Origin and Detection of Bacterial Species Associated with Lettuce and Salad Vegetables by Peter James Ng B Sc. (Hons) Food Science and Technology (The University of New South Wales, Australia) A thesis submitted for the degree of Doctor of Philosophy in Food Science and Technology School of Chemical Sciences and Engineering The University of New South Wales 2007 ORIGINALITY STATEMENT ‘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 material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement 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.’ Signed …………………………………………….............. Date …………………………………………….............. 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 ……………………………………………........................... i ACKNOWLEDGEMENTS During the duration of this study, there were many people and associations involved who need to be acknowledged. Firstly, I would like to acknowledge my supervisor, Professor Graham Fleet, and my co- supervisor, Dr Gillian Heard, for their assistance, support and guidance during the course of this study. Secondly, the assistance of the Australian Research Council (ARC) and Harvest FreshCuts/Vegco Pty. Ltd., Wacol, Australia for funding of this research. The donation of concentrated pesticides by the manufacturers, BASF Australia Ltd., Nufarm Australia Ltd. and Syngenta Crop Protection Pty. Ltd., Australia. The technical staff of the Department of Food Science and Technology, especially Camillo Taraborrelli, for their assistance and support. I would also like to acknowledge, my fellow postgraduate research colleagues, Ai Lin, Sungsook, Hugh, Pat and Lidia for their friendship and support during the long days and nights taken to complete this research. Finally, thanks to my family, for their assistance and support during the course of this research. ii PUBLICATIONS RELATED TO THIS THESIS Ng, P.J., Fleet, G.H., Heard, G.M. 2005. Pesticides as a source of microbial contamination of salad vegetables. International Journal of Food Microbiology 101: 237-250 iii TABLE OF CONTENTS ORIGINALITY STATEMENT.........................................................................................i COPYRIGHT STATEMENT ............................................................................................i AUTHENTICITY STATEMENT .....................................................................................i ACKNOWLEDGEMENTS ..............................................................................................ii PUBLICATIONS RELATED TO THIS THESIS...........................................................iii TABLE OF CONTENTS.................................................................................................iv ABSTRACT......................................................................................................................1 CHAPTER 1: INTRODUCTION ....................................................................................3 CHAPTER 2: LITERATURE REVIEW ..........................................................................8 2.1 Australian fresh-cut, vegetable salad industry....................................................8 2.2 Production chain...............................................................................................10 2.3 Microbiology of lettuce and fresh cut salads....................................................14 2.4 Microflora of lettuce and ready-to-eat salads...................................................14 2.5 Significance of microflora of lettuce and ready-to-eat salads..........................24 2.6 Sources of microbial contamination of vegetable produce ..............................28 2.6.1 Irrigation water..........................................................................................28 2.6.2 Irrigation method.......................................................................................29 2.6.3 Soil ............................................................................................................30 2.6.4 Fertilisers and Manure ..............................................................................31 2.6.5 Pesticides...................................................................................................32 2.6.6 Pests and Farm Animals............................................................................33 2.6.7 Handling operations ..................................................................................35 2.7 Methods for studying the microbial ecology of lettuce and related produce ...37 iv 2.7.1 Culture-dependent microbiological analysis of vegetables.......................37 2.7.2 Molecular strategies for monitoring bacterial communities .....................40 2.7.3 Factors affecting the performance of molecular methods for analysis of bacteria......................................................................................................43 2.8 Antimicrobials and lettuce................................................................................45 2.8.1 Natural antimicrobials...............................................................................45 2.8.2 Methods for the extraction of antimicrobial compounds from plant materials ....................................................................................................46 CHAPTER 3: PESTICIDES AS A SOURCE OF MICROBIAL CONTAMINATION OF VEGETABLE CROPS .............................................................................................49 3.1 Introduction ......................................................................................................49 3.2 Materials and Methods .....................................................................................51 3.2.1 Pesticides...................................................................................................51 3.2.2 Microorganisms and their cultivation .......................................................51 3.2.3 Analysis of pesticide concentrates for microorganisms............................53 3.2.4 Antimicrobial properties of pesticide solutions ........................................53 3.2.5 Growth and survival of pathogenic and spoilage organisms in pesticide solutions ....................................................................................................54 3.2.6 Growth and survival of natural populations from agricultural water in pesticide solutions.....................................................................................54 3.2.7 Identification of bacterial isolates .............................................................55 3.3 Results ..............................................................................................................57 3.3.1 Microbial content and antimicrobial activity of pesticides.......................57 3.3.2 Survival and growth of microorganisms inoculated into pesticide solutions ...................................................................................................................57 v 3.3.3 Growth and survival of microorganisms in pesticides reconstituted in different sources of agricultural water ......................................................61 3.3.4 Identification of bacterial species that grow in pesticides ........................69 3.4 Discussion ........................................................................................................73 CHAPTER 4: THE BACTERIAL ECOLOGY OF LETTUCE AS DETERMINED BY CULTURAL AND PCR-DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE) ANALYSIS ......................................................................................................78 4.1 Introduction ......................................................................................................78