Final Thesis File

Understanding the Importance of Microbial Biogeography to Australian Winemaking Di Liu ORCID iD: https://orcid.org/0000-0003-3241-1421 Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy – Agricultural Sciences Faculty of Veterinary and Agricultural Sciences The University of Melbourne August 2020 ABSTRACT Microbes are a vital part of ecosystems and play key roles in the essential processes of the functioning. In agriculture, microbial ecology has wide reaching impacts on crop growth and quality commodity production. As a high value agricultural product, wine is a useful model for elucidating the effects of microbial ecology from the vineyard to the winery. Microbial growth and metabolism is an inherent component of wine production, influencing grapevine health and productivity, conversion of sugar to ethanol during fermentation, and the flavour, aroma and quality of finished wines. Recent advances in genetic sequencing and metagenomic approaches has extended our understanding of microbial distribution patterns and established the unique biogeography model in viticulture. While the contributions of microbial biogeography to wine metabolites and regional distinctiveness (known as terroir, a well-recognised and celebrated character in wine industry), and by which mechanisms, remain tenuous. This thesis focuses on the microbial biogeography of wine, the interplay between microbial patterns and affecting factors, and how these patterns drive wine quality and styles. I begin by investigating the distribution patterns of bacteria and fungi at large scale, and their roles in shaping wine characteristics. Samples were collected from vineyard soil, grape must, and wine ferments across six geographically separated wine-producing regions in southern Australia (~ 400 km). Soil and grape must microbiota exhibited distinctive regional patterns, as well as wine aroma profiles. Associations among soil and wine microbiota, abiotic factors (weather and soil properties), and wine regionality were modelled, highlighting that fungal communities was the most important driver of wine aroma profiles. Source tracking wine-related fungi in the vineyard suggests that soil is a source reservoir of grape- and must-associated fungi which might be translocated via xylem sap. I then move on to elucidate the fungal ecology within vineyards. Fungal communities were characterised over space and time that associated with the grapevine (grapes, flowers, leaves, roots, root zone soil) during the annual growth cycle (flowering, fruit set, veraison, and harvest). Fungi were significantly influenced by the grapevine habitat and plant development stage, with little influences i from the geographic location (<5 km). The developmental stage of veraison, where grapes undergo a dramatic change in metabolism and start ripening process, saw a distinct shift in fungal communities. A core fungal microbiota of grapevines (based on abundance-occupancy models) existed over space and time which drove the seasonal community succession. Beyond coinciding with the changing plant metabolism and physiology, strong correlations with solar radiation and water status suggests that the core microbiota changes with respect to the changing environments during plant development. I further investigate fungal contributions to wine aroma profiles by quantifying multiple layers of fungi, combining metagenomics and population genetics. Fungal communities were characterised associated with Pinot Noir and Chardonnay grape must/juice and ferments coming from three wine estates (including 11 vineyards) in the Mornington Peninsula wine region. At this scale (< 12 km), fungal communities, yeast populations, and Saccharomyces cerevisiae populations differentiated between geographic origins (estate/vineyard), with influences from the grape variety. During spontaneous fermentation, growth and dominance of S. cerevisiae reshaped the fungal community and structured the biodiversity at strain level. Associations between fungal microbiota and wine metabolites highlights the primary role of S. cerevisiae in determining wine aroma profiles at sub- regional scale. Overall, this thesis provides a significant body of knowledge to the microbial ecology field. Using vineyards, grapes, and wine as a model system, these findings relate microbial biogeography, environments, and quality agricultural commodity production. It provides fundamental perspectives to conserve the biodiversity and functioning for sustainable agriculture under the changing climate. ii DECLARATION This is to certify that: i. the thesis comprises only my original work towards the Ph.D. except where indicated in the Preface, ii. due acknowledgement has been made in the text to all other material used, and iii. the thesis is fewer than 100,000 words in length, exclusive of tables, figures, bibliographies and appendices. Di Liu August 2020 iii PREFACE Dr Kate Howell, Prof Deli Chen, Dr Pangzhen Zhang, and Dr Qinglin Chen were supervisors of this PhD project, and were involved in experimental design and manuscript editing. Dr Pangzhen Zhang built connections with the wine industry. Chapter 5 was performed in collaboration with Institut National de la Recherche Agronomique (INRA), France. Dr Jean-Luc Legras performed microsatellite analysis and assisted in the interpretation of the data. Sample collection, yeast isolation, genomic DNA extraction and sequencing, wine volatile analysis, data analysis, interpretation and writing of the manuscript were my responsibility. The candidate’s principal supervisor, Dr Kate Howell has signed The University of Melbourne’s declaration for a thesis with publication form. For all published work in this thesis, the respective co- authors have signed The University of Melbourne’s co-author authorisation form to certify that the candidate’s contribution to the publication was greater than 50%. The manuscript intended to be submitted for publication is formatted according to the journal’s formatting requirements. Supplemental data included in this thesis refers to supplemental files of individual published/to be submitted articles. Addendum refers to the data and discussion that was omitted from the published article. This thesis comprises the following original manuscripts, where I was the lead author: Review article: • Liu, D., Zhang, P., Chen, D., and Howell, K.S. (2019). From the vineyard to the winery: how microbial ecology drives regional distinctiveness of wine. Frontiers in Microbiology 10, 2679. Candidate’s contribution: 80% Work description: DL did the systematic literature review and wrote the first draft of the manuscript. KH revised and added to the draft. All authors edited the manuscript. iv Research articles: • Liu D., Chen Q., Zhang P., Chen D., Howell K.S. (2020). The fungal microbiome is an important component of vineyard ecosystems and correlates with regional distinctiveness of wine. mSphere 5: e00534-20. Candidate’s contribution: 80% Work description: DL and PZ designed the experiment. DL collected samples, conducted experiments, data analysis, data interpretation, and wrote the draft. KH and QC revised and added to the draft. All authors edited the manuscript. • Liu, D. and Howell, K.S.. (2020). Community succession of the grapevine fungal microbiome in the annual growth cycle. Environmental Microbiology. Accepted Author Manuscript. doi:10.1111/1462-2920.15172 Candidate’s contribution: 80% Work description: Both authors designed the experiment and structured the article. DL collected samples, conducted experiments, data analysis, data interpretation, and wrote the draft. KH revised and added to the draft. • Liu, D., Legras, J.-L., Zhang, P., Chen, D., and Howell, K.S. Diversity and dynamics of microbes during spontaneous fermentation and its associations with unique aroma profiles in wine. International Journal of Food Microbiology. Accepted Author Manuscript. Candidate’s contribution: 70% Work description: DL and KH designed the experiment. DL collected samples, conducted experiments (except microsatellite analysis), data analysis, data interpretation, and wrote the draft. JLL performed microsatellite analysis and assisted in the interpretation of the data. KH revised and added to the draft. All authors edited the manuscript. v There are also two co-authored papers published during my PhD, which are attached in the appendices to the thesis: • Winters, M., Panayotides, D., Bayrak, M., Rémont, G., Gonzalez Viejo, C., Liu, D., Le, B., Liu, Y., Luo, J., Zhang, P., and Howell, K. (2019). Defined co‐cultures of yeast and bacteria modify the aroma, crumb and sensory properties of bread. Journal of applied microbiology 127: 778-793. Candidate’s contribution: Assistance in wine aroma determination method establishment, data analysis, data interpretation, figure 2 plotting, and draft editing. • Zhang, P., Wu, X., Needs, S., Liu, D., Fuentes, S., Howell, K., (2017). The influence of apical and basal defoliation on the canopy structure and biochemical composition of Vitis vinifera cv. Shiraz grapes and wine. Frontiers in chemistry 5: 48. Candidate’s contribution: Assistance in wine aroma determination, data analysis, data interpretation, and draft editing. vi ACKNOWLEDGEMENTS My PhD work was jointly sponsored by The University of Melbourne and Australia Grape and Wine Authority (Wine Australia). The work was performed at The University of Melbourne and supported by vignerons across Victoria. I give my sincere thanks to Wine Australia and our industry partners who allowed

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