UNS W >013910086 U N SW - 8 AUG 2008 LIBRARY Investigation of yeasts and yeast-like fungi associated with Australian wine grapes using cultural and molecular methods by Ai Lin Beh A thesis submitted as a fulfillment for the degree of Doctor of Philosophy University of New South Wales School of Chemical Sciences and Engineering Sydney, Australia 2007 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Beh First name: Ai Lin Other name/s: Abbreviation for degree as given in the University calendar: PhD School: University of New South Wales Faculty: Faculty of Engineering School of Chemical Sciences and Title: Investigation of the yeasts and yeast-like Engineering fungi associated with Australian wine grapes (Food Science and Technology) using cultural and molecular methods Abstract 350 words maximiim: (PLEASE TYPE) This thesis presents a systematic investigation of yeasts associated with wine grapes cuJtivated in several Australian vineyards during the 2001-2003 vintages. Using a combination of cultural and molecular methods, yeast populations of red (Cabernet sauvignon, Merlot, Tyrian) and white (Sauvignon blanc. Semillen) grape varieties were examined throughout grape cultivation. The yeast-like ftingus, Aiireohasidiumpiillulans, was the most prevalent species found on grapes. Various species of Cryptococcus, Rhodolorula and Sporobohmyces were frequently isolated throughout grape maturation. Ripe grapes showed an increased incidence of Hameniaspora and Melschnikowia species for the 2001-2002 season, but not for the drought affected, 2002-2003 season. Atypical, hot and dry conditions may account for this difference in yeast flora and have limited comparisons of data to determine the influences of vineyard location, grape variety and pesticide applications on the yeast ecology. More systematic and controlled studies of these variables are required. Damaged grape berries harboured higher yeast jropulations and species diversity than intact healthy berries. PCR-DGGE analysis was less sensitive than plate culture for describing the diversity of yeast species on grapes; it detected prevalent species, but subdominant populations below 10^ CFU/g were not detected. In some cases, PCR-DGGE revealed the presence of yeasts {Candida galii, C. zemphnim) not isolated by culture. Fermentative wine species (Kluyveromyce.s, Tonilaspora, Saccharomyces) were rarely isolated, and only detected by enrichment cultures. Significant morphological and genetic variability were detected among A. pullulam and other black yeasts isolates from grapes. Taxonomic characterization of 61 strains by ITS-RFLP and rDNA sequencing revealed that they belonged to several distinct species within the generic groupings of Aureohasidium, Hormonema and Kahatiella. Isolates were strong producers of extracellular enzymes and polysaccharides that could have oenological significance, and, using a plate assay, some were antagonistic towards Badllus ihuringiemis, several wine yeasts, and some spoilage and mycotoxigenic fiingi found on grapes. Growth of Saccharomyces cerevisiae was not inhibited by these organisms in grape juice. A species-specific probe was developed for the identification of the wine s{X)ilage yeast, Zygosaccharomyces hailii in a microtitre plate hybridization assay. The probe detected 10^ cells/ml in wine, reliably differentiating Z bailii from other Zygosaccharomyces and other wine-related yeasts. Declziration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing to the Registrar. Requests for a longer period of restriction may be considered in exceptional circumstances if accompanied by a letter of support from the Supervisor or Head of School. Such requests must be submitted with the thesis/dissertation. FOR OFFICE USE ONLY Date of completion of requirements for Award: h rr / , Registrar and Deputy . ^[n/^ Principal THIS SHEET IS TO BE GLUED TUOO THE INSIDE FRONT COVER OF TTH1 E THESIS N:\FLORENCE \ABSTRACT DECLARATION I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of materials which have been accepted for the award of any other degree of diploma at UNSW or any other educational institution, except where due acknowledgment is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged. Ai Lin Beh COPYRIGHT STATEMENT 'I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed Date AUTHENTICITY STATEMENT 'I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.' Signed Date ACKNOWLEDGEMENTS The following is an expression of thanks and an acknowledgement to the people and institutions who have helped me make this work possible. I would like to express my gratitude to my supervisor, Prof Graham Fleet, for his guidance on this project, and for my development as a scientist. I thank him for his support, patience and his many anecdotes that inspired and amused me throughout my studies. He has given me so many opportunities to learn and grow, and I feel very privileged to have been a part of his team. I would like to give special thanks to my co-supervisor. Dr. Gillian Heard, who offered her perceptiveness, words of wisdom and encouragement when at times, all seemed lost, but also for the cracking of the whip when needed. This study was made possible by the financial support of the Australian Grape and Wine Research Development and Corporation (GWRDC) and in part, by the Australian Department of Agriculture, Fisheries and Forestry (DAFF). I am grateful to these organizations for the opportunity to undertake in this project. The brief visit to the Rosenstiel School of Marine and Atmospheric Science (RSMAS), University of Miami, was a major highlight in my project. I would like to express my warmest thanks to Mara Diaz, Sara Cotton, Traci Kiesling and Jack Fell for sharing with me their expertise in, and enthusiasm for molecular diagnostics. I greatly appreciate all of your patient and cheerful instruction in the development of capture probes for the microplate hybridization assay. I am grateful to the yeast culture curators at the Australian Wine Research Institute (AWRI), Centraalbureau voor Schimmelcultures (CBS), Food Science Australia (FRR), National Agricultural Utilization Research (NRRL) and the ZIM Culture Collection of Industrial Microorganisms; they generously provided me with reference cultures for this work. I would also like to acknowledge the viticulturalists at Rosemount Estate and McWilliam's Wines for their kind assistance with sampling grapes for our study. To my lab mates Sungsook, Pete, Hugh, Lidia, Pat, Victoria and Michelle, I thank you for your friendship over the years. Your warmth and good humour have made the long days in the Food Micro lab filled with fun, memorable moments. I will miss your happy Ill faces and our discourses (both fruitful and frivolous) that were held over many samplings of fermented foods and beverages. Thanks are extended to the staff of Food Science and Technology for providing valuable ideas, advice and assistance during my studies. Finally, I would like to thank my family. Without their love, support and encouragement, 1 would not have been able to complete this work. I am indebted to my parents for the sacrifices