Exploring the Molecular Basis of Microbial Wine-Terroir From Deep Soil Horizons to Grapevines and Wines Alex Gobbi Phd Thesis by Alex Gobbi Department of Environmental Science (ENVS), Aarhus University Environmental Microbiology and Biotechnology (EMBI) Environmental Microbial Genomics (EMG) Supervisor: Prof. Lars Hestbjerg Hansen Co-Supervisor: Dr. Lea Ellegaard-Jensen “Getting a PhD is like performing a guitar solo in front of an audience, learning how to play the instrument while playing it” Samuel Butler & Alex Gobbi “A man cannot discover new oceans unless he has the courage to lose sight of the shore.” Andre Gide Cover Designed by: Alex Gobbi February 2019 Copyrights statement: - PCoA Plot from Manuscript 1 produced by Alex Gobbi - Phylogenetic Tree from Manuscript 4 by Marie Rønne Aggerback - Topsoil image and grapevine tree pictures are freely available on internet and “no copyright violation is intended” according to the Google copyrights policy. Acknowledgements I’d like to acknowledge my supervisor Prof. Lars Hestbjerg Hansen, for three main reasons; first of all because he believed in me, at the time of my application, giving the chance of joining his group for this amazing adventure that has been MICROWINE. Second, because he supported me during my work, when necessary. Third, and most important to me; because he let me free to learn and make mistakes (only small ones ) on my own. This, needlessy to say, allowed me to understand what I like and what I don’t, as well as what I am good at and which are my lacks in this kind of job. As I mentioned, I have been part of this fantastic group called the MICROWINE Network, a project funded by Marie-Curie Foundation within the Horizon 2020 Programme. This experience, all those journeys, projected me into an astonishing Network of crazy people and experienced PIs which became, during these three years, colleagues, mentors and dear friends. I want to thank Elisa, my girlfriend, for accepting my choice regardless the sacrifices I have exposed our relationship to. My gratitude for you extends also to the working environment, since you have been, for more than a year, a tireless help in the lab and your skills have been appreciated and acknowledged by several members of MICROWINE. Voglio ringraziare i miei genitori per aver accettato la mia scelta di partire e per essermi stati vicino ogni giorno, in ogni momento, da ben prima dell’inizio di questa esperienza. Grazie. Back in Denmark, I want to thank my office-mates Alexander, Amaru, Ifigeneia and Olivia with whom I shared the coffee-free room and where every-day chats suddenly could turn into meaningful scientific or philosophical discussion. Thanks to my colleagues which always welcomed and help me if I needed; in particular a big thank goes to Lea for the co-supervision, to the IT-Crew of Tue (s), Patrick, Zohaib, Toke and Marie for the collaborations and to all the other PhD students; I wish you good luck for your work . Thanks to all the technicians, Tina, Tanja and Pia for keeping the lab clean and efficient, thanks also for the kindness of the everyday chats along the corridor. Thanks to the other PIs Witold, Niels, Anne, Carsten and Anders with whom I felt comfortable whenever I needed to ask for an advice. Last but not least, thanks to Klaus, our best secretary ever. You are the real Santa Klaus for all the newcomers and it has been a pleasure to be your office-neighbour. Finally I want to thank Alberto, Adrian, Merche, Ana, Veronica, Javier, Eneas, Nuria, Francisco, Ava, Berta, Hector, Charles, Luis and Ignacio, from Biome Makers, for welcoming and hosting me in Valladolid and California and all the wine-makers who allowed me to get samples from their precious vineyards. I want to thank the guys that run the Friday Bar in Risø every week, for the consistent supply of smiles, beers, pizzas and pool challenges. Thanks in particular to Simon, Megha and Ilaria for doing such a big part of the job. I want to thank my friends in Italy. Thanks to Stefano, Valentina, Jennyfer, Giada, Alessandro, Irene, Francesca, Giuditta, Zambo, Tale and Davide which I feel close even though they are always far. I wish I could make a pocket-size version of you all and stuff it in my luggage. More than this, I wish I could be there for you if you need it. However, although my everyday thoughts goes also to the friends I left home, I am grateful to all the new people I have met here and, despite they come from far, are closer than many. A big hug goes to Susana, Valentina, Jay, Jin, Marta, Kristian, Jibran, Monica, Ulla, Mar all the others that I must admit I have sinfully forgotten. tina, Dario, Giulia, Michał and to In the end, thanks to the Sønderlundsvej Crew, all the people that have been living with me, sharing their life into our old house for a long or short period of time. This living experience made me understand that we don’t need much more than a roof above our head to feel like home. The fact that we will never feel the same as in our country does not mean we cannot feel equally good in other ways. So thanks to Ifigeneia, Christina, Daina, Robertino and the fresh (almost) newcomers Michele and Ginesta. I wish you good luck for your studies. Meanwhile, thanks for the guitar lessons, the Netflix nights on Lars’s couch, the dinners together, to the many Gin-Tonics and to the legendary hammock in the living room. Abstract Microbes are crucial in each stage of the wine production process. The correlation between microbes and geography, incorporated in the notion of terroir in wine, can be associated to the unique impact the microbial community can have on wine taste and flavour. Applying Next Generation Sequencing technology and in particular meta- barcoding, we revealed the dynamics and the composition of different microbiomes related with the wine-milieu. During this 3-years long microbiological journey, we extended the concept of the microbial terroir on a global scale, sequencing soil-DNA from a hundred of vineyards worldwide in four continents. This biogeographical correlation exists at different scales; between different wine-regions in the same country and between fields within the same region. We revealed unique biomarkers in remote volcanic vineyards in Azores and temperature-driven microbial gradients from North Ebro Valley to Ribera Del Duero, passing from La Rioja, in Spain. We discriminated the microbial communities between closely related vineyards in Pfalz, Germany, an area famous for the production of Riesling. Further, we characterized the microbiota of Danish vineyards, young-winemaker country, within the regions of Zealand and Funen. Throughout several fruitful industrial and academic collaborations, we improved the existing DNA extraction methods for analysing the microbes in deep-soil cores taken within vineyards, by testing DNA-enhancer such as G2. Hence, with this new protocol, we characterize the vertical stratification of the microbial community associated with different types of soil in three countries (Spain, Australia and Denmark). In another collaborative work, we characterize the inner-mycobiome of grapevine trunks, looking at the impacts of plant-fungal-disease such as Esca and different fungicide treatments. At the same time, we also characterize the microbial community resident on the phyllosphere and its dependence on the grape cultivar. Furthermore, by combining NGS and qPCR in a commercial vineyard of Western Cape, South Africa, we measured the dynamics of the leaves’ microbiota during the complete growing season. Therefore, we investigated the impact of two different fungicide treatments: a traditional copper spray compared with the application of Lactobacillus plantarum MW-1 as a biocontrol agent. All of these contributions to the existing scientific literature will be valuable for microbial ecologist interested in the agricultural part of the winemaking industry. This information will also help the winemakers, raising awareness on biodiversity and the importance it can have within the modern wine industry. Resumé Mikroorganismer er af afgørende betydning i enhver fase af vinproduktionen. Sammenhængen mellem mikroorganismer og geografi, elementer som blandt andre indgår i vin-miljøets begreb terroir, kan skyldes den unikke påvirkning, som det mikrobielle samfund har på vinens overordnede smag og dens smagsnuancer. Ved anvendelse af Next Generation Sequencing (NGS) teknologi og især metabarcoding afslørede vi dynamikken og sammensætningen af forskellige mikrobiomer relateret til vin-miljøet. I løbet af denne 3-årige lange mikrobiologiske rejse udvidede vi begrebet mikrobielle terroir på globalt plan på baggrund af vores verdensomspændende sekventering af DNA fra hundredevis af vingårde jorde - fordelt på fire kontinenter. Vi fandt at disse biogeografiske korrelationer eksisterer på forskellige skalaer; mellem forskellige vinregioner i samme land og mellem marker inden for samme region. Vi afslørede unikke biomarkører i fjerntliggende vinmarker på Azorerne med vulkaniske jorde og temperaturdrevne mikrobielle gradienter fra nordlige Ebro dalen til Ribera Del Duero, der passerer fra La Rioja i Spanien. Vi sammenlignede de mikrobielle samfund mellem nært beslægtede vinmarker i Pfalz, Tyskland, et område berømt for produktion af Riesling. Endvidere har vi karakteriseret mikrobiota fra danske vinmarker inden for regionerne Sjælland og Fyn - i et relativt nystartet vin-producerende land. Gennem flere frugtbare industrielle og akademiske samarbejder forbedrede vi de eksisterende DNA-ekstraktionsmetoder til analyse af mikrober i jordkerner taget inden for vinmarker f.eks. ved at teste effekten af G2 DNA-enhancer. Med den nyudviklede protokol karakteriserede vi de mikrobielle samfund i forskellige jordlag for forskellige jordtyper i tre lande (Spanien, Australien og Danmark). I et andet samarbejdsarbejde karakteriserede vi det indre mycobiome af vinstammer, og undersøgte virkningerne af plantesygdommen Esca og effekterne af fungicidbehandlinger på denne.