______________________________________________________________ Understanding the flesh browning disorder of ‘Cripps Pink’ apples Hannah Jill James B.Sc.Hort. (Hons.) The University of Sydney A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Faculty of Agriculture, Food and Natural Resources The University of Sydney New South Wales Australia MMVII _____________________________________________________________________ Declaration of originality The text of this thesis contains no material that has been accepted as part of the requirements for any degree or diploma in any university, or any material previously published or written unless reference to this material is made. Hannah Jill James October 2007 Conference presentations James, H., G. Brown, E. Mitcham, D. Tanner, S. Tustin, I. Wilkinson, A. Zanella, and J. Jobling. 2005. Flesh browning in Pink Lady™ apples: maturity at harvest is critical but how accurately can it be measured? Proceedings of the International Symposium on Harnessing the Potential of Horticulture in the Asian-Pacific Region. Acta Horticulturae. 694, 399-403. James, H., G. Brown, E. Mitcham, D. Tanner, S. Tustin, I. Wilkinson, A. Zanella, and J. Jobling. 2005. Flesh browning in Pink Lady™ apples: research results have helped to change market specifications for blush colour which is an added bonus for growers. Proceedings of the International Conference Postharvest Unlimited Downunder 2004. Acta Horticulturae. 687, 175-180. James, H., J. Jobling, D. Tanner, S. Tustin, and I. Wilkinson. 2005. Climatic conditions during growth relate to risk of Pink Lady™ apples developing flesh browning during storage. Proceedings of the 9th International Controlled Atmosphere Research Conference. In Press. James, H., J. Jobling, and D. Tanner. 2006. Investigating structural and physiological differences between radial and diffuse types of flesh browning of Cripps Pink apples. Proceedings of the 27th International Horticultural Congress. In Press. ii _____________________________________________________________________ Acknowledgements Foremost I would like to thank Dr Jenny Jobling for her supervision, support, encouragement and inspiration. I am indebted to Jenny for the considerable time and effort that she has devoted to this project and to the revision of this thesis. Thanks are also due to Dr Stephen Morris and Dr Michael Forbes-Smith of Sydney Postharvest Laboratory and Ms Vicki Eggleston and Mr Cameron Graves of Food Science Australia for their invaluable assistance collecting, counting, moving, sorting, waxing, cutting and assessing tens of thousands of apples over the last three years. I would also like to acknowledge the scientific and technical staff in the NANO Major National Research Facility at the Electron Microscope Unit at the University of Sydney, for their guidance on specimen preparation and the use of facilities. I also acknowledge the Faculty of Agriculture, Food and Natural Resources for providing the Thomas Lawrence Pawlett postgraduate scholarship and Sydney Postharvest Laboratory for further financial assistance. This research is a part of the Horticulture Australia Ltd funded project ‘Understanding the flesh browning disorder of Pink Lady™ apples’ (Project number AP02009). iii _____________________________________________________________________ Abstract The Flesh Browning (FB) disorder of ‘Cripps Pink’ apples presents a significant threat to the established market identity of the ‘Cripps Pink’ apple in Australian and export markets. Climatic conditions during fruit growth and development predispose ‘Cripps Pink’ apples to developing the FB disorder during storage. The FB disorder can be classified into two distinct disorders based on their physiological and structural differences and by seasonal climatic conditions. The diffuse type of FB (DFB) is a chilling injury, occurring in districts or seasons accumulating less than 1100 growing degree days (GDD) above 10oC between full bloom and harvest. In these climatic conditions, ‘Cripps Pink’ apples have delayed postharvest ethylene production. Diffuse FB effects fruit cortex tissue and is characterised as cellular collapse. Storing fruit at 3oC can reduce the incidence of DFB. The radial type of FB (RFB) is primarily a senescent disorder, occurring in districts or seasons accumulating greater than 1400 GDD above 10oC between full bloom and harvest. In these climatic conditions, postharvest ethylene production is not delayed. Radial FB affects the cells adjacent to the vascular tissue of the fruit and is characterised by damaged cell walls. Storing fruit at 1oC can reduce the incidence of RFB. Harvest maturity and the level of CO in 2 the storage atmosphere are additive influences on the development of RFB. Seasons or districts accumulating more than 1700 GDD have a very low risk for developing RFB. Seasonal climatic conditions can provide a guide for predicting the risk of developing RFB and DFB during storage. iv _____________________________________________________________________ Table of Contents Declaration of originality ii Acknowledgements iii Abstract iv Table of contents v List of figures ix List of tables xiii Abbreviations xvi Chapter 1: General introduction 1.1 The 'Cripps Pink' cultivar 1 1.2 The flesh browning disorder of 'Cripps Pink' apples 2 1.3 Thesis outline 3 1.4 References 5 Chapter 2: The determination of maturity and the physiology of ripening of ‘Cripps Pink’ apples grown in contrasting climatic conditions 2.1 Abstract 7 2.2 Introduction 7 2.2.1 Fruit ripening 8 2.2.1.1 Ethylene and fruit ripening 8 2.2.2 Fruit maturity 11 2.2.2.1 Prediction of maturity 12 2.2.2.1.1 Average harvest date 12 2.2.2.1.2 Days from full bloom 13 2.2.2.2 Determination of quality and maturity 13 2.2.2.2.1 Total soluble solids 14 2.2.2.2.2 Flesh firmness 14 2.2.2.2.3 Skin colour 15 2.2.2.2.4 Starch content 15 2.2.2.2.5 Respiration rate and rate of ethylene 17 production 2.2.2.2.6 Limitations in the determination of maturity 18 2.2.2.3 Variability in maturity 18 2.2.2.3.1 Rootstock 18 2.2.2.3.2 Within tree variation 19 2.2.2.3.3 Crop load 20 2.2.2.3.4 Irrigation 20 2.2.2.3.5 Orchard location 21 2.2.2.4 Maturity and storage disorders of apples 21 2.3 Aims 21 2.4 Materials and methods 21 2.4.1 Fruit sources 21 2.4.1.1 Batlow (New South Wales) 22 2.4.1.2 The Huon Valley (Tasmania) 22 2.4.2 Fruit numbers 22 2.4.3 Maturity and quality 22 2.4.3.1 Internal ethylene concentration 22 2.4.3.2 Flesh firmness 23 2.4.3.3 Background colour 23 2.4.3.4 Starch pattern index 22 2.4.4 Ripening evaluation 23 2.4.5 Statistical analysis 24 v _____________________________________________________________________ 2.5 Results 24 2.5.1 Maturity 24 2.5.1.1 Batlow 24 2.5.1.1 The Huon Valley 26 2.5.2 Ripening 27 2.5.2.1 Respiration rate 27 2.5.2.2 Rate of ethylene production 28 2.5.2.2.1 Batlow 29 2.5.2.2.2 The Huon Valley 31 2.5.3 Greenlife 32 2.5.3.1 Batlow 34 2.5.3.2 The Huon Valley 38 2.6 Discussion 41 2.7 Summary 48 2.8 References 48 Chapter 3: The determination of the structure and physiology of flesh browning of ‘Cripps Pink’ apples and the influence of mineral nutrition on the development of the disorder 3.1 Abstract 56 3.2 Introduction 56 3.2.1 Plant cells 57 3.2.1.1 Cell wall 57 3.2.1.2 Cellular membranes 58 3.2.1.3 Mitochondria 59 3.2.1.4 Vacuole 60 3.2.2 Orchard conditions influencing apple structure 61 3.2.2.1 Mineral nutrition 61 3.2.2.1.1 Boron 62 3.2.2.1.2 Calcium 62 3.2.2.1.3 Magnesium 63 3.2.2.1.4 Phosphorus 63 3.2.2.1.5 Potassium 63 3.2.2.1.6 Other minerals and mineral interactions 64 3.2.2.2 Nutritional requirements of apples 65 3.2.2.3 Rootstock 65 3.2.2.4 Crop load 66 3.2.2.5 Climate 66 3.2.3 Impact of cell structure on storage disorders 67 3.2.3.1 Senescence 67 3.2.3.2 High CO and low O injury 67 2 2 3.2.3.3 Chilling injury 68 3.3 Aims 68 3.4 Materials and methods 68 3.4.1 Fruit sources 68 3.4.2 Crop load 69 3.4.3 Distribution of flesh browning 69 3.4.4 Scanning electron microscopy 69 3.4.5 Mineral analysis 70 3.4.6 Density 70 3.4.7 Statistical analysis 70 3.5 Results 71 3.5.1 Distribution of flesh browning 71 3.5.2 Structure of flesh browning 72 vi _____________________________________________________________________ 3.5.2.1 Radial flesh browning 72 3.5.2.2 Diffuse flesh browning 73 3.5.2.3 CO injury 74 2 3.5.3 Mineral nutrition and flesh browning 75 3.6 Discussion 78 3.7 Summary 82 3.8 References 82 Chapter 4: The influence of storage conditions on the development of radial and diffuse types of flesh browning of 'Cripps Pink' apples 4.1 Abstract 86 4.2 Introduction 87 4.2.1 Storage conditions 88 4.2.1.1 Storage temperature 88 4.2.1.2 Storage atmosphere 89 4.2.1.3 Fruit maturity 90 4.2.2 Postharvest losses 90 4.2.2.1 Injury in cool storage 90 4.2.2.1.1 Mechanism of chilling injury 91 4.2.2.1.2 Chilling injury of apples 93 4.2.2.2 Injury in controlled atmosphere storage 94 4.2.2.2.1 Mechanism of CO injury 95 2 4.2.2.2.2 CO injury of apples 96 2 4.2.2.2.3 Mechanism of O injury 98 2 4.2.2.2.4 O injury of apples 99 2 4.2.2.3 Senescence 99 4.2.2.3.1 Mechanism of senescence 99 4.2.2.3.2 Senescent disorders of apples 100 4.2.3 Postharvest treatments for the prevention physiological disorders 101 4.2.3.1 Diphenylamine 101 4.2.3.2 1-Methylcyclopropene 101 4.2.4 The interactions of storage disorders of apples 102 4.2.5 Flesh browning of 'Cripps Pink' apples 102 4.3 Aims 102 4.4 Materials and methods 103 4.4.1 Fruit sources 103 4.4.1.1 Batlow 103 4.4.1.2 Manjimup 103 4.4.1.3 Goulburn Valley 103 4.4.1.4 Huon Valley 103 4.4.2 Harvest maturity 104 4.4.3 Pre-storage wax 104 4.4.4 Storage conditions 104 4.4.4.1 Controlled atmosphere 104 4.4.4.2 Storage temperature 104 4.4.5 Maturity and Quality evaluation 105 4.4.6 Flesh browning determination 105 4.4.7 Statistical analysis 105 4.5 Results 106 4.5.1 Response of flesh browning to controlled atmosphere storage 106 4.5.1.1 Radial flesh browning 106 4.5.1.1.1 Batlow 106 4.5.1.1.2 Manjimup 108 4.5.1.1.3 Goulburn Valley 109 vii _____________________________________________________________________ 4.5.1.2 Diffuse flesh browning 111 4.5.1.2.1 Huon Valley 111 4.5.2 Response of flesh browning to storage temperature 112 4.5.2.1 Radial flesh browning 112 4.5.2.2 Diffuse flesh browning 115 4.5.3 Relationship between storage temperature and fruit quality 117 4.5.3.1 Flesh firmness 117 4.5.3.2 Skin greasiness 118 4.5.3.3 Internal ethylene concentration 119 4.5.4 Development of flesh browning over time 119 4.5.4.1 Radial flesh browning 120 4.5.4.2 Diffuse flesh browning 120 4.6 Discussion 121 4.6.1 Radial flesh browning 122 4.6.2 Diffuse flesh browning 126 4.7 Summary 129 4.8 References 129 Chapter 5: The influence of climatic conditions during fruit growth on the development of radial and diffuse types of flesh browning of ‘Cripps Pink’ apples 5.1 Abstract 135 5.2 Introduction 135 5.2.1 Apple growth and development 135 5.2.1.1 Bud induction, flowering and fruit set 136 5.2.1.2 Fruit development 137 5.2.1.2.1 Early fruit development 137 5.2.1.2.2 Late fruit development and maturation 137 5.2.2 Climatic conditions influencing apple development 138 5.2.2.1 First 50 days after full bloom 138 5.2.2.2 Last 60 days before harvest 139 5.2.3 Climatic conditions influencing storage disorders of apples 139 5.3 Aims 141 5.4 Materials and methods 141 5.4.1 Cumulative growing degree days 141 5.4.2 Density 142 5.4.3 Flesh browning determination 142 5.4.4 Statistical analysis 142 5.5 Results 143 5.5.1 Seasonal accumulation of growing degree days 143 5.5.2 Relationship between GDD>10oC in the entire season (full bloom to 145 harvest) and type of flesh browning 5.5.3 145 Relationship between specific climatic times, fruit density and the incidence of flesh browning 5.5.4 Relationship between GDD>10oC in the entire season (full bloom to 147 harvest) and the incidence of flesh browning 5.6 Discussion 150 5.6.1 Radial flesh browning 150 5.6.2 Diffuse flesh browning 152 5.6.3 Climatic data for the prediction of developing flesh browning during storage 154 5.7 Summary 155 5.8 References 156 Chapter 6: General discussion 160 viii _____________________________________________________________________ List of figures Chapter 1 Figure 1.1 The flesh browning disorder of 'Cripps Pink' apples. Left: 3 diffuse flesh browning (DFB), centre: radial flesh browning (RFB), right: CO injury. 2 Chapter 2 Figure 2.1 Respiration rate (mL.kg-1.hr-1) of 'Cripps Pink' apples grown 28 in Batlow (New South Wales) in 2004. Figure 2.2 Respiration rate (mL.kg-1.hr-1) of 'Cripps Pink' apples grown 28 in the Huon Valley (Tasmania) in 2004. Figure 2.3 Ethylene production at 20oC of 'Cripps Pink' apples grown in 29 Batlow (New South Wales) in 2004. Figure 2.4 Ethylene production at 20oC of 'Cripps Pink' apples grown in 30 Batlow (New South Wales) in 2005. Figure 2.5 Ethylene production at 20oC of 'Cripps Pink' apples grown in 30 Batlow (New South Wales) in 2006. Figure 2.6 Ethylene production at 20oC of 'Cripps Pink' apples grown in 31 the Huon Valley (Tasmania) in 2004. Figure 2.7 Ethylene production at 20oC of 'Cripps Pink' apples grown in 31 the Huon Valley (Tasmania) in 2005. Figure 2.8 Days of greenlife against starch pattern index for 'Cripps 33 Pink' apples, data from Batlow (New South Wales), 2004, 2005 and 2006 seasons and the Huon Valley (Tasmania) 2004 and 2005 seasons combined. Figure 2.9 Days of greenlife against starch pattern index for 'Cripps 34 Pink' apples, data from Batlow (New South Wales), 2004, 2005 and 2006 seasons combined and the Huon Valley (Tasmania) 2004 and 2005 seasons combined. Figure 2.10 Days of greenlife against starch pattern index for 'Cripps 37 Pink' apples, data from Batlow (New South Wales), 2004, 2005 and 2006 seasons. Figure 2.11 Days of greenlife against starch pattern index for 'Cripps 40 Pink' apples, data from the Huon Valley (Tasmania), 2004 and 2005 seasons. Figure 2.12 Relationship between fruit growth and development, 47 respiration rate and ethylene production (adapted from Little and Holmes, 2000). Inset graph of days from optimal harvest at 20oC (greenlife) indicating hypothesised patterns of synchronised and unsynchronised ripening based on data collected from fruit grown in Batlow (New South Wales) and the Huon Valley (Tasmania). ix _____________________________________________________________________ Chapter 3 Figure 3.1 Area of tissue affected by radial flesh browning and diffuse 72 flesh browning at the stem, middle and calyx positions of the 'Cripps Pink' apples. Figure 3.2 Scanning electron microscope (SEM) micrographs showing 73 ultrastructure of apple vascular tissue (left) and cortex tissue (right) of 'Cripps Pink' apple grown in Batlow (New South Wales) following 9 months or air storage at 0oC. Fruit had no visual symptoms of radial flesh browning (RFB). Figure 3.3 Scanning electron microscope (SEM) micrographs showing 73 ultrastructure of apple vascular tissue (left) and cortex tissue (right) of 'Cripps Pink' apple grown in Batlow (New South Wales) following 9 months or air storage at 0oC. Fruit had visual symptoms of radial flesh browning (RFB); sections of tissue were taken from the affected areas. Figure 3.4 Scanning electron microscope (SEM) micrographs showing 74 ultrastructure of apple vascular tissue (left) and cortex tissue (right) of 'Cripps Pink' apple grown in the Huon Valley (Tasmania) following 9 months of air storage at 0oC. Fruit had no visual symptoms of diffuse flesh browning (DFB). Figure 3.5 Scanning electron microscope (SEM) micrographs showing 74 ultrastructure of apple vascular tissue (left) and cortex tissue (right) of 'Cripps Pink' apple grown in the Huon Valley (Tasmania) following 9 months of air storage at 0oC. Fruit had visual symptoms of diffuse flesh browning (DFB); sections of tissue were taken from the affected areas. Figure 3.6 Scanning electron microscope (SEM) micrographs showing 75 ultrastructure of CO2 injury cavity formation of 'Cripps Pink' apple grown in Batlow (New South Wales) following 9 months of air storage at 0oC. Chapter 4 Figure 4.1 Effect of storage atmosphere, pre storage wax treatment and 107 fruit maturity on radial flesh browning of 'Cripps Pink' apples grown in Batlow (New South Wales) after 4 months storage at 0oC. Figure 4.2 E ffect of storage atmosphere, pre storage wax treatment and 107 fruit maturity on radial flesh browning of 'Cripps Pink' apples grown in Batlow (New South Wales) after 7 months storage at 0oC. Figure 4.3 E ffect of storage atmosphere, pre storage wax treatment and 108 fruit maturity on radial flesh browning of 'Cripps Pink' apples grown in Manjimup (Western Australia) after 4 months storage at 0oC. Figure 4.4 E ffect of storage atmosphere, pre storage wax treatment and 109 fruit maturity on radial flesh browning of 'Cripps Pink' apples grown in Manjimup (Western Australia) after 7 months storage at 0oC. Figure 4.5 Effect of storage atmosphere, pre storage wax treatment and 110 fruit maturity on radial flesh browning of 'Cripps Pink' apples grown in the Goulburn Valley (Victoria) after 4 months storage at 0oC. x