI STUDI DI UDINE in agreement with FONDAZIONE EDMUND MACH PhD School in Agricultural Science and Biotechnology Cycle XXX Doctoral Thesis Chemical characteristics of wine made by disease tolerant varieties PhD Candidate Supervisor Silvia Ruocco Dr. Urska Vrhovsek Co-Supervisor Prof. Doris Rauhut DEFENCE YEAR 2018 To the best gift that life gave us: to you Nonna Rosa CONTENTS Abstract 1 Aim of the PhD project 2 Chapter 1 Introduction 3 Preface to Chapter 2 17 Chapter 2 The metabolomic profile of red non-V. vinifera genotypes 19 Preface to Chapter 3 and 4 50 Chapter 3 Study of the composition of grape from disease tolerant varieties 56 Chapter 4 Investigation of volatile and non-volatile compounds of wine 79 produced by disease tolerant varieties Concluding remarks 140 Summary of PhD experiences 141 Acknowledgements 142 Abstract Vitis vinifera L. is the most widely cultivated Vitis species around the world which includes a great number of cultivars. Owing to the superior quality of their grapes, these cultivars were long considered the only suitable for the production of high quality wines. However, the lack of resistance genes to fungal diseases like powdery and downy mildew (Uncinula necator and Plasmopara viticola) makes it necessary the application of huge amounts of chemical products in vineyard. Thus, the search for alternative and more sustainable methods to control the major grapevine pathogens have increased the interest in new disease tolerant varieties. Chemical characterisation of these varieties is an important prerequisite to evaluate and promote their use on the global wine market. The aim of this project was to produce a comprehensive study of some promising new disease tolerant varieties recently introduced to the cultivation by identifying the peculiar aspects of their composition and measuring their positive and negative quality traits. A multi- targeted approach using different analytical techniques (GC-MS, UPLC-MS, NMR and FTIR analysis) was adopted to investigate the main classes of volatile and non-volatile compounds which play a key role in the organoleptic and sensory properties of wine. The findings of this study provide a clear picture of the chemical profile of wine made from a selection of mildew tolerant varieties. Knowledge gained would serve to evaluate their use for quality wine production as well as to suggest the most appropriate winemaking style, allowing the improvement of the wine quality and valorisation of the characteristics of each grapevine variety. Considering that grape quality is crucial for wine quality, the chemical composition of the grapes from mildew tolerant varieties was also investigated. Additionally, bearing in mind the role of wild Vitis genotypes as a source of genetic resistance to biotic and abiotic stresses, the metabolomic profile of red grapes from non-V. vinifera genotypes was explored. By evaluating these wild genotypes, it was possible to assess the value of this grape germplasm and the information acquired could provide wider choices to the breeders. To the best of our knowledge this survey is the most extended metabolomic profiling study on non-V. vinifera genotypes. 1 Aim of the PhD project Recently, the continuous use of plant protection products and the growing awareness of their negative consequences on environment and human health have led to search for alternative and low impact strategies to control the major grapevine pathogens. Disease tolerant varieties of Vitis vinifera, which combine high wine quality and resistance to pathogens, have the potential to reduce the application of chemical products significantly as well as to reduce the production costs. Therefore, they represent one of the most promising tools for a more sustainable viticulture. To promote their use and diffusion for wine production, it is necessary to gain more information about their qualitative traits. The main aim of the project was to characterise the chemical composition of wine produced by some promising disease tolerant grape varieties grown in Italy and Germany in different vintages. To identify and quantify the main classes of compounds involved in determining the organoleptic and sensory properties of wine, the targeted analysis was taken into account. Both volatile and non-volatile profile of the wines under study was investigated. Since grape quality is a crucial prerequisite for wine quality, the chemical composition of the grapes from mildew tolerant varieties was also determined. Considering the role of wild Vitis genotypes as a source of genetic resistance to biotic and also abiotic stresses, it was very interesting to explore the composition of their grapes. This thesis focuses on the three following topics: study of the grape metabolomic composition, in terms of polyphenols and lipids, of seven non-V. vinifera genotypes in different vintages (chapter 2); analysis of the composition of grape from a selection of some promising disease tolerant varieties cultivated in Italy and Germany (chapter 3); analysis of the volatile and non-volatile profile of wine produced by disease tolerant varieties grown in Italy and Germany in three different vintages (chapter 4). 2 CHAPTER 1 Introduction 3 General introduction Grapevine is one of the most widespread and cultivated fruit crops worldwide since ancient times. Today the world’s total area under vines accounts for about 7.5 million hectares, of which over 3.3 million hectares of vineyards are distributed in Europe (from ‘OIV Statistical Report on World Vitiviniculture’ – 2017 release). The high versatility of grapes deriving from their use in production of wine, grape juice, jam, vinegar and other products has also made grapevine one of the most economically important plant species in the world. 1.1 The Genus Vitis The grape is a member of the family Vitaceae, within the genus Vitis which involves about 60 species in total classified in two sub-genera, Euvitis and Muscadinia (Table 1). The species of these two subgenera differ in anatomic, taxonomist and cytological traits. Euvitis species possess 38 chromosomes (2n=2x=38), shredding bark, non-prominent lenticels, pyriform seeds and nodal diaphragms; Muscadinia species contain 40 chromosomes (2n=2x=40), non-shredding bark, prominent lenticels, naviform seeds and no diaphragm interrupting the pith at nodes (Reisch, Owens, & Cousins, 2012). Subgenus Euvitis comprises the vast majority of the species which are the most important in viticulture while only three species, mainly distributed in the southern United States and eastern Mexico, are included in the subgenus Muscadinia. Crosses between two subgenera are feasible even if the resulting progeny is often characterised by poor fertility probably because of an imprecise separation of chromosomes during meiosis. Within the same subgenus, species can be crossed producing a fertile progeny (Mullins, Bouquet, & Williams, 1992). Among all the species of the genus Vitis, the most renewed is the European grape Vitis vinifera which is native of the Mediterranean basin, southern and central Europe, northern Africa, and southwest and central Asia. The domestication of V. vinifera subsp. vinifera (or sativa) from its wild ancestor (V. vinifera ssp. sylvestris) probably occurred approximately 5000 years ago somewhere in Asia Minor or Armenia (Alleweldt & Pissingham, 1988). During this process grapes underwent several dramatic changes in order to increase the sugar content for better fermentation and yield, and also to have a more regular production. In particular, the modifications occurred in 4 berry and bunch size, in seed morphology and in its reproductive structure changing from dioecious wild plants to hermaphrodite ones (This, Lacombe, & Thomas, 2006). Table 1. Classification and geographical distribution of Vitis species. Order Rhamnales Family Vitaceae Genus Vitis Subgenus Euvitis Subgenus Euvitis Series Species Origin Series Species Origin I. Candicansae V. candicans North Am. (East) VIII. Flexuosae V. flexuosa Asia V. doaniana North Am. (East) V. thunbergii Asia V. longii North Am. (East) V. betulifolia Asia V. coriacea North Am. (East) V. reticulata Asia V. simpsonii North Am. (East) V. amurensis Asia V. champinii North Am. (East) V. piasekii Asia II. Labruscae V. labrusca North Am. (East) V. embergeri Asia V. coignetiae Asia V. pentagona Asia III. Caribaeae V. caribaea North Am. (South) V. chunganensis Asia V. blancoii North Am. (East) V.chingii Asia V. lanata Asia V. piloso-nerva Asia IV. Arizonae V. arizonica North Am. (West) V. balsalsaeana Asia V. californica North Am. (West) V. hancockii Asia V. girdiana North Am. (West) V. hexamera Asia V. treleasei North Am. (West) V. pedicellata Asia V. Cinereae V. cinerea North Am. (East) V. retordii Asia V. berlandieri North Am. (East) V. seguinii Asia V. baileyana North Am. (East) V. silvestrii Asia V. bourgeana North Am. (South) V. tsoii Asia VI. Aestivalae V. aestivalis North Am. (East) V. byroniifolia Asia V. lincecumii North Am. (East) IX. Spinosae V. armata Asia V. bicolor North Am. (East) V. davidii Asia V. gigas North Am. (East) V. romanetii Asia V. rufotomentosa North Am. (East) X. Ripariae V. riparia North Am. (East) V. bourquina North Am. (East) V. rupestris North Am. (East) VII. Cordifoliae V. cordifolia North Am. (East) XI. Viniferae V. vinifera Eurasia V. rubra North Am. (East) Subgenus Muscadinia V. monticola North Am. (East) V. rotundifolia North Am. (East) V. ilex North Am. (East) V. munsoniana North Am. (East) V. helleri North Am. (East) V. popenoei