POSTHARVEST DISEASE CONTROL OF MELONS USING SYSTEMIC ACQUIRED RESISTANCE AND OTHER SAFE METHODS ANOWARUL ISLAM BOKSHI MScAgr The University of Sydney A thesis presented in fulfillment of the requirements for the degree of Doctor of Philosophy Faculty of Agriculture, Food and Natural Resources The University of Sydney February 2008 ABSTRACT The goal of this research was to test commercially viable alternative methods to fungicides for controlling storage diseases of melons that are safe for human health and the environment. Initially, experiments were conducted on melons to develop a protocol for optimum conditions of disease development during storage for different pathogens and for different stages of fruit maturity. For all pathogens tested, the study found that humidity greater than 90% and temperatures above 20° C support infection and rapid growth of disease. Differences in the rate of infection and extent of disease development after the inoculation of different storage pathogens was observed between rockmelon and honeydew melons, indicating differences in host pathogen interactions. Among the tested pathogens, Alternaria spp. was the least aggressive in infection and disease severity, growing considerably slower than Fusarium acuminatum which was moderately aggressive and Rhizopus spp. which was very aggressive, in comparison. Green half-slip melons showed greater resistance to pathogen attack than green full-slip fruit, while yellow full-slip melons were highly susceptible to pathogen attack. Therefore, the laboratory experiments for postharvest treatments of rockmelons were performed using green full-slip fruit challenged with F. acuminatum. Evaluation of physical and safe chemical methods of postharvest treatment to control postharvest diseases of melons showed that none of the treatments alone was as effective as the commercially available fungicide. Hot water solutions of safe compounds considerably increase their efficacy against postharvest rots, however, symptoms of phytotoxicity on the i rind after dipping made their use unacceptable. Iodine was the only safe chemical tested which did not cause any phytotoxicity on melons. When combined with hot water, iodine showed the best control of storage rots and was as good as the fungicides carbendazim or guazatine. Hot water iodine dipping of fruit also delayed ripening and fruit were firmer during storage for a longer period of time. Systemic acquired resistance (SAR) was evaluated as a method of controlling powdery mildew in glasshouse grown rockmelon seedlings by treating with the activators 2,6- dichloroisonicotinic acid (INA) or benzothiadiazole (BTH) or water. Increased resistance due to application of INA or BTH, was observed by the reduction of powdery mildew on pre-inoculated detached leaves and also on intact leaves from natural infections. Heightened resistance due to spraying with elicitors of SAR, was further evident by the increased activities of the pathogenesis related proteins (PR proteins), peroxidase and accumulation of phenolics or antifungal compounds during and after challenge inoculation. Field grown rockmelons were treated with INA or BTH or BABA ((cid:1)-aminobutyric acid) or water at various stages of plant growth and evaluated for increased resistance against pre and postharvest diseases. Both powdery mildew and downy mildew were significantly less on the SAR elicitor treated plants. Preharvest treatment with SAR elicitors also reduced storage diseases of the harvested rockmelon fruit. The reduction in postharvest disease was similar whether plants were treated once, three weeks before harvest, or given four sprays during the growing season beginning at anthesis. A further postharvest dip with 500 ppm of guazatine gave substantial reduction of storage rots of melons. Enhanced activities of chitinase and peroxidase, two major PR-proteins, compared to the control, indicated induction of defence had occurred in the foliage and fruit as a result of SAR. Over the course of four field and one glasshouse experiments slight phytotoxicity was observed in ii plants frequently sprayed with INA or BTH, but no phytotoxicity was seen after a single spray during the late stages of fruit development. The combination of SAR elicitor treatment and use of a safe postharvest dip provided substantial control of storage rots of rockmelons. The best treatment for control of storage rots involved application of BTH (50 ppm) two weeks before harvest, combined with a hot iodine dip (55° C) of fruit, achieving equivalent or better disease control than use of guazatine fungicide dip. iii DECLARATION OF ORIGINALITY The contents and subject matter of this thesis are the original work conducted by the author, except where otherwise acknowledged. None of the work has been previously submitted either in whole or in part, for a higher degree at this or any other institute. Anowarul I Bokshi iv ACKNOWLEDGEMENTS There have been many without whom I could not have finished my project. However, I wish to acknowledge a few of them for their outstanding support and assistance during my study. It is my great pleasure to thank Dr. Robyn McConchie for her enormous support with continuous advice and guidance, right from the initiation of the project and on the preparation of this thesis. I would like to specially thank to Dr Jenny Jobling for her cheerful support and encouragement during my research. My grateful thanks to Dr. Kerrie McDonald for her initiatives and effort to enrich the molecular biology laboratory with sophisticated instruments and helping enthusiastically with the biochemical assays. Also thanks to Prof. Brian Deverall for his enthusiastic advice on a key portion of this project regarding systemic induced resistance and disease reactions. I would like specially thank to Prof. Burgess Lester and his student Tran Nguyen Ha for their generous advice and diagnosis of the plant pathogens involved in the research. I express my deep gratitude to Dr Stephen Morris for his encouragement, scholastic guidance with constructive analysis and valuable suggestions given with great enthusiasm throughout the whole period of the project. To Michael Forbes Smith and Penelope Symes for their support for a comfortable working environment at Sydney Postharvest Laboratory. I would like to thank friends and colleagues at the Sydney Postharvest Laboratory and Food Science Australia, CSIRO North Ryde for their continuous support over the last four years. Also grateful thanks to Glen Foxwell for his help and assistance in conducting field experiments at Camden, Ivan Desailly and Jarka Geisler for their help in the laboratory. My thanks also to all staff and students in the Faculty of Agriculture, Food and Natural Resources and to all my friends for their help and encouragement during my study. At Griffith, Gary Amaro and his family and workers allowed me unlimited access to their farm and postharvest processing facilities, as well as helping with supplying and transporting melons to Sydney for my research. Their enthusiastic support during the field experiments definitely helped make life easier in a remote country area. v Finally, I would like to gratefully acknowledge my wife, Anjuara Begum for her continuous inspiration and also taking care of me and all the family during my study, giving me minimum burden in family management. Also grateful thanks to all of my family and friends who shared my difficulties and offered sympathy and compassion during my research. Last but not least, my grateful acknowledgement of the F.H. Loxton foundation, which provided the scholarship for my degree and the Australian Centre for International Agricultural Research (ACIAR) for funding my research project. vi CONFERENCES ATTENDED AND PUBLICATIONS FROM THIS THESIS 1. Conferences attended and presented during PhD: 28-30 September 2005: Australasian Postharvest Conference, Rotorua, New Zealand. 1-3 September 2004: International Symposium on Harnessing the Potential of Horticulture in the Asia Pacific Region, Coolum, Australia. 1-3 October 2003: Australasian Postharvest Conference, Brisbane, Australia. 29 September – 2 October 2002: Australian Society of Horticultural Science Conference, Sydney Australia. 2. Publications; independent but related Bokshi, A.I., Morris, S.C., McDonald, K., and McConchie, R.M. 2007. Environmentally Safe Control of Postharvest Diseases of Melons by Integrating Heat Treatment, Safe Chemicals and Systemic Acquired Resistance. New Zealand Journal of Crop and Horticultural Science, 35: 179–186. Bokshi, A.I., Morris, S.C., McConchie, R. and Deverall, B.J. 2006. Pre-harvest application of INA, BABA or BTH to control post-harvest storage diseases of melons by inducing systemic acquired resistance (SAR). Journal of Horticultural Science and Biotechnology, 81: 700-706. Bokshi, A.I., Morris, S.C., McDonald, K., and McConchie, R.M. 2005. Application of INA and BABA control pre and postharvest diseases of melons through induction of systemic acquired resistance. Acta Horticulturae, 694: 416-419. Proceedings of the International Symposium on Harnessing the Potential of Horticulture in the Asia-Pacific Region. Bokshi, A.I., Morris, S.C., An Li, Feng, Z., McDonald, K. and McConchie, R. 2005. Evaluation of conventional fungicides, heat treatment and safe compounds in hot solutions for the control of postharvest diseases of melons. Acta Horticulturae, 694: 411-415. Proceedings of the International Symposium on Harnessing the Potential of Horticulture in the Asia-Pacific Region. McDonald, K.L., McConchie, R.M., Bokshi, A.I. and Morris, S.C. 2004. Heat treatment: A natural way to inhibit postharvest diseases in rockmelon. Acta Horticulturae, 682: 2029- 2033. Proceedings of the V International Postharvest Symposium. vii TABLE OF CONTENTS Page ABSTRACT i DECLARATION OF ORIGINALITY iv ACKNOWLEDGEMENTS v CONFERENCES ATTENDED AND PUBLICATIONS FROM THESIS vii TABLE OF CONTENT viii LIST OF TABLES xiv LIST OF FIGURES xv LIST OF PLATES xvii LIST OF ABBREVIATIONS xx CHAPTER 1: INTRODUCTION 1 CHAPTER 2: LITERATURE REVIEW 7 2.1 The melons 7 2.2 Australian melon industry 7 2.3 Prospects of exporting Australian melons 8 2.4 Problems associated with exporting Australian melons 10 2.5 Postharvest losses of melons 11 2.5.1 Losses from diseases and storage conditions 11 2.5.2 Losses from field diseases 12 2.6 Postharvest storage diseases: facts and factors 13 2.6.1 Effect of temperature on disease development 13 2.6.2 Effect of humidity on disease development 14 2.6.3 Effect of harvest maturity on disease development 16 2.7 Current practices of postharvest treatment of melons 17 2.8 Problems with current practices of postharvest treatments 18 2.9 Alternatives to fungicides for postharvest treatment 20 2.9.1 Biological Agents 20 2.9.2 Physical Treatment (heat/ hot water treatment) 21 2.9.2.1 Postharvest disease control with hot water 22 2.9.2.2 Hot water enhances quality of fresh produce 23 viii 2.9.2.3 Mode of action of hot water treatment 25 2.9.2.4 Factors affecting the efficacy of hot water treatment 27 2.9.2.5 Prospects and problems of hot water treatment 29 2.9.3 Postharvest disease control by safe chemicals 31 2.9.3.1 Safe chemicals for postharvest dip 31 2.9.4 Combination of hot water and safe chemicals for postharvest dips 35 2.10 Plant resistance mechanisms 36 2.10.1 Inherent resistance mechanisms in the plant 36 2.10.2 Induction of resistance in the plant 39 2.10.3 Mechanisms involving SAR 44 2.11 Agents for induction of SAR 55 2.11.1 Biological agents of SAR 55 2.11.2 Chemical elicitors of SAR 58 2.11.3 Potentials and problems of SAR by chemicals 67 CHAPTER 3: ENVIRONMENT AND BIOLOGY FOR DISEASE 70 DEVELOPMENT OF MELONS 3.1 INTRODUCTION 70 3.2 MATERIALS AND METHODS 72 3.2.1 Source of fruit 73 3.2.2 Collection and preservation of pathogenic fungal strains 73 3.2.3 Culture and preparation of inoculum 73 3.2.4 Temperature and humidity effect on storage rots 74 3.2.5 Inoculation and incubation 76 3.2.6 Rot assessment and weight loss 76 3.2.7 Effect of fruit storage on rot development 76 3.2.8 Effect of harvest maturity on storage rots 78 3.2.9 Statistical analyses 79 3.3 RESULTS 79 3.3.1 Temperature and humidity effect on storage rots 79 3.3.2 Storage rots on melon cultivars at different conditions 87 3.3.3 Effect of maturity of rockmelon on storage rots 88 3.4 DISCUSSION 90 3.5 SUMMARY 93 ix