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Heat Treatments for Controlling Postharvest Diseases and Chilling Injury in Florida Citrus

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HEAT TREATMENTS FOR CONTROLLI NG POSTHARVEST DISEASES AND CHILLING INJURY IN FLORIDA CITRUS By KARTHIK-JOSEPH JOHN-KARUPPIAH A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Karthik-Joseph John-Karuppiah

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This document is dedicated to my parents Dr. J. John Karuppiah and Mrs. J. Geetha.

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ACKNOWLEDGMENTS I would like to express my sincere thanks and gratitude to Dr. Mark A. Ritenour, my committee chair, for giving me the opportunity to work on postharvest physiology and for his valuable advice and guidance throughout my master's program. I thank my committee member Dr. Jeffrey K. Brecht who initially taught me to organize and conduct experiments. I would like to extend my thanks and gratitude to Dr. T. Gregory McCollum, my committee member, for teaching me numerous laboratory techniques and for helping me to statistically analyze my results. I would like to thank Kim Cordasco and Michael S. Burton for their valuable help in conducting my experiments. I thank my parents Dr. J. John Karuppiah and Mrs. J. Geetha, my sister R. Denniz Arthi and her family for their love and encouragement during my whole life. I thank my friends S.K. Ashok Kumar, A. Nithya Devi, M. Janakiraman, R. Rajesh and other friends in India and in USA for their friendship and support. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................xi ABSTRACT.....................................................................................................................xiii CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW....................................................1 Curing...........................................................................................................................3 Vapor Heat....................................................................................................................4 Hot Water Dipping.......................................................................................................4 Hot Water Brushing......................................................................................................7 Commodity Responses to Heat Treatments..................................................................8 Scald......................................................................................................................8 Electrolyte Leakage...............................................................................................9 Respiration and Juice Quality................................................................................9 Peel Color............................................................................................................10 Flesh and Peel Firmness......................................................................................11 Structural Changes of Epicuticular Wax.............................................................11 Host-Pathogen Interaction..........................................................................................12 Stem-End Rot......................................................................................................13 Anthracnose.........................................................................................................14 Green Mold..........................................................................................................14 Minor Postharvest Diseases.................................................................................15 Physiological Disorders.......................................................................................15 Objectives...................................................................................................................17 2 EFFECTS OF ETHYLENE TREATMENT ON RUBY RED GRAPEFRUIT QUALITY AFTER SHORT-DURATION HOT WATER DIP TREATMENTS.....18 Introduction.................................................................................................................18 Materials and Methods...............................................................................................18 Fruit.....................................................................................................................18 Heat Treatment and Degreening..........................................................................18 v

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Respiration Rate and Ethylene Production..........................................................19 Peel Scalding.......................................................................................................20 Total Soluble Solids and Titratable Acidity........................................................20 Statistical Analysis..............................................................................................20 Results and Discussion...............................................................................................21 3 PHYSIOLOGICAL RESPONSES OF VALENCIA ORANGES TO SHORT-DURATION HOT WATER DIP TREATMENT......................................................26 Introduction.................................................................................................................26 Materials and Methods...............................................................................................26 Fruit.....................................................................................................................26 Experiment 1.......................................................................................................26 Experiment 2.......................................................................................................27 Experiment 3.......................................................................................................27 Experiment 4.......................................................................................................27 Peel Scalding.......................................................................................................28 Peel Color............................................................................................................28 Electrolyte Leakage.............................................................................................28 Preparation of Flavedo Samples for Protein, Phenolic and Peroxidase Assays..29 Lowry Assay for Protein.....................................................................................30 Estimation of Total Phenolics.............................................................................30 Peroxidase Assay.................................................................................................31 Statistical Analysis..............................................................................................31 Results and Discussion...............................................................................................31 Experiment 1.......................................................................................................31 Experiment 2.......................................................................................................33 Experiment 3.......................................................................................................36 Experiment 4.......................................................................................................40 4 CONTROL OF POSTHARVEST DISEASES IN RUBY RED GRAPEFRUIT BY HOT WATER DIP TREATMENT......................................................................44 Introduction.................................................................................................................44 Materials and Methods...............................................................................................44 Fruit.....................................................................................................................44 Experiment 1.......................................................................................................45 Experiment 2.......................................................................................................45 Experiment 3.......................................................................................................46 Peel Scalding.......................................................................................................47 Residue Analysis.................................................................................................47 Weight Loss.........................................................................................................47 Peel Color............................................................................................................48 Total Soluble Solids and Titratable Acidity........................................................48 Percent Juice........................................................................................................48 Peel Puncture Resistance.....................................................................................48 Statistical Analysis..............................................................................................48 vi

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Results and Discussion...............................................................................................49 Experiment 1.......................................................................................................49 Experiment 2.......................................................................................................52 Experiment 3.......................................................................................................54 5 CONTROL OF CHILLING INJURY IN RUBY RED GRAPEFRUIT BY HOT WATER DIP TREATMENT.....................................................................................57 Introduction.................................................................................................................57 Materials and Methods...............................................................................................57 Fruit.....................................................................................................................57 Heat Treatment....................................................................................................58 Chilling Injury.....................................................................................................58 Peel Scalding.......................................................................................................58 Weight Loss.........................................................................................................58 Peel Color............................................................................................................59 Total Soluble Solids and Titratable Acidity........................................................59 Peel Puncture Resistance.....................................................................................59 Percent Juice........................................................................................................59 Statistical Analysis..............................................................................................59 Results and Discussion...............................................................................................59 6 CONCLUSIONS........................................................................................................63 APPENDIX A ANALYSIS OF VARIANCE FOR CHAPTER 2......................................................66 B ANALYSIS OF VARIANCE FOR CHAPTER 3......................................................67 Experiment 1...............................................................................................................67 Experiment 2...............................................................................................................68 Experiment 3...............................................................................................................69 Experiment 4...............................................................................................................70 C ANALYSIS OF VARIANCE FOR CHAPTER 4......................................................72 Experiment 1...............................................................................................................72 Experiment 2...............................................................................................................73 Experiment 3...............................................................................................................74 D ANALYSIS OF VARIANCE FOR CHAPTER 5......................................................75 LIST OF REFERENCES...................................................................................................77 BIOGRAPHICAL SKETCH.............................................................................................85 vii

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LIST OF TABLES Table page 3-1 Peel scalding of Valencia oranges on the heat-treated side of the fruit after 12 d at 10 C.............................................................................................................32 3-2 Peel hue, chroma and L* values of Valencia oranges after 14 d of storage at 10 C.....................................................................................................................36 3-3 Peel hue, chroma and L* values of the heat-treated and untreated sides of Valencia oranges after 8 d of storage at 10 C......................................................39 3-4 Total phenolics from flavedo of Valencia oranges immediately after hot water dip and after 2, 4, and 7 d of storage at 10 C..........................................................42 3-5 Total protein content from flavedo of Valencia oranges immediately after hot water dip and after 2, 4, and 7 d of storage at 10 C................................................43 4-1 Peel scalding (percent of fruit scalded) of Ruby Red grapefruit after 30 s HW dip treatments followed by post-dip treatments. Fruit were evaluated after 4 weeks of storage at 10 C......................................................................................50 4-2 Peel scalding incidence (percent of fruit scalded) of Ruby Red grapefruit after 30 s HW dip treatments followed by post-dip treatments. Fruit were evaluated after 4 weeks of storage at 16 C..............................................................................53 4-3 Peel scalding severity (percent of fruit surface scalded) of Ruby Red grapefruit after 30 s HW dip treatments followed by post-dip treatments. Fruit were evaluated after 4 weeks of storage at 16 C.............................................................54 5-1 Peel puncture resistance in Newtons of Ruby Red grapefruit after 30 s dip treatments. Fruit were evaluated after 4 weeks of storage at 5 or 16 C..................62 5-2 Weight loss (%) from Ruby Red grapefruit after 30 s dip treatments. Fruit were evaluated after 7 weeks of storage at 5 or 16 C......................................................62 A-1 Analysis of variance for respiration rates of Ruby Red grapefruit during the first six days in storage at 20 C...............................................................................66 A-2 Analysis of variance for ethylene production of Ruby Red grapefruit during the first six days in storage at 20 C...............................................................................66 viii

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A-3 Analysis of variance for percent of fruit scalded, total soluble solids and titratable acidity of Ruby Red grapefruit after 10 d of storage at 20 C...............................66 B-1 Analysis of variance for percent of fruit scalded after 12 d of storage at 10 C, electrolyte leakage of treated and untreated sides of Valencia oranges immediately after treatment.....................................................................................67 B-2 Analysis of variance for peel scalding of Valencia oranges after 14 d of storage at 10 C.....................................................................................................................68 B-3 Analysis of variance for electrolyte leakage of treated and untreated sides of Valencia oranges immediately after treatment and after 14 d of storage at 10 C.....................................................................................................................68 B-4 Analysis of variance for peel scalding of Valencia oranges after 8 d of storage at 10 C.....................................................................................................................69 B-5 Analysis of variance for electrolyte leakage of treated and untreated sides of Valencia oranges immediately after treatment and after 8 d of storage at 10 C.........................................................................................................................69 B-6 Analysis of variance for peel scalding of Valencia oranges after 4 and 7 d of storage at 10 C........................................................................................................70 B-7 Analysis of variance for electrolyte leakage from flavedo of Valencia oranges immediately after treatment and after 2, 4 and 7 d of storage at 10 C....................70 B-8 Analysis of variance for peroxidase activity in flavedo of Valencia oranges immediately after treatment and after 2, 4 and 7 d of storage at 10 C....................70 B-9 Analysis of variance for total phenolics in flavedo of Valencia oranges immediately after treatment and after 2, 4 and 7 d of storage at 10 C....................71 B-10 Analysis of variance for total protein in flavedo of Valencia oranges immediately after treatment and after 2, 4 and 7 d of storage at 10 C....................71 C-1 Analysis of variance for peel scalding, total decay and chilling injury of Ruby Red grapefruit. Peel scalding was evaluated after 4 weeks of storage at 10 C. Total decay and chilling injury were evaluated after 12 weeks of storage at 10 C.....................................................................................................................72 C-2 Analysis of variance for quality of Ruby Red grapefruit after 4 weeks of storage at 10 C........................................................................................................72 C-3 Analysis of variance for peel scalding and total decay of Ruby Red grapefruit. Peel scalding was evaluated after 4 weeks and total decay was evaluated after 12 weeks of storage at 16 C....................................................................................73 ix

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C-4 Analysis of variance for imazalil residue of Ruby Red grapefruit immediately after treatment..........................................................................................................74 C-5 Analysis of variance for peel scalding and stem-end rot of Ruby Red grapefruit. Peel scalding was evaluated after 4 weeks and stem-end rot was evaluated after 12 weeks of storage at 16 C....................................................................................74 D-1 Analysis of variance for peel scalding, chilling injury index and total decay of Ruby Red grapefruit. Peel scalding was evaluated at 4 weeks of storage. Chilling injury and total decay were evaluated after 7 weeks of storage.................75 D-2 Analysis of variance for quality of Ruby Red grapefruit after 4 weeks of storage......................................................................................................................75 D-3 Analysis of variance for weight loss in Ruby Red grapefruit after 4 and 7 weeks of storage.......................................................................................................76 x

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LIST OF FIGURES Figure page 2-1 Rates of respiration of Ruby Red grapefruit for the first six d after 62 C hot water treatment for 60 s and stored at 20 C............................................................22 2-2 Peel scalding of Ruby Red grapefruit after 10 d of storage at 20 C. Fruit were dipped in 62 C water for 60 s..................................................................................22 2-3 Peel scalding (% of fruit scalded) of Ruby Red grapefruit due to 62 C hot water dip for 60 s after 10 d of storage at 20 C......................................................23 2-4 Total soluble solids content in Ruby Red grapefruit on the 7 th and 10 th d after 62 C hot water treatment for 60 s...........................................................................24 2-5 Titratable acidity in Ruby Red grapefruit on the 7 th and 10 th d after 62 C hot water treatment for 60 s............................................................................................25 3-1 Electrolyte leakage from flavedo of Valencia oranges immediately after 56 C hot water treatment...................................................................................................32 3-2 Peel scalding (percent of fruit scalded) of Valencia oranges on the heat-treated side of the fruit after 14 d of storage at 10 C..........................................................34 3-3 Peel scalding (percent of fruit surface scalded) of Valencia oranges on the heat-treated side of the fruit after 14 d of storage at 10 C......................................35 3-4 Electrolyte leakage from flavedo of the heat-treated sides of Valencia oranges immediately after treatment and after 14 d of storage at 10 C...............................35 3-5 Peel scalding of Valencia oranges after 8 d of storage at 10 C. Fruit were dipped in 66, 68, or 70 C water for 60 s.................................................................37 3-6 Peel scalding (percent of fruit scalded) of Valencia oranges on the heat treated side of the fruit after 8 d of storage at 10 C. Fruit were dipped in 66, 68, or 70 C water for 60 s..................................................................................................38 3-7 Peel scalding (percent of fruit surface scalded) of Valencia oranges on the heat-treated side of the fruit after 8 d of storage at 10 C........................................38 xi

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3-8 Electrolyte leakage from flavedo of the heat-treated sides of Valencia oranges immediately after treatment and after 8 d of storage at 10 C.................................39 3-9 Peel scalding (percent of fruit scalded) of Valencia oranges after heat treatment at 60 and 66 C for 60 s. Scald was evaluated after 4 and 7 d of storage at 10 C.........................................................................................................................41 3-10 Electrolyte leakage from flavedo of Valencia oranges immediately after hot water dip for 60 s and after 2, 4, and 7 d of storage at 10 C...................................41 3-11 Peroxidase activity from flavedo of Valencia oranges immediately after hot water dip for 60 s and after 2, 4, and 7 d of storage at 10 C...................................42 4-1 Total decay of Ruby Red grapefruit after 30 s HW dip treatments followed by post-dip treatments. Fruit were evaluated after 12 weeks of storage at 10 C.........51 4-2 Chilling injury of Ruby Red grapefruit after 30 s HW dip treatments followed by post-dip treatments. Fruit were evaluated after 12 weeks of storage at 10 C....51 4-3 Total decay of Ruby Red grapefruit after 30 s HW dip treatments followed by post-dip treatments. Fruit were evaluated after 12 weeks of storage at 16 C.........53 4-4 Imazalil residue in Ruby Red grapefruit immediately after the 30 s dip treatments at 25 and 56 C.......................................................................................55 4-5 Stem-end rot for different treatment temperatures after 12 weeks of storage at 16 C.........................................................................................................................56 5-1 Chilling injury in Ruby Red grapefruit after 30 s dip treatments of inner and outer canopy fruit. Fruit were evaluated after 6 weeks of storage at 5 C plus 1 week at 16 C. Chilling injury was rated from 0 (none) to 3 (severe)..................61 5-2 Total decay in Ruby Red grapefruit after 30 s dip treatments of inner and outer canopy fruit. Fruit were evaluated after 7 weeks of storage at 5 or 16 C...............61 xii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science HEAT TREATMENTS FOR CONTROLLING POSTHARVEST DISEASES AND CHILLING INJURY IN FLORIDA CITRUS By Karthik-Joseph John-Karuppiah August 2004 Chair: Mark A. Ritenour Cochair: Jeffrey K. Brecht Major Department: Horticultural Science For years, heat treatment has been tested and used on various fresh horticultural commodities as a non-chemical method of reducing postharvest diseases, insects, and physiological disorders such as chilling injury (CI). In citrus, the increasing demand for fruit with less or no synthetic fungicide residues has led to the development and increased use of hot water (HW) treatments, especially in Mediterranean climates such as those found in Israel and California. However, the efficacy of these treatments on citrus under subtropical Florida conditions is unclear. In evaluation of physiological effects of HW dip on Florida citrus, rind discs of Valencia oranges dipped in 66, 68 or 70 C water for 60 s had higher electrolyte leakage immediately after HW dip than non-treated fruit. In another experiment, HW-dipped fruit (66 C for 60 s) had higher electrolyte leakage and lower peroxidase activity than non-treated fruit. In all these treatments, 100% of fruit developed scalding. Dipping Valencia oranges in 60 C water for 60 s caused about 20% scalding, but did not xiii

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increase electrolyte leakage. Higher electrolyte leakage and lower peroxidase activity were only observed when peel injury was severe. Hence, these parameters cannot be used as early indicators of heat injury. Treating grapefruit with ethylene (2-3 ppm for 3 d) before or after HW dip at 62 C for 60 s did not affect the response of grapefruit to HW dip. Washing and coating grapefruit with shellac immediately after HW dip at 56 or 59 C reduced peel scalding by 45% or 37%, respectively, compared with fruit that were not washed and coated. Dipping grapefruit at 56 or 59 C for 30 s followed by washing and shellac coating reduced incidence of CI to 2% or 1%, respectively, compared with 50% CI for fruit dipped at 25 C followed by no post-dip treatment. In another experiment, HW dip at 56 or 59 C for 30 s developed 18% or 32%, respectively, less CI after storage at 5 C for 6 weeks plus 1 week at 16 C compared with fruit dipped at 25 C. Hot water dip was more effective in reducing CI of inner canopy fruit (32%) compared with outer canopy fruit (10%). After 12 weeks of storage, November-harvested grapefruit dipped in water at 56 or 59 C for 30 s developed 25% or 18% decay, respectively, compared with 40% decay in fruit dipped at 25 C water. In an experiment using February-harvested fruit, dipping in heated solutions of 3% or 6% sodium carbonate increased the incidence of stem-end rot by 50% compared with fruit dipped in water alone. Dipping fruit in heated solutions of 125 or 250 ppm imazalil did not have any effect in reducing decay after 12 weeks of storage. This could have been due to low incidence of decay in February-harvested fruit. Dipping grapefruit in 59 C water for 30 s induced significant peel scalding in all the experiments whereas treatment at 56 C for 30 s did not induce peel scalding. Hot water dip treatment at 56 C for 30 s followed by washing and shellac coating was effective in reducing postharvest decay and CI without causing damage to the peel. xiv

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CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW The origin of almost all citrus species was probably the southern slopes of the Himalayas in northeastern India and adjacent Burma. The origin of trifoliate orange and kumquat is eastern China. In Florida, sweet oranges could have been introduced in 1565 when the colony at St. Augustine was established. The important species of citrus are Citrus sinensis (sweet orange), C. paradisi (grapefruit), C. reticulata (mandarin), C. aurantifolia (sour lime) and C. grandis (pummelo). Citrus production in the United States is second only to Brazil. However, the U.S. ranks first worldwide in grapefruit production. Commercial citrus production in the U.S. is limited to the states of Florida, California, Texas and Arizona. Florida accounts for 74% of the total U.S. citrus production, California produces 23% and Texas and Arizona contribute the remaining 3% (NASS, 2003). Though Florida is the number one grapefruit producer in the world, less than 50% of the grapefruit produced in Florida is suitable for the fresh market primarily due to cosmetic defects (NASS, 2003). In 2002-03, 65% of fresh grapefruit was exported from the U.S., of which 36% was exported to Japan (Fla. Dept. Citrus, 2004). Because of the increased attention on the importance of fresh fruits and vegetables as part of good human nutrition, consumption of fresh fruits and vegetables continues to increase. Furthermore, worldwide marketing and shipment of produce has increased the requirement for maintaining fruit and vegetable quality throughout extended shipping and storage durations. For a 50% packout of fresh Florida grapefruit, meaning that 50% of the 1

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2 fruit are suitable for fresh market, harvesting and postharvest handling accounts for 63% of the total cost of production and postharvest handling (University of Florida, 2003; University of Florida, 2004). Fresh grapefruit shipped from Florida to Japan must maintain its quality throughout the almost 30-d transit, plus an additional 1 to 3 months in storage at destination warehouses. During this time, as with most commodities, decay represents one of the greatest sources of product and economic loss. Grapefruit exported from the U.S. must meet U.S. grade standards of U.S. Fancy, U.S. #1 Bright, or U.S. #1. The tolerance level for decay for these grades is only 3% (USDA, 1997). So, reducing postharvest decay of citrus is of critical importance for maintaining Floridas competitiveness, especially in international markets. The susceptibility of fresh horticultural commodities to postharvest diseases increases during prolonged storage as a result of physiological changes in the fruits and vegetables that enable pathogens to develop (Eckert and Ogawa, 1988). Postharvest chemical treatments are very effective in controlling decay and are widely used on citrus. Recently there has been an increased demand for fresh horticultural commodities with less or no chemical residues. A number of fungicides are no longer registered for use on fresh citrus, including those that were used to effectively control postharvest diseases. There are only three fungicides (imazalil, thiabendazole and sodium o-phenylphenate) currently registered for postharvest use on citrus and there are problems like development of resistant pathogenic strains and environmental concerns in disposing the chemicals. To minimize preor postharvest treatments of fresh citrus with synthetic fungicides, research efforts are currently focused on enhancement of host resistance to pathogens through

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3 physical, chemical, or biological agents (Ben-Yehoshua et al., 1988, 1997; Wilson et al., 1994). Of the physical treatments, prestorage heat treatment appears to be a promising method of decay control for fresh horticultural commodities in general (Couey, 1989; Klein and Lurie, 1991; Schirra and Ben-Yehoshua, 1999). Many fresh horticultural products can tolerate temperatures of 50 C to 60 C for up to 10 min, but shorter exposure at this temperature can control many postharvest diseases (Barkai-Golan and Phillips, 1991). Heat treatments may be applied to fruits and vegetables via: 1) long-duration (e.g., 3-7 d) curing at warm temperatures and high relative humidity (RH) (Kim et al., 1991), 2) longor short-duration hot water (HW) dips (Schirra and Ben-Yehoshua, 1999), 3) vapor heat (Hallman et al., 1990a; Paull, 1994), 4) hot dry air (Lurie, 1998a, b; Schirra and Ben-Yehoshua, 1999) or 5) short-duration (< 1 min) HW sprays (Fallik et al., 1996). Curing Curing involves heat treatment for relatively long periods (i.e., 3-7 d). The first curing experiments on citrus fruit were conducted by Fawcett in 1922 to reduce Phytophthora citrophthora. In this case, fruit were held for 1-3 d at 30-36 C in a water-saturated atmosphere. Ben-Yehoshua et al. (1987 a, b) showed that curing of seal-packaged citrus fruit at 36 C and saturated humidity for 3 d effectively reduced decay without damage during subsequent storage at 17 C for 35 d. Curing citrus fruits increases their resistance to green mold development (Brown and Barmore, 1983; Kim et al., 1991). Postharvest curing at 34-36 C for 48-72 h effectively controls citrus decay

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4 and reduces chilling injury (CI) symptoms (Ben-Yehoshua et al., 1987b; Del Rio et al., 1992). Curing lemons for 3 d at 36 C reduced the decline of anti-fungal compounds (that inhibit germ tube elongation) in the flavedo tissue, reduced the loss of citral, and thus suppressed decay development (Ben-Yehoshua et al., 1995). In spite of its beneficial effects on reducing decay and CI in different citrus fruits, curing is not widely utilized on a commercial scale for citrus. Practical implementation of curing is difficult because of the long treatment durations, which result in fruit damage, and the high cost of heating large volumes of fruit for up to 3 d (Schirra et al., 2000). Vapor Heat At temperatures higher than what is normally used for curing, the major obstacle to widespread use of heat to reduce postharvest disease or insect infestation is the sensitivity of many fruits to the high temperatures required for effective treatment (Couey, 1989). For example, navel oranges, lemons, and avocados grown in California were easily damaged by the vapor heat treatment (Sinclair and Lindgren, 1955). Visible heat damage was reported for grapefruit exposed to forced vapor at 46 C for 3.75 h (Hallman et al., 1990b). Flavor and appearance of grapefruit air-heated at 46 C for 3 h with controlled atmosphere were inferior to those of non-heated fruit (Shellie et al., 1997). Vapor heat treatment at 59 C for 180 s resulted in severe peel scalding of grapefruit (over 77% of the fruit surface) (Ritenour et al., 2003). Hot Water Dipping Hot water dip treatments are applied for only a few seconds to minutes at temperatures higher than those used for vapor heat or hot air. Hot water dips have been

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5 used for many years as non-chemical methods to control postharvest decay in various fruits and vegetables (Barkai-Golan and Phillips, 1991; Couey, 1989; Lurie, 1998b). Fawcett (1922) conducted the first studies using HW treatments and first reported control of decay in oranges. For citrus, HW dips (2-3 min at 50-53 C) were shown to be as effective as curing for 72 h at 36 C in controlling postharvest decay and CI in various citrus fruits and are much less expensive, mainly because of shorter treatment duration (Rodov et al., 1993, 1995a). Dipping grapefruit in water at 53 C for 3 min resulted in about 50% reduction in decay (Rodov et al., 1995a). Ben-Yehoshua et al. (2000) reported that the effective temperature range for 2 min grapefruit dip treatments is between 51 and 54 C; temperatures above 54 C caused brown discoloration of the peel and temperatures below 51 C were not effective in reducing decay. Hot water dips at 52 C for 3 min have been shown to reduce green mold in organic lemon inoculated with the spores of Penicillium digitatum (Lanza et al., 2000). In Bianchetto and Verdello lemons, there was only 1% and 0% decay, respectively, in the HW dip-treated fruit, which was as effective as non-heated imazalil treatment (1g a.i./L), which had no decay. The untreated Bianchetto and Verdello fruit developed 86% and 75% decay, respectively. Most work on HW treatments of citrus has evaluated their effectiveness in reducing decay from Penicillium molds. While these represent the most important cause of citrus postharvest decay worldwide (Eckert and Brown, 1986), stem-end rot (SER; primarily from Lasiodiplodia theobromae) commonly is the primary cause of postharvest decay in Florida. Ritenour et al. (2003) reported that grapefruit dipped in water at 56 C for 120 s

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6 developed SER in only 8% of the fruit compared with 33% of the fruit with SER in fruit dipped in ambient water. However, they found very few instances where water temperatures and dipping times that reduced SER did not also injure the fruit. Fungicides in heated solutions (50-60 C) are more effective at controlling decay than non-heated solutions. Heated solutions leave higher fungicide residues on the fruit than non-heated solutions and are therefore effective even at much lower concentrations (Cabras et al., 1999). Heated solutions of thiabendazole (TBZ) and imazalil are more effective at controlling postharvest decay in citrus than non-heated solutions (McDonald et al., 1991; Schirra and Mulas, 1995a, 1995b; Wild, 1993). Heated solutions of compounds generally recognized as safe (GRAS) like sulfur dioxide, ethanol, and sodium carbonate have been found to be efficient in controlling green mold in citrus (Smilanick et al., 1995, 1997). For example, navel and Valencia oranges inoculated with Penicillium digitatum spores, and after 24 h immersed in 2%, 4% or 6% sodium carbonate solutions for 1 or 2 min at 35.0, 40.6, 43.3, or 46.1 C, had 40%-70% less green mold than in fruit dipped in water alone (Smilanick et al., 1997). Treatments of 4% or 6% sodium carbonate at 40.6 or 43.3 C provided better decay control than did 2% sodium carbonate at 35.0 or 46.1 C. In contrast to the report of Smilanick et al. (1997), Palou et al. (2001) found that temperature of the sodium carbonate solution had more effect than concentration of the sodium carbonate. Treating oranges in 3% or 4% sodium carbonate solution for 150 s at 45 C reduced the incidence of green mold and blue mold to 1% and 14%, respectively, whereas the hot water without sodium carbonate reduced the incidence of green mold and blue mold to 12% and 27%, respectively. The untreated fruit had 100% decay incidence.

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7 Palou et al. (2002) evaluated the effects of dipping Clementine mandarins in 0%, 2% or 3% sodium carbonate solutions at 20, 45 or 50 C for 60 or 150 s. Fruit were artificially inoculated with Penicillium digitatum 2 h prior to treatment. Treatment with 45 or 50 C water alone did not effectively control the decay. Adding sodium carbonate to the solution enhanced decay control compared with water alone at all temperatures and immersion periods. Dipping the fruit in 3% sodium carbonate solution at 50 C for 150 s completely controlled decay development. At lower temperatures, sodium carbonate solution was more effective than water alone and reduced the risk of peel damage by heat. Hot Water Brushing Recently, interest has been focused on short-duration HW rinsing and brushing of fresh fruits and vegetables (Fallik et al., 1996). In this method, HW is sprayed over the produce as it moves along a set of brush rollers, thus, simultaneously cleaning and disinfecting the produce (Porat et al., 2000a). Hot water brushing (10-30 s at 55-64 C; Israeli patent 116965) has been commercially used in Israel with bell peppers (Fallik et al., 1999), mangoes (Prusky et al., 1999), kumquat (Ben-Yehoshua et al., 1998), citrus (Porat et al., 2000a), and several other crops to reduce postharvest decay. Hot water brushing of grapefruit for 20 s at 56, 59 or 62 C, reduced decay by 80%, 95%, and 99%, respectively, compared with brushing in ambient water (Porat et al., 2000a). In separate experiments, Oroblanco citrus fruit (a pummelo-grapefruit hybrid) were either dipped in water for 2 min at 52 C or HW brushed (10 s at 52, 56 or 60 C) (Rodov et al., 2000). After air-drying and waxing, dipped fruit developed significantly less decay than did the non-treated fruit. Decay development was less after HW brushing

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8 for 10 s at 56 or 60 C than after HW brushing at 52 C, but was higher than HW-dipped fruit. For grapefruit, HW brushing at 62 C for 20 s significantly reduced green mold even when the fruit were inoculated with Penicillium digitatum; only 22% or 32% of the inoculated wounds were infected with green mold if inoculated 1 or 3 d after HW treatment, respectively, compared with 50% or 59% infection if inoculated on the HW treatment day or 7 d later, respectively (Pavoncello, 2001). For grapefruit brushed and rinsed for 20 s with 20, 53, 56, 59, or 62 C water and then inoculated with Penicillium digitatum 24 h later, treatments at 53 C did not significantly reduce decay, whereas treatments at 56, 59, or 62 C reduced postharvest decay by 20%, 52%, or 69%, respectively, compared with the untreated fruit (Porat et al., 2000b). Since inoculation occurred after HW brushing in these experiments, they demonstrate that HW treatment reduce decay by enhancing grapefruit resistance to the decay organisms. Commodity Responses to Heat Treatments When fruit are exposed to high temperatures, there is potential risk of injury. Symptoms of heat injury can be external (i.e., peel scalding, pitting, etc.) or internal (i.e., softening, discoloration, tissue disintegration, off-flavors, etc.) (Lurie, 1998a). For example, Miller et al. (1988) dipped grapefruit in 43.5 C water for 4 h and observed resulting peel discoloration after 3 weeks. Scald Peel scalding is a brown discoloration of the flavedo that can be caused by heat treatment. Schirra and Dhallewin (1997) dipped Fortune mandarins in 50, 54, 56, or 58 C water for 3 min and then stored at 6 C for 30 d followed by 3 d at 20 C. After

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9 storage, 10%, 70% or 100% of the fruit developed peel scalding after HW dip in 54, 56, or 58 C water, respectively. Dipping Tarocco oranges in 53 C water for 3 min caused little peel scald in fruit harvested in February and none in fruit harvested in March (Schirra et al., 1997). Fruit harvest before February developed 20-30% peel scald, whereas 70% of fruit harvested in April developed peel scald. In preliminary experiments, HW brushing at 60 C damaged Shamouti oranges. In contrast, HW brushing treatment at 56 C for 20 s is non-damaging in all tested cultivars of citrus (Porat et al., 2000a). Ritenour et al. (2003) showed a time temperature interaction for peel scalding in grapefruit. Fruit dipped in water at 56 C for 120 s, 59 C for 20-120 s or 62 C for 10-120 s developed significant peel scalding after 33 d in storage at 10 C. Hot water dips at 62 C for 60 or 120 s resulted in 100% peel scalding. Electrolyte Leakage Fortune mandarins dipped in 50, 52, 54, 56 or 58 C water for 3 min were stored at 6 C for 30 d followed by 3 d at 20 C (Schirra and Dhallewin, 1997). Immediately after treatment, there was no significant difference in electrolyte leakage, while at the end of storage period, fruit dipped in water at 56 or 58 C had greater electrolyte leakage than did fruit dipped in water at 50, 52 or 54 C. However, Schirra et al. (1997) reported no change in electrolyte leakage due to HW dips of Tarocco oranges at 53 C for 3 min. Respiration and Juice Quality Respiration is the process of breakdown of organic materials to simple end products, providing energy required for various metabolic processes and results in the loss of food reserves (Kader, 2002). Increased respiration allows enhanced metabolic

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10 rates to occur in the commodity and thus supports more rapid senescence. Temperature affects the respiration rate of fruits and vegetables by increasing the demand for energy to drive metabolic reactions. The respiration rate thus increases with increase in temperature of the product. After 33 d in storage, the respiration rate was higher in Fortune mandarins previously dipped in 56 or 58 C water (Schirra and Dhallewin, 1997). The rate of ethylene production was also higher in fruit treated at 58 C. Total soluble solids (TSS) and titratable acidity (TA), however, did not show much difference between the untreated and the heat-treated (50, 54, 56 and 58 C for 3 min) fruit. Dipping Tarocco oranges in 53 C water for 3 min did not influence the respiration rate, ethylene production, TSS and TA (Schirra et al., 1997). Hot water brushing at 56 C for 20 s did not affect juice TSS and TA in Minneola tangerines, Shamouti oranges and Star Ruby red grapefruit (Porat et al., 2000a). In general, respiration rate was higher immediately after heat treatment, but later decreased to levels like that of the non-treated fruit. Juice TSS and TA were not affected by heat treatments. Peel Color No significant difference could be found in rind color between untreated Fortune mandarin fruit and those treated in water at 50 or 54 C for 3 min (Schirra and Dhallewin, 1997). L* values were lower for mandarins treated at 58 C, a* values were lower for fruit treated at 54, 56 and 58 C, and b* values were lower for fruit treated at 56 and 58 C. Hot water brushing at 60 C for 10 s followed by waxing slowed the yellowing process in Oroblanco (Rodov et al., 2000). There was a delay of about 2 weeks in the change of rind color as compared with the non-treated fruit.

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11 Flesh and Peel Firmness Firmness of citrus fruit depends mainly on turgidity and weight loss (Rodov et al., 1997). Heat treatment causes the redistribution of natural epicuticular wax on the fruit surface and closes many microscopic cuticular cracks (Rodov et al., 1996). This could be the reason for better maintenance of firmness in heat-treated citrus fruit. However, in further experiments, maintenance of citrus fruit firmness by heat treatment was not accompanied by reduction in weight loss (Rodov et al., 2000). Heat treatment could have inhibited enzymatic action involved in softening or enhanced cell wall strengthening processes like lignification. Hot water dips for 2 min at 52 C maintained firmness by inhibiting fruit softening Oroblanco. Hot water brushing at 60 C for 10 s also maintained fruit firmness but brushing at 52 or 56 C did not. Structural Changes of Epicuticular Wax A number of deep surface cracks that form an interconnected network on peel surface are observed on the epicuticular wax of non-heated apples (Roy et al., 1999). Changes in the structure of the epicuticular wax are quite similar following different types of heat treatment (Schirra et al., 2000). After heat treatment at 38 C for 4 d, the cuticular cracks on apples disappeared, probably due to the melting of wax platelets (Roy et al., 1994). Changes in epicuticular wax structure have been noticed in several types of produce when heat-treated, such as after a 2-min water dip at 52 C of Oroblanco (Rodov et al., 1996), a 2-min water dip at 50-54 C of Fortune mandarins (Schirra and Dhallewin, 1996, 1997), and a 20 s HW brushing at 56-62 C of grapefruit (Porat et al., 2000a).

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12 Heat treated Marsh grapefruit showed that during long term storage the cuticular cracks became wider and deeper (Dhallewin and Schirra, 2000). Also during long term storage, severe alterations occurred in the outer stomatal chambers, thus becoming important invasion sites for wound pathogens (Eckert and Eaks, 1988). Host-Pathogen Interaction Inoculum levels are directly related to decay potential of a particular commodity (Trapero-Casas and Kaiser, 1992; Yao and Tuite, 1989). The effect of heat on decay-causing organisms can be influenced by factors like moisture content of spores, age of the inoculum, and inoculum concentration (Barkai-Golan and Phillips, 1991), as well as factors inherent within the host (i.e., physiological maturity and stress) (Klein and Lurie, 1991). Schirra et al. (2000) reported that heat treatments have a direct effect on fungal pathogens by slowing germ tube elongation or by inactivating or killing the germinating spores. Heat treatments may also have an indirect effect on decay development by inducing anti-fungal substances within the commodity that inhibit fungal development or by promoting the healing of wounds on the commodity (Schirra et al., 2000). Oil glands of citrus flavedo contain compounds, such as citral in lemons that have anti-fungal activity (Ben-Yehoshua et al., 1992; Kim et al., 1991; Rodov et al., 1995b). Heat treatments enhance wound healing by promoting the synthesis of lignin-like compounds and these compounds act as physical barriers to the penetration of pathogens (Schirra et al., 2000). High concentration of scoparone was found in heat-treated citrus and this could have anti-fungal properties (Kim et al., 1991). Heat treatments may also influence decay susceptibility by altering surface features of the commodity. For example, HW brushing of Minneola tangerines at 56 C for 20 s

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13 smoothed the fruit epicuticular waxes, which covered and sealed the stomata and microscopic cracks on the fruit (Porat et al., 2000a). This could reduce the entry of pathogens and thus reduce decay development. Heat treatments can also damage tissues and thus make them more susceptible to invasion by pathogens. For example, HW and vapor heat treatments used to control Caribbean fruit fly resulted in increased decay compared with the non-treated fruit (Hallman et al., 1990a; Miller et al., 1988). The long duration of treatment caused damage to the tissue and this led to increased decay (Jacobi and Wong, 1992). Water dips for 4.5 h at 43.5 C increased decay of Marsh grapefruit compared with fruit dipped in ambient water and the non-treated fruit (Miller et al., 1988). Heat-treated fruit developed 45% decay in 2 weeks whereas fruit dipped in ambient water and untreated fruit developed only 6% and 1% decay, respectively. Grapefruit dipped in 62 C water for 30 s developed only 5% SER after 82 d in storage, whereas increasing the treatment duration to 120 s caused significant peel scalding (100%) and increased incidence of SER (23%) (Ritenour et al., 2003). Stem-End Rot Stem-end rot of citrus fruits is caused by either Lasiodiplodia theobromae (previously known as Diplodia natalensis and commonly called Diplodia stem-end rot) or Phomopsis citri. The pathogen infects the calyx of immature citrus fruit and remains quiescent (Eckert and Brown, 1986). After harvest, decay develops when the fungus grows from the calyx into the fruit. The fungus does not spread from fruit to fruit in packed containers. Stem-end rot may also begin in wounds in the rind and at the stylar end of the fruit.

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14 Diplodia SER is the most serious postharvest disease in Florida citrus. The warm and humid conditions in Florida favor the development of SER. Development of Diplodia SER is hastened during degreening with ethylene and it is more serious in early season degreened fruits (Brooks, 1944; McCornack, 1972). Phomopsis SER is more prevalent later in the season when degreening is not necessary. Stem-end rots appear as a leathery, pliable area encircling the button or stem-end of citrus fruit. The affected area is buff colored to brown. Diplodia proceeds more rapidly through the core than the rind and the decay often appears at both ends of the fruit. It usually develops unevenly in the rind and forms finger-like projections. Phomopsis SER spreads through the core and in a nearly even rind pattern from the stem-end without finger-like projections. Anthracnose Anthracnose of citrus fruits is caused by Colletotrichum gloeosporioides. This fungus also infects immature fruit before harvest and remains quiescent (Eckert and Brown, 1986). Degreening with ethylene enhances anthracnose development and so it is more severe in early season fruit (Brown, 1975, 1978). The lesions initially appear silvery gray and leathery. The rind becomes brown to grayish black and softens as the rot progresses. In humid conditions, pink masses of spores may form on the lesions. Infections do not spread to adjacent healthy fruit. Green Mold Green mold of citrus fruits is caused by Penicillium digitatum. The fungus enters the fruit only through injuries in the skin (Eckert and Brown, 1986). Initially the decay appears as soft, water-soaked spots. Later the white fungal mycelium appears on the surface and produces a mass of powdery olive green spores. As the disease advances, the

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15 colored sporulating area is surrounded by a white margin. The fungus produces enzymes that break down the cell walls and macerate the tissues as the mycelium grows. Finally the decayed fruit becomes soft, shrunken, and shriveled and is entirely covered with green spores. Green mold is the major decay causing organism in citrus fruits worldwide. But in Florida this disease is less severe than SER and anthracnose. Minor Postharvest Diseases Some of the other citrus postharvest diseases that are of less importance are black rot (Alternaria citri), blue mold (Penicillium italicum) and sour rot (Galactomyces citri-aurantii). Physiological Disorders Physiological disorders are caused by nutritional imbalances, improper harvesting and handling practices or storage at undesirable temperatures (Grierson, 1986). Some of the common physiological disorders in citrus fruits are CI, stem-end rind breakdown, postharvest pitting, blossom-end clearing, and oleocellosis. Chilling injury occurs mainly in tropical and subtropical commodities when held below a critical threshold temperature, but above their freezing point (Kader, 2002). Grapefruit develops CI when stored at temperatures below 10-12 C (Chace et al., 1966). Symptoms of CI become more noticeable when the fruit are transferred to non-chilling temperatures. Grapefruit harvested early and late in the season are more susceptible to CI than are fruit harvested in mid-season (Grierson and Hatton, 1977). Fruit from the outer-canopy are more susceptible to CI than are fruit from the inner-canopy (Purvis, 1980). Common symptoms of CI are surface pitting, discoloration of the skin or water-soaked area of the rind (Grierson, 1986).

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16 Chilling injury in many horticultural commodities has been successfully reduced by high temperature prestorage conditioning or by curing treatments (Wang, 1990). Changes in respiratory rate, soluble carbohydrates, and starch content in Fortune mandarin due to conditioning (3 d at 37 C) were evaluated by Holland et al. (2002). They found that changes in carbohydrate concentration were mainly due to the consumption of carbohydrates for respiration, which was highest in the heat-treated fruit. There was no relation between CI and changes in carbohydrates. The sucrose levels in untreated fruit were 57-79% lower than the heat-treated fruit. But there were losses in glucose, fructose, and starch in heat-treated fruit. So sucrose could be involved in heat-induced chilling tolerance of citrus fruit. Rodov et al. (1995a) studied the effect of curing and HW dip treatments on CI development of grapefruit. Curing (36 C for 72 h), HW dip (53 C for 3 min) or hot imazalil dip (53 C for 3 min) each reduced CI by about 40%. When grapefruit were sealed in plastic film (D-950 film, Cryovac) after HW dip treatment, CI was reduced by about 60%. But curing and sealing the fruit did not provide better decay control than curing alone. Tarocco oranges, harvested monthly from November to April and then dipped in 53 C water for 3 min, were stored at 3 C for 10 weeks followed by 1 week at 20 C (Schirra et al., 1997). The fruit harvested between November and January were more susceptible to CI than fruit harvested in February, March or April. Heat treatment reduced the development of CI, especially in fruit that were more susceptible to CI. Tarocco oranges harvested monthly from December to April were dipped in water or TBZ solution (200 ppm) at 50 C for 3 min (Schirra et al., 1998). Dipping fruit in HW

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17 alone reduced CI only in fruit that were harvested during January or April. But, dipping the fruit in heated TBZ solution significantly reduced CI throughout the season. Valencia oranges dipped in water, benomyl (500 mg/L) or TBZ (1000 mg/L) for 2 min at ambient temperature or 53 C developed less CI after 15 weeks of storage at 1 C when dipped at 53 C (Wild and Hood, 1989). In another experiment, grapefruit dipped in water, TBZ (1000mg/L) or benomyl (500 mg/L) for 2 min at 14 C or 50 C developed significantly less CI after 8 weeks storage at 1 C when dipped at the higher temperature (Wild, 1993). Objectives Determine the effects of ethylene treatment of grapefruit on response to hot water dip treatment Determine the physiological responses of oranges to hot water dip treatment Determine if postharvest diseases in Florida grapefruit can be reduced by hot water dip treatment Determine if hot water dip treatment can reduce chilling injury in Florida grapefruit

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CHAPTER 2 EFFECTS OF ETHYLENE TREATMENT ON RUBY RED GRAPEFRUIT QUALITY AFTER SHORT-DURATION HOT WATER DIP TREATMENTS Introduction Degreening with ethylene is a common practice with early season grapefruit that enhances rind color without affecting the fruits internal quality. But ethylene treatment can have adverse effects like making the fruit susceptible to Diplodia stem-end rot (Brooks, 1944; McCornack, 1972). The objective of this experiment was to determine if ethylene treatment affects the response of grapefruit to hot water (HW) dip treatments. Materials and Methods Fruit Commercially mature (TSS:TA 7:1) and healthy Ruby Red grapefruit were harvested randomly from 1-1.5 m above ground level on healthy trees, evenly spaced around the tree and distributed through the inner and outer canopy on 22 Nov. 2002 at the Indian River Research and Education Center research grove in Fort Pierce, Fla. The trees received standard commercial care. The fruit were transported to Gainesville, Fla. on the day of harvest and stored at 10 C overnight before receiving their respective treatments the following day. Heat Treatment and Degreening The fruit were exposed to one of the following treatments. 1. Control (no HW dip or degreening) 2. Degreening only 3. Hot water dip only 18

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19 4. Hot water dip followed by degreening 5. Degreening followed by HW dip Each treatment had three replicates of 20 fruit each. Hot water dips were done at 62 C for 60 s. Hot water dip was administered using a laboratory scale fruit heating system (Model HWH, Gaffney Engineering, Gainesville, Fla.) capable of maintaining water temperatures up to 65 C, to a stability within + 2.0 C of the initial water temperature for the first 4 min after submerging up to 32 kg of fruit with an initial fruit temperature of 20 C. Following treatment, the fruit were air dried and stored at 20 C. Degreening was accomplished by treating fruit with 2-3 ppm ethylene for 3 d at 29 C and 95% relative humidity (RH). Fruit that were not degreened were held at 20 C and 95% RH while fruit from the other treatments were being degreened. Initial total soluble solids (TSS) and titratable acidity (TA) were measured from three replicates of 10 fruit each. Following treatments, fruit were stored at 20 C, with 95% RH. Rates of respiration and ethylene production were measured for 6 d. On the 7 th d of storage, half the fruit were evaluated for peel scalding, TSS and TA, and on the 10 th d, the remaining 10 fruit per replication were evaluated. Respiration Rate and Ethylene Production Rates of respiration and ethylene production were measured by the static system. Three fruit per replicate were weighed and sealed together in a 3 L container for 2 h. Gas samples (0.5 mL) were withdrawn through a rubber septum using a syringe and the percentage of carbon dioxide determined using a Gow-Mac gas chromatograph (Series 580, Bridgewater, N.J.) equipped with a thermal conductivity detector. The respiration rate was calculated using the following formula:

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20 Respiration rate (mL CO 2 kg -1 h -1 ) = % CO 2 void volume (mL) Sample weight (kg) sealed time (h) 100 Ethylene production was measured by injecting a 1 mL gas sample into a HP 5890 gas chromatograph (Hewlett Packard, Avondale, Pa.) equipped with a flame ionization detector. The rate of ethylene production was calculated using the following formula: L C 2 H 4 kg -1 h -1 = ppm C 2 H 4 void volume (mL) Sample weight (kg) sealed time (h) 1000 Peel Scalding Peel scalding was evaluated on each fruit and the percentage of fruit showing any peel scalding was calculated. Total Soluble Solids and Titratable Acidity A 13 mm thick cross sectional slice was removed from the equatorial region of each fruit and the peel removed. The samples were macerated in a blender and then centrifuged at 4,000 g n for 20 min. The supernatant solution was used for measuring TSS and TA. Total soluble solids were measured using a Mark II refractometer (Model 10480, Reichert-Jung, Depew, N.Y.) and the values expressed as Brix. Titratable acidity was measured with an automatic titrimeter (Fisher Titrimeter II, No. 9-313-10, Pittsburg, Pa.) using 6.00 g of juice that was diluted with 50 mL of distilled water and titrated with 0.1 N NaOH to an endpoint of pH 8.2. Titratable acidity was expressed as percentage citric acid. The TA was calculated as follows: TA (% citric acid) = mL NaOH Normality of NaOH 0.064 100 6 g of juice Statistical Analysis Percentage data were transformed to arcsine values and analyzed by ANOVA using SAS (PROC GLM) for PC (SAS Institute Inc, Cary, N.C.). When differences were

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21 significant (P < 0.05), individual treatment means were separated using Duncans Multiple Range Test (P = 0.05). Means presented are untransformed values. Results and Discussion Dipping grapefruit in 62 C water for 60 s before or after degreening did not result in consistent differences in the respiration rate (Figure 2-1). Respiration increased dramatically in fruit from all treatments on the 6 th d of storage, but there were no significant differences between treatments. There were no decay of fruit and hence the sudden increase can be due to mistake in calibration of the gas chromatograph. Ethylene production was low (<0.1 Lkg -1 hr -1 ) and not significantly different among treatments throughout the entire 6-d monitoring period (data not shown). Schirra and Dhallewin (1997) have shown that ethylene production from Fortune mandarins was stimulated by treatment at 58 C for 3 min. But, Schirra et al. (1997) reported that a heat treatment at 53 C for 3 min did not affect the respiration rate and ethylene production of Tarocco oranges. Peel scalding (Figire 2-2) did not develop on fruit that were not dipped in heated water, regardless if they were degreened or not (Figure 2-3). However, 50% of the fruit receiving the HW dip treatments developed peel scalding after 10 d in storage. Scalding was not significantly affected by the degreening treatments. There were no consistent differences in TSS (Figure 2-4) or TA (Figure 2-5). Porat et al. (1999) reported that ethylene degreening does not affect the juice TSS and TA of Shamouti oranges. Neither did HW dips at 53 C for 3 min have any effect on the TSS and TA of Tarocco oranges (Schirra et al., 1997). Heat treating grapefruit before or after degreening did not have any effect on the incidence of peel scalding, rates of respiration and ethylene production, or

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22 0246810121416123456Days after treatmentRespiration rate (mL CO2/kg.h) Ctrl DWHT HTWD HTBD HTAD LSD0.05 Figure 2-1. Rates of respiration of Ruby Red grapefruit for the first six d after 62 C hot water treatment for 60 s and stored at 20 C. Vertical bar represents the 5% LSD value. Ctrl Control (no HW dip or degreening), DWHT Degreening without heat treatment, HTWD Heat treatment without degreening, HTBD Heat treatment before degreening and HTAD Heat treatment after degreening. Figure 2-2. Peel scalding of Ruby Red grapefruit after 10 d of storage at 20 C. Fruit were dipped in 62 C water for 60 s.

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23 on juice quality (TSS and TA). From these results it may be concluded that degreening grapefruit with ethylene had no effect on the response to HW dip treatment. 0102030405060CtrlDWHTHTWDHTBDHTADTreatment (62 C for 60 s)Scalding (% of fruit)bbaaa Figure 2-3. Peel scalding (% of fruit scalded) of Ruby Red grapefruit due to 62 C hot water dip for 60 s after 10 d of storage at 20 C. Bars with different letters are significantly different by Duncans multiple range test at P 0.05. Ctrl Control (no HW dip or degreening), DWHT Degreening without heat treatment, HTWD Heat treatment without degreening, HTBD Heat treatment before degreening and HTAD Heat treatment after degreening.

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24 0246810127th Day10th Day Days after treatmentTotal Soluble Solids (Brix) Ctrl DWHT HTWD HTBD HTADbabbbaaaaa Figure 2-4. Total soluble solids content in Ruby Red grapefruit on the 7 th and 10 th d after 62 C hot water treatment for 60 s. Bars within each day with different letters are significantly different by Duncans multiple range test at P 0.05. Ctrl Control (no HW dip or degreening), DWHT Degreening without heat treatment, HTWD Heat treatment without degreening, HTBD Heat treatment before degreening and HTAD Heat treatment after degreening.

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25 0.00.20.40.60.81.01.21.47th Day10th Day Days after treatmentTitratable Acidity (% citric acid) Ctrl DWHT HTWD HTBD HTADabaabbababcbccab Figure 2-5. Titratable acidity in Ruby Red grapefruit on the 7 th and 10 th d after 62 C hot water treatment for 60 s. Bars within each day with different letters are significantly different by Duncans multiple range test at P 0.05. Ctrl Control (no HW dip or degreening), DWHT Degreening without heat treatment, HTWD Heat treatment without degreening, HTBD Heat treatment before degreening and HTAD Heat treatment after degreening.

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CHAPTER 3 PHYSIOLOGICAL RESPONSES OF VALENCIA ORANGES TO SHORT-DURATION HOT WATER DIP TREATMENT Introduction When fruit are dipped in hot water (HW), there is a potential risk of damaging the tissues. Tissue damage may be associated with changes in peel electrolyte leakage, peroxidase activity, total phenolics or total protein content. If so, such changes could even serve as early, quantitative measures of heat injury. The following experiments were conducted to evaluate the physiological responses of Valencia oranges to HW dips. Materials and Methods Fruit Valencia oranges were harvested at the Indian River Research and Education Center research grove in Fort Pierce, Fla. between June and August 2003. Inner-canopy fruit harvested from 1-1.5 m above ground level were used in all experiments. The fruit were harvested in the morning and were treated later the same day. Experiment 1 Harvested fruit were dipped in water at 56 C for 10, 20, 30, 60 or 120 s. Hot water dips were conducted in a temperature controlled water bath (Optima series immersion circulators, Boekel Scientific, Feasterville, Pa.). Fruit were dipped such that half the surface of each fruit along the meridian was immersed in the water. Control fruit were not dipped. Each treatment had three replicates of three fruit each. Electrolyte leakage from flavedo tissue was measured using the procedure described below on the treated and 26

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27 untreated sides of each fruit immediately after treatment. After taking discs of peel for electrolyte leakage measurement, the fruit were stored at 10 C (90% RH). This resulted in decay development on the 12 th d after treatment at which time scald was evaluated and the experiment terminated. Experiment 2 Because treatments in the previous experiment did not result in measurable differences, fruit were dipped in water at 50, 60, 70, 80 or 90 C for 60 s. Dipping was conducted as described in experiment 1. Each treatment had three replicates of three fruit each and these were duplicated so that one set of three replicates was used to measure electrolyte leakage immediately after treatment, and the other set of three replicates was stored at 10 C (90% RH). After 14 d of storage, fruit were evaluated for scald, peel color, and electrolyte leakage. Experiment 3 Based on results from the second experiment and to focus on the temperature range where changes in electrolyte leakage occurred, harvested fruit were dipped in water at 60, 62, 64, 66, 68 or 70 C for 60 s. Hot water dip was administered as described in experiment 1. Each treatment had three replicates of five fruit each and these were duplicated so that one set of three replicates was used to measure electrolyte leakage immediately after treatment, and the other set of three replicates was stored at 10 C (90% RH). After 8 d of storage, fruit were evaluated for scald, peel color, and electrolyte leakage. Experiment 4 Because peel electrolyte leakage and scalding changed significantly between 60 and 66 C, harvested fruit were dipped in water at 60 or 66 C for 60 s and peel color,

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28 total phenolics, protein content, and peroxidase activity were evaluated in addition to electrolyte leakage and peel scalding over time. Dipping was conducted as described in experiment 1 except that the whole fruit were completely submerged. Each treatment had three replicates of five fruit each and these were duplicated four times so that one set was evaluated at each of four sampling times: immediately after treatment, and after storage at 10 C (90% RH) for 2, 4, or 7 d. Peel Scalding Peel scalding was evaluated on each fruit and expressed on a 0 to 10 scale with 0 representing no scalding and 10 representing scald on 100% of the fruit surface. The percentage of fruit with any peel scalding (rated as a 1 or above) was also calculated. Peel Color Peel color was measured using a Minolta Chroma Meter (CR-300 series, Minolta Co. Ltd., Japan) at three equidistant locations on each fruit along the equator of the fruit and expressed as L*, a* and b* values. The hue and chroma values were calculated from a* and b* values using the following formula: Hue = arc tangent (b*a* -1 ) Chroma = (a* 2 + b* 2 ) 1/2 Electrolyte Leakage Electrolyte leakage of flavedo tissue was determined following the procedure of McCollum and McDonald (1991). Discs of peel tissue were taken from the equator of each fruit using a 13-mm cork borer. Two discs were taken per fruit during the first three experiments, and only one during the fourth experiment. The albedo was removed using a razor blade and the flavedo discs were incubated in 25 mL of 0.4 M mannitol solution for 4 h at room temperature with constant shaking. After 4 h, the initial conductivity was

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29 measured using a conductivity meter (D-24, Horiba Ltd., Japan). The samples were then frozen overnight. Following thawing and warming to room temperature total conductivity was measured. Electrolyte leakage was calculated using the following formula: Electrolyte leakage (%) = Initial conductivity (Sm -1 ) 100 Total conductivity (Sm -1 ) Preparation of Flavedo Samples for Protein, Phenolic and Peroxidase Assays Flavedo was peeled from one meridian half of each fruit using a fruit peeler, frozen in liquid nitrogen, crushed into small pieces with a mortar and pestle, and then freeze dried. After drying, the samples were ground into a fine powder and stored at -20 C. Acetone-washed flavedo powder was prepared by shaking 1 g of freeze dried flavedo in 25 mL of acetone for 1 h. The samples were filtered through Whatman No.1 filter paper under vacuum and washed with 25 mL acetone before being collected in beakers and dried overnight. Protein was extracted by adding 25 mL borate buffer (0.1 M, pH 8.8) to 0.3 g of the acetone-washed powder and shaking at 5 C for 1 h. Extracts were then filtered through 4-6 layers of cheesecloth and the volume brought up to 25 mL with the borate buffer before centrifugation at 25,000 g n for 10 min. Supernatants were decanted, brought to 70% saturation with solid ammonium sulfate and shaken overnight at 5 C. Samples were centrifuged at 25,000 g n for 10 min the following day and the supernatant discarded. The pellets were dissolved in 4 mL borate buffer. Desalting was done by dialysis using dialysis tubes with molecular weight cut-off = 6,000-8,000.

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30 Lowry Assay for Protein For protein measurement, standards for the Lowry assay (Lowry et al., 1951) were prepared from 0, 4, 8, 12, 16, and 20 g of bovine serum albumin. Samples were prepared by adding 190 L of water to 10 L of protein extract. Working solution of copper-tartrate-carbonate (CTC) was prepared by mixing 5 mL of CTC, 5 mL of NaOH (0.8 M), and 10 mL of water. The CTC working solution (200 L) was added to the protein solution, and the mixture was vortexed and allowed to stand for 10 min. Working solution of Folin-Ciocalteu was prepared by mixing one volume of Folin-Ciocalteu phenol reagent with five volumes of water. Then 100 L of Folin-Ciocalteu working solution was added to the protein and CTC solution, and the mixture was vortexed and allowed to stand for 30 min. Absorbance was read at 750 nm using a microplate reader (Ultramark, BioRad, Hercules, Calif.). The results were expressed as mg of protein per g dry weight of the tissue. Estimation of Total Phenolics For phenolics estimation (Swain and Hillis, 1959), methanol (10 mL) was added to 200 mg of freeze dried flavedo acetone powder and kept on a shaker overnight. The extracts were filtered through Whatman No.1 filter paper under vacuum. To 0.2 mL of filtered extract, Folin-Ciocalteu reagent (0.4 mL) and 0.5 M ethanolamine (0.9 mL) were added and the mixture vortexed. After 20 min, 200 L aliquot aliquots were applied to a microplate reader (Ultramark, Bio-Rad Laboratories, Hercules, Calif.) and absorbance was measured at 630 nm. Gallic acid solutions in methanol at 0, 50, 100, 150, and 200 ppm were used as standards. The results were expressed as mg of total phenolics per g dry weight of the tissue.

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31 Peroxidase Assay For peroxidase assay (Worthington, 1972), protein extract (100 L) was added to 1.4 mL of 0.0025 M 4-aminoantipyrine and 1.5 mL of 0.0017 M H 2 O 2 and mixed well by shaking. The absorbance was read in a UV-visible recording spectrophotometer (UV160U, Shimadzu, Japan) at 510 nm over a period of time. The spectrophotometer was set under kinetic program to have a lag time of 30 s, rate time of 180 s and interval time of 30 s. The results were expressed as peroxidase activity per minute per g dry weight of the tissue. Statistical Analysis Percentage data were transformed to arcsine values and analyzed by ANOVA using SAS (PROC GLM) for PC (SAS Institute Inc, Cary, N.C.). When differences were significant (P < 0.05), individual treatment means were separated using Duncans Multiple Range Test (P = 0.05). Means presented are untransformed values. Results and Discussion Experiment 1 Electrolyte leakage on the heat-treated sides of fruit from each treatment averaged between 33% and 42%, and was not significantly different from each other (Figure 3-1). Schirra and Dhallewin (1997) reported that HW dip at 50, 52, 54, 56 or 58 C for 3 min of Fortune mandarin did not affect the electrolyte leakage immediately after treatment. However, they observed higher electrolyte leakage after 30 d of storage in fruit dipped at 56 or 58 C. After 12 d of storage, only fruit dipped in 56 C water for 120 s developed peel scalding (Table 3-1). Of these fruit, about 67% were scalded and an average of 89% of the fruit surface was scalded, but this did not result in a significant change in

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32 electrolyte leakage. Therefore, HW dip at 56 C for 10-120 s did not result in significant changes in electrolyte leakage. Table 3-1. Peel scalding of Valencia oranges on the heat-treated side of the fruit after 12 d at 10 C. Treatment (56 C)Scalding (% of fruit surface) Scalding (% of fruit)0 s0.00 bz0.00 b10 s0.00 b0.00 b20 s0.00 b0.00 b30 s0.00 b0.00 b60 s0.00 b0.00 b120 s89.00 a66.67 aSignificance** z Values within each column followed by different letters are significantly different by Duncans multiple range test at P 0.05. Significant at P 0.05. 0102030405060700 s10 s20 s30 s60 s120 sTreatment duration (56 C)Electrolyte leakage (%) Treated side Untreated side LSD0.05 Figure 3-1. Electrolyte leakage from flavedo of Valencia oranges immediately after 56 C hot water treatment. Vertical bar represents the 5% LSD value.

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33 Experiment 2 To further evaluate the effect of HW dip on electrolyte leakage, the range of treatment temperatures was increased to between 50 and 90 C, all for 60 s. Hot water dip at 60, 70, 80 or 90 C for 60 s resulted in all fruit developing peel scalding (Figure 3-2). An average of 64% of the fruit surface was scalded in fruit dipped at 60 C, which was significantly less than the 100% of the fruit surface scalded in fruit dipped at 70, 80 or 90 C (Figure 3-3). Scalding incidence was still high (56%) on fruit dipped at 50 C for 60 s, but was significantly lower than on fruit dipped at higher temperatures (Figure 3-2); 20% of the fruit surface was scalded when dipped at 50 C (Figure 3-3). The untreated half of the fruit did not develop peel scalding in any of the treatment temperatures (data not shown). Electrolyte leakage from fruit dipped at 50 or 60 C was not significantly different from the non-treated fruit when evaluated immediately after the treatments, or after 14 d of storage (Figure 3-4). Fruit dipped in 70, 80 or 90 C water had higher electrolyte leakage (79-97%) compared with the non-treated fruit (35%), 50 C (35%), or 60 C (39%) treated fruit. Electrolyte leakage from 50 and 60 C-dipped fruit was not significantly different from the non-treated fruit, although the 60 C HW dip resulted in significant peel scalding (Figures 3-2 and Figure 3-3). Schirra and Dhallewin (1997) reported that HW dip at 54, 56 or 58 C for 3 min did not result in significant difference in electrolyte leakage immediately after treatment though the scalding incidence was 10%, 70%, or 100%, respectively, after 33 d in storage. Thus, flavedo electrolyte leakage is not an early indicator of peel scalding. Electrolyte leakage of flavedo from the untreated sides of fruit from all treatments did not result in significant differences from the non-treated fruit throughout the 14 d experiment (data not shown).

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34 020406080100120Ctrl50 C60 C70 C80 C90 CTreatment temperature (60 s)Scalding (% of fruit)aaaacb Figure 3-2. Peel scalding (percent of fruit scalded) of Valencia oranges on the heat-treated side of the fruit after 14 d of storage at 10 C. Bars with different letters are significantly different by Duncans multiple range test at P 0.05. Ctrl Control (no HW dip). Peel hue and chroma values did not show consistent treatment differences (Table 3-2). The L* values from fruit treated at 70, 80 or 90 C, which had scalding on 100% of the peel surface, were significantly lower than the non-treated fruit. Though not significantly lower, L* values also tended to decline at dipping temperatures above 50 or 60 C treatment. Schirra and Dhallewin (1997) reported that Fortune mandarins dipped at 58 C for 3 min had lower L* values after 33 d in storage. Differences in treatment duration and orange cultivar studied likely explain the slight differences in results.

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35 020406080100120Ctrl50 C60 C70 C80 C90 CTreatment temperature (60 s)Scalding (% of fruit surface)cbaaad Figure 3-3. Peel scalding (percent of fruit surface scalded) of Valencia oranges on the heat-treated side of the fruit after 14 d of storage at 10 C. Bars with different letters are significantly different by Duncans multiple range test at P 0.05. Ctrl Control (no HW dip). 0102030405060708090100Ctrl50 C60 C70 C80 C90 CTreatment temperature (60 s)Electrolyte leakage (%) Initial 14th dayLSD0.05 Figure 3-4. Electrolyte leakage from flavedo of the heat-treated sides of Valencia oranges immediately after treatment and after 14 d of storage at 10 C. Vertical bar represents the 5% LSD value. Ctrl Control (no HW dip).

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36 Table 3-2. Peel hue, chroma and L* values of Valencia oranges after 14 d of storage at 10 C. Treatment (60 s)Treated sideUntreated sideTreated sideUntreated sideTreated sideUntreated sideCtrl75.109 b cz75.10955.60755.60756.090 a56.09050 C77.630 a b77.14153.67154.70755.240 a b56.09760 C79.236 a77.60051.14654.56354.09 a b c55.78370 C73.128 c d77.23049.83757.93051.047 c d56.64480 C70.28 d71.94949.05457.49850.197 d54.79190 C73.619 c d75.29751.43858.12852.373 b c d57.596Significance*nsnsns*nsL*HueChroma z Values within each column followed by different letters are significantly different by Duncans multiple range test at P 0.05. Significant at P 0.05. ns Not significant at P 0.05. Ctrl Control (no HW dip). Experiment 3 Because flavedo electrolyte leakage increased markedly following dip treatments between 60 and 70 C, experiments were conducted further evaluating this range of temperatures. There was a distinct increase in peel scalding at treatment temperatures of 64 C and above (Figure 3-5, Figure 3-6); fruit dipped at 60 or 62 C developed no scalding, whereas all fruit dipped at 66, 68 or 70 C developed scalding after 8 d of storage. Parallel increases in the percentage of fruit surface scalded at higher dip temperatures were also observed (Figure 3-7). Electrolyte leakage immediately after the treatments also increased significantly at treatment temperatures of 64 C and above (Figure 3-8); values for fruit dipped in 66, 68, or 70 C water were 45%, 53%, and 78%, respectively, higher than non-treated fruit. After 8 d of storage, electrolyte leakage was significantly higher only in fruit dipped at 68 or 70 C, which were 23% and 36% higher, respectively, than the non-treated fruit. Electrolyte leakage on the untreated side of fruit did not vary significantly immediately after treatments or after 8 d of storage (data not shown). While scalding developed in fruit

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37 dipped at 64 C or higher, increased electrolyte leakage was only detected in tissue exposed to 66 C or higher immediately after treatment, and at 68 C or higher after 8 d storage. Thus, increased electrolyte leakage was only observed if severe scalding developed, and is not suitable as an early indicator of peel scalding. After 8 d of storage, there was no significant change in hue angle between the treated and untreated tissue (Table 3-3). However, chroma and L* values for fruit dipped at 66, 68 or 70 C were significantly lower than for fruit not dipped. These results are similar to the previous experiment where HW dips at higher temperatures resulted in lower L* values. But the chroma values in the previous experiment were not affected by HW dips. The untreated sides of the fruit did not have any significant changes in the chroma and L* values. Figure 3-5. Peel scalding of Valencia oranges after 8 d of storage at 10 C. Fruit were dipped in 66, 68, or 70 C water for 60 s.

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38 020406080100120Ctrl60 C62 C64 C66 C68 C70 CTreatment temperature (60 s)Scalding (% of fruit)cccbaaa Figure 3-6. Peel scalding (percent of fruit scalded) of Valencia oranges on the heat treated side of the fruit after 8 d of storage at 10 C. Fruit were dipped in 66, 68, or 70 C water for 60 s. Bars with different letters are significantly different by Duncans multiple range test at P 0.05. Ctrl Control (no HW dip). 020406080100120Ctrl60 C62 C64 C66 C68 C70 CTreatment temperature (60 s)Scalding (% of fruit surface)dddcbba Figure 3-7. Peel scalding (percent of fruit surface scalded) of Valencia oranges on the heat-treated side of the fruit after 8 d of storage at 10 C. Bars with different letters are significantly different by Duncans multiple range test at P 0.05. Ctrl Control (no HW dip).

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39 0102030405060708090100Ctrl60 C62 C64 C66 C68 C70 CTreatment Temperature (60 s)Electrolyte leakage (%) Initial 8th day LSD0.05 Figure 3-8. Electrolyte leakage from flavedo of the heat-treated sides of Valencia oranges immediately after treatment and after 8 d of storage at 10 C. Vertical bar represents the 5% LSD value. Ctrl Control (no HW dip). Table 3-3. Peel hue, chroma and L* values of the heat-treated and untreated sides of Valencia oranges after 8 d of storage at 10 C. Ctrl Control (no HW dip). Treatment (60 s)Treated sideUntreated sideTreated sideUntreated sideTreated sideUntreated sideCtrl75.87775.87770.252 a bz70.25263.913 a63.91360 C66.15765.50871.247 a71.91763.880 a64.80962 C77.35877.74670.859 a b69.18663.895 a63.82664 C77.57677.40766.957 b c64.88462.011 a61.53166 C75.55979.14164.957 c69.83559.047 b63.4468 C77.46578.95863.320 c68.32259.529 b62.93370 C74.34875.68663.844 c71.06658.529 b63.317Sinificancensns*ns*nsHueChromaL* z Values within each column followed by different letters are significantly different by Duncans multiple range test at P 0.05. Significant at P 0.05. ns Not significant at P 0.05.

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40 Experiment 4 In testing for other potential physiological changes in heat-treated Valencia oranges, all fruit dipped in 66 C water for 60 s developed peel scalding within 7 d, whereas only 20% of the fruit developed scalding when dipped in 60 C water (Figure 3-9). In experiment 2, all the fruit dipped at 60 C for 60 s were scalded after 14 d of storage and in experiment 3, none of the fruit dipped at 60 C for 60 s were scalded after 8 d of storage. The differences in sensitivity to heat injury could be due to the differences in the time of harvest which was between 1 st week of June and 1 st week of August. Again, only severely scalded fruit from the 66 C water treatment developed significantly higher electrolyte leakage throughout the 7 d storage period (Figure 3-10). Electrolyte leakage averaged 19% higher in 66 C-treated fruit than in untreated fruit. These results are consistent with the previous experiments. Peroxidase activity of fruit treated at 66 C was lower than the non-treated fruit and 60 C treatment (Figure 3-11). Higher electrolyte leakage and lower peroxidase activity were observed only on fruit that were dipped at temperatures of 66 C or greater, which resulted in significant peel scalding. The HW dip treatments did not significantly affect total phenolics or total protein contents in the peel (Table 3-4 and Table 3-5). Peel browning is generally caused by the oxidation of phenols mainly by the enzymes polyphenol oxidase (PPO) and peroxidase (Lattanzio et al., 1994). The total phenolics did not change with heat treatment and there was lower peroxidase activity. So the flavedo browning was likely due to oxidation by PPO. Martnez-Tellez and Lafuente (1993) have reported that chilling-induced browning had no correlation with PPO and peroxidase activities. There are also non-enzymatic browning reactions in which colored complexes are formed by the interactions between phenolics and heavy metals (Lattanzio

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41 0204060801001204 days7 daysDays after TreatmentScalding (% of fruit) Ctrl 60 C 66 Cbabbab Figure 3-9. Peel scalding (percent of fruit scalded) of Valencia oranges after heat treatment at 60 and 66 C for 60 s. Scald was evaluated after 4 and 7 d of storage at 10 C. Bars within each day with different letters are significantly different by Duncans multiple range test at P 0.05. Ctrl Control (no HW dip). 0102030405060700 day2 days4 days7 daysDays after TreatmentElectrolyte Leakage (%) Ctrl 60 C 66 Cbbbbbbbbaaaa Figure 3-10. Electrolyte leakage from flavedo of Valencia oranges immediately after hot water dip for 60 s and after 2, 4, and 7 d of storage at 10 C. Bars within each day with different letters are significantly different by Duncans multiple range test at P 0.05. Ctrl Control (no HW dip).

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42 et al., 1994). These could have also contributed to the peel browning caused by HW treatment. 05101520250 day2 days4 days7 daysDays after TreatmentPeroxidase activity (/min/g dry wt.) Ctrl 60C 66CLSD0.05 Figure 3-11. Peroxidase activity from flavedo of Valencia oranges immediately after hot water dip for 60 s and after 2, 4, and 7 d of storage at 10 C. Vertical bar represents the 5% LSD value. Ctrl Control (no HW dip). Table 3-4. Total phenolics from flavedo of Valencia oranges immediately after hot water dip and after 2, 4, and 7 d of storage at 10 C. Ctrl Control (no HW dip). Treatment (60 s)0 day2 days4 days7 daysCtrl3.1013.4993.5103.74660 C3.3364.0713.9003.33366 C3.8083.1173.3203.695SignificancensnsnsnsTotal phenolics (mg/g dry wt.) ns Not significant at P 0.05.

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43 Table 3-5. Total protein content from flavedo of Valencia oranges immediately after hot water dip and after 2, 4, and 7 d of storage at 10 C. Ctrl Control (no HW dip). Treatment (60 s)0 day2 days4 days7 daysCtrl18.96921.24125.56923.73260 C20.46819.09623.54323.89766 C18.90614.81216.45919.501SignificancensnsnsnsTotal protein (mg/g dry wt.) ns Not significant at P 0.05.

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CHAPTER 4 CONTROL OF POSTHARVEST DISEASES IN RUBY RED GRAPEFRUIT BY HOT WATER DIP TREATMENT Introduction Short duration, hot water (HW) dip treatments appear to be a promising method to control postharvest decay in citrus (Rodov et al., 1995a). Heated solutions of sodium carbonate have been more effective in controlling decay than non-heated solutions (Palou et al., 2002). The following experiments were conducted to evaluate the effectiveness of HW dip, hot sodium carbonate dip, and hot imazalil dip for decay control in Florida grapefruit. The effect of washing or coating the fruit immediately after HW dip was also studied. Materials and Methods Fruit Three experiments were conducted between November 2003 and May 2004. For the first experiment, fruit were harvested during November 2003 and for the last two experiments, fruit were harvested during February 2004 at the Indian River Research and Education Center research grove in Fort Pierce, Fla. Commercially mature (TSS:TA 7:1) and healthy Ruby Red grapefruit were harvested randomly from 1-1.5 m above ground level on healthy trees, evenly spaced around the tree and distributed through the inner and outer canopy. Harvested fruit were stored at room temperature overnight and heat treatment was done on the next day. 44

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45 Experiment 1 Harvested fruit were dipped in 25, 53, 56, 59 or 62 C water for 30 s. There were three post-dip treatments: 1. Hot water dip only 2. Hot water dip, followed immediately by a 1 min dip in water at ambient (~25 C) temperature 3. Hot water dip, followed immediately by washing and coating (simulated commercial packinghouse treatment) The HW dip treatments were conducted using stainless steel tanks holding ~ 95 L of rapidly stirred water. Heating was accomplished using a large gas burner with the temperature varying by ~ + 1 C during each treatment. Fruit were treated by placing them into perforated plastic crates that allowed water circulation past the fruit. Each treatment had four replicates of 40 fruit each. Fruit were washed over a brush bed and then coated with shellac (FMC Foodtech., Lakeland, Fla.) to simulate commercial handling. Fungicides were not used. Following treatment, the fruit were stored at 10 C (90% RH). Ten fruit from each replicate were randomly selected, marked and weighed to follow weight loss during storage. Initial analyses of peel color, total soluble solids (TSS), titratable acidity (TA), peel puncture resistance (PPR), and percent juice were done using four replications of 10 fruit each. Fruit were evaluated for peel scalding 1 and 4 weeks after treatment. After 4 weeks of storage, marked fruit were weighed, and peel color, TSS, TA, PPR, and percent juice were determined. Decay was evaluated after 4, 8, and 12 weeks in storage. Experiment 2 Harvested fruit were dipped in water at 25, 56 or 59 C for 30 s. There were three post-dip treatments:

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46 1. Hot water dip only 2. Hot water dip, followed immediately by washing (~25 C water) through a commercial packing line 3. Hot water dip, followed immediately by washing (~25 C water) and coating (simulated commercial packinghouse treatment) The HW dips were administered as described in experiment 1. Each treatment had four replicates of 40 fruit each. Fungicides were not used. Following treatment, the fruit were stored at 16 C (90% RH). Ten fruit from each replicate were randomly selected, marked, and weighed to follow weight loss measurement during storage. Fruit were evaluated for peel scalding 1, 2, and 4 weeks after treatment. After 4 weeks of storage, marked fruit were weighed. Decay was evaluated after 4, 8, and 12 weeks in storage. Experiment 3 There were two dip temperatures and six chemical treatments: 1. Water alone 2. 3% sodium carbonate solution 3. 6% sodium carbonate solution 4. 125 ppm imazalil solution 5. 250 ppm imazalil solution 6. 3% sodium carbonate + 125 ppm imazalil solution Harvested fruit were dipped at 25 or 56 C for 30 s for each of the above treatments. The HW dips were administered as described in experiment 1. Each treatment had four replicates of 40 fruit each. There were five additional fruit in each replication for the imazalil treatments, which were taken for residue analysis after coating the fruit with shellac. Fruit were washed and shellac coated immediately after HW dip treatment. Following treatment, fruit were stored at 16 C (90% RH). Ten fruit from each replicate

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47 were randomly selected, marked, and weighed to follow weight loss during storage. Initial analyses of peel color, TSS, TA, PPR and percent juice were done using four replications of 10 fruit each. Fruit were evaluated for peel scalding 1 and 4 weeks after treatment. After 4 weeks of storage, marked fruit were weighed and peel color, TSS, TA, PPR, and percent juice were determined. Decay was evaluated after 4, 8, and 12 weeks in storage. Peel Scalding Peel scalding was evaluated as described in chapter 3. Residue Analysis Residue analysis was done by Decco, Ceraxagri, Inc., Fort Pierce, Fla. Fruit were cut into quarters and one section from each fruit was used. The samples were weighed and then blended. To the blended samples, DI water (100 mL), 5N NaOH (10 mL) and NaCl (50 g) were added and the homogenate was weighed. After homogenizing for 3 min, 50 g of the sample was taken in centrifuge tubes and 10 mL of iso-octane was added. After centrifuging at 1800 g n for 10 min, 5 mL of supernatant solution was decanted into a 25 mL beaker. The samples were dried with anhydrous sodium sulfate. A 2 L sample of the extract was injected into the gas chromatograph with electron capture (EC) or nitrogen phosphorus (NP) detector. The residue level was calculated from the following formula: Imazalil (ppm) = Chromatogram reading 10 Total weight Weight of blended fruit taken Initial weight Weight Loss Fruit were weighed on the 1 st d after treatment and then again on the 4 th week after treatment. Weight loss was calculated as follows:

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48 Weight loss (%) = Initial weight (g) Final weight (g) 100 Initial weight (g) Peel Color Peel color was measured as described in chapter 3. Total Soluble Solids and Titratable Acidity The fruit were cut into halves along the equator and juice was extracted using a test juice extractor (Model 2700, Brown Citrus Systems Inc., Winter Haven, Fla.). Juice TSS (Brix) was measured using a refractometer (Abbe-3L, Spectronic Instruments Inc., Rochester, N.Y.) and the juice TA (% citric acid) was measured by titrating 40 mL of juice samples to pH 8.3 with 0.3125 N NaOH using an automatic titrimeter (DL 12, Mettler-Toledo Inc., Columbus, Ohio). Percent Juice Percent juice was calculated from the total weight of fruit and total weight of juice. Percent juice = Juice weight (g) 100 Fruit weight (g) Peel Puncture Resistance Peel puncture resistance was measured at two equidistant spots along the equator of each fruit using a texture analyzer (Model TAXT2i, Stable Micro Systems, Godalming, England) with a 2 mm diameter, flat-tipped, cylindrical probe. The analyzer was set such that the probe traveled at a speed of 2 mms -1 and the maximum force exerted to puncture the peel was recorded. Peel puncture resistance was expressed in Newtons. Statistical Analysis Percentage data were transformed to arcsine values and analyzed by ANOVA using SAS (PROC GLM) for PC (SAS Institute Inc, Cary, N.C.). When differences were

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49 significant (P < 0.05), individual treatment means were separated using Duncans Multiple Range Test (P = 0.05). Means presented are untransformed values. Results and Discussion Experiment 1 Dipping fruit in 25, 53, or 56 C water for 30 s did not cause any peel scalding after 4 weeks of storage at 10 C (Table 4-1). Fruit dipped in 59 C water for 30 s developed 17%-31% peel scalding and fruit dipped in 62 C water for 30 s developed 46%-72% peel scalding depending on the post-dip treatment (Table 4-1). Schirra and Dhallewin (1997) reported peel scalding in Fortune mandarins after HW dips at 56 or 58 C for 3 min. Washing and shellac coating of fruit immediately after HW dip reduced the development of peel scalding by 45% or 37% in fruit dipped at 59 or 62 C, respectively, compared with fruit that were not washed and coated. Dipping fruit in ambient water immediately after HW dip reduced the development of scalding by only 6% or 5% in fruit dipped at 59 or 62 C, respectively, compared with fruit that were not dipped in ambient water after HW dips. So, washing and coating fruit immediately after HW dip can significantly reduce the heat damage. Grapefruit dipped in 56 or 59 C water followed by washing and shellac coating showed the best result in reducing decay to 25% or 18%, respectively, after 12 weeks of storage at 10 C (Figure 4-1). Fruit dipped in 25 C water followed by no post-dip treatment developed 75% decay. Decay development in fruit dipped in ambient water after HW dip at 62 C did not differ from fruit that received no post-dip treatment after HW dip at 62 C. But fruit treated at other temperatures followed by dipping in ambient water developed higher decay than fruit that received no post-dip treatment. Fruit dipped in 62 C water were injured by the treatment, which negated the beneficial effect of

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50 reducing decay. Hot water (Miller et al., 1988) and vapor heat (Hallman et al., 1990) treatments of grapefruit that caused scalding were reported to also result in increased decay, which was suggested to be a result of the damaged tissue being more susceptible to pathogen invasion. Though others have reported the development of chilling injury (CI) of Florida grapefruit stored at 10 C (Grierson and Hatton, 1977), it is fairly uncommon commercially because of the almost universal use of wax coatings that restrict gas exchange to varying degrees and reduce chilling sensitivity. Early season fruit (September-November) are more susceptible to CI than fruit harvested during the middle of the season (December-February) (Grierson and Hatton, 1977; Schirra et al., 2000). The fruit utilized for the current studies were still very chilling sensitive and developed CI during storage at 10 C. Dipping fruit in 56 or 59 C water followed by washing and Table 4-1. Peel scalding (percent of fruit scalded) of Ruby Red grapefruit after 30 s HW dip treatments followed by post-dip treatments. Fruit were evaluated after 4 weeks of storage at 10 C. Dip Fruit were dipped in ambient water for 1 min and Shellac Fruit were washed over a brush bed and coated with shellac. NoneDipShellac25 C0.000.000.0053 C0.000.000.0056 C0.000.000.0059 C30.6328.7516.8862 C71.8868.1345.63Post-dip treatmentTmnt. temp. LSD 0.05 = 14.37 shellac coating reduced CI to 2% or 1%, respectively, after 12 weeks of storage at 10 C (Figure 4-2). Fruit dipped in 25 C water followed by no post-dip treatment developed 50% CI. Hot water dip did not affect the TSS, TA or the amount of juice (data not

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51 010203040506070809010025 C53 C56 C59 C62 CTreatment temperature (30 s)Total decay (%) None Dip ShellacLSD0.05 Figure 4-1. Total decay of Ruby Red grapefruit after 30 s HW dip treatments followed by post-dip treatments. Fruit were evaluated after 12 weeks of storage at 10 C. Vertical bar represents the 5% LSD value. Dip Fruit were dipped in ambient water for 1 min and Shellac Fruit were washed over a brush bed and coated with shellac. 010203040506070809010025 C53 C56 C59 C62 CTreatment temperature (30 s)Chilling injury (%) None Dip ShellacLSD0.05 Figure 4-2. Chilling injury of Ruby Red grapefruit after 30 s HW dip treatments followed by post-dip treatments. Fruit were evaluated after 12 weeks of storage at 10 C. Vertical bar represents the 5% LSD value. Dip Fruit were dipped in ambient water for 1 min and Shellac Fruit were washed over a brush bed and coated with shellac.

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52 shown). There were no consistent differences in weight loss or peel puncture resistance (data not shown). Experiment 2 Fruit dipped in 59 C water for 30 s had significantly more scalding than the fruit that were dipped in 25 C water after 1, 2, and 4 weeks of storage at 16 C. After 4 weeks in storage, the incidence of peel scalding for fruit dipped in 59 C water for 30 s was 3%-5% depending on the post-dip treatment (Table 4-2) and peel scalding severity ranged from 8%-14% of the fruit surface (Table 4-3). In the previous experiment using November-harvested fruit, HW dips at 59 C resulted in 17%-31% peel scalding incidence after 4 weeks of storage. So, the late season (February-harvested) fruit used in the second experiment may have been more resistant to heat damage. Hot water dips at 56 C water for 30 s followed by no post-dip treatment resulted in only 2% incidence of peel scalding whereas fruit that were shellac coated after HW dipping at 56 C did not develop any peel scalding. Washing alone or washing and coating the fruit immediately after HW dip did not have a significant effect on the development of peel scalding. Since the level of scalding was very low compared with the previous experiment, the effect of coating the fruit on heat damage could not be seen. None of the HW or post-dip treatments were effective in controlling decay (Figure 4-3). Heat treatment and washing alone or washing and coating the fruit did not result in significant reduction in postharvest decay. The incidence of total decay was very low in this season compared with the previous experiment. This could be a reason why significant effect of treatments could not be seen.

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53 Table 4-2. Peel scalding incidence (percent of fruit scalded) of Ruby Red grapefruit after 30 s HW dip treatments followed by post-dip treatments. Fruit were evaluated after 4 weeks of storage at 16 C. Wash Fruit were washed over a brush bed and Shellac Fruit were washed over a brush bed and coated with shellac. NoneWashShellac25 C0.000.000.0056 C1.883.130.0059 C2.503.755.00Post-dip treatmentTmnt. temp. LSD 0.05 = 2.05 010203040506070809010025 C56 C59 CTreatment temperature (30 s)Total decay (%) None Wash ShellacLSD0.05 Figure 4-3. Total decay of Ruby Red grapefruit after 30 s HW dip treatments followed by post-dip treatments. Fruit were evaluated after 12 weeks of storage at 16 C. Vertical bar represents the 5% LSD value. Wash Fruit were washed over a brush bed and Shellac Fruit were washed over a brush bed and coated with shellac.

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54 Table 4-3. Peel scalding severity (percent of fruit surface scalded) of Ruby Red grapefruit after 30 s HW dip treatments followed by post-dip treatments. Fruit were evaluated after 4 weeks of storage at 16 C. Wash Fruit were washed over a brush bed and Shellac Fruit were washed over a brush bed and coated with shellac. NoneWashShellac25 C0.000.000.0056 C6.6711.250.0059 C7.5013.759.17Post-dip treatmentTmnt. temp. LSD 0.05 = 2.05 Experiment 3 Fruit dipped in heated solutions of imazalil had greater residue levels immediately after dipping than fruit dipped in non-heated solutions of imazalil (Figure 4-4). Fruit dipped in heated solutions of imazalil had 0.6-1 ppm of residue whereas fruit dipped in ambient temperature solution had only 0.1 ppm of residue. Salustiana oranges dipped in imazalil solution at 50 C had 8-fold greater residue than when dipping was done at 20 C (Cabras et al., 1999). Hence, lower concentrations of imazalil could be more effective when the application occurs at higher temperatures. After 4 weeks of storage, no significant scalding was observed in fruit dipped at 56 C for 30 s. Only 0.2% of the fruit were scalded. Most decay was due to stem-end rot (SER). After 12 weeks of storage at 16 C, HW dipping at 56 C for 30 s resulted in lower SER than the non-heated solutions for all the imazalil and sodium carbonate solutions except for the water dip treatment (Figure 4-5). Ritenour et al. (2003) reported that a HW dip at 56 C for 30 s decreased SER to 20% after 12 weeks in storage compared with 33% SER in fruit dipped in ambient water. Treatments with 3% or 6% sodium carbonate solutions at 25 C increased the incidence of SER to 27% or 23%,

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55 respectively, compared with water dip treatment at 25 C, which developed 6% SER. Treatments with 3% or 6% sodium carbonate solutions at 56 C also increased the incidence of SER (to 14% or 19%, respectively) compared with the water dip treatment at 56 C, which developed 11% SER. This is in contrast to the effect of hot sodium carbonate solutions on green and blue molds. Smilanick et al. (1997) and Palou et al. (2001, 2002) have shown that sodium carbonate solutions, especially heated solutions, significantly reduce the development of green and blue molds in citrus. The SER may be enhanced by the high pH (10-11) of the sodium carbonate solutions. Treating the fruit with imazalil solution did not result in significant reduction in SER though the fruit dipped in heated imazalil solutions had higher residue levels. Since the incidence of natural decay was low in fruit harvested during February, the effect of imazalil could not be observed. 0.00.20.40.60.81.01.2125 ppm125 ppm + 3% SC250 ppmImazalil concentrationImazalil residue (ppm) 25 C 56 CLSD0.05 Figure 4-4. Imazalil residue in Ruby Red grapefruit immediately after the 30 s dip treatments at 25 and 56 C. Vertical bar represents the 5% LSD value. SC Sodium carbonate.

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56 01020304050607080901000% SC3% SC6% SC125 ppmimazalil125 ppmimazalil + 3% SC250 ppmimazalilTreatment (30 s)Stem-end rot (%) 25 C 56 C LSD0.05 Figure 4-5. Stem-end rot for different treatment temperatures after 12 weeks of storage at 16 C. Bars with different letters are significantly different by Duncans multiple range test at P 0.05. SC Sodium carbonate.

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CHAPTER 5 CONTROL OF CHILLING INJURY IN RUBY RED GRAPEFRUIT BY HOT WATER DIP TREATMENT Introduction Grapefruit develops chilling injury (CI) when stored at temperatures below 10-12 C (Chace et al., 1966). Grapefruit harvested early and late in the season are more susceptible to CI than are fruit harvested in mid-season (Grierson and Hatton, 1977). Fruit from the outer-canopy are more susceptible to CI than are fruit from the inner-canopy (Purvis, 1980). Heat treatments have been successful in reducing CI in many citrus varieties (Rodov et al., 1995a; Schirra et al., 1997). The following experiment was conducted to study the effect of short duration, hot water (HW) dip treatments in controlling CI in Florida grapefruit. Materials and Methods Fruit Commercially mature (TSS:TA 7:1) and healthy Ruby Red grapefruit were harvested randomly from 1-1.5 m above ground level on healthy trees, evenly spaced around the tree on 3 Nov. 2003 at the Indian River Research and Education Center research grove in Fort Pierce, Fla. Fruit were harvested separately from the inner and outer canopies of the tree. The harvested fruit were stored at room temperature overnight and heat treatment was done on the next day. 57

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58 Heat Treatment Fruit were dipped in water at 25, 53, 56 or 59 C for 30 s. Hot water dips were administered as described in chapter 4. Each treatment had four replicates of 30 fruit each. After the HW dip, half of the fruit from each treatment and canopy position were stored at 5 C (90% RH) and the other half stored at 16 C (90% RH). Five fruit from each replicate were randomly selected, marked, and weighed to follow weight loss during storage. Initial analyses of peel color, total soluble solids (TSS), titratable acidity (TA), peel puncture resistance (PPR) and percent juice were done using four replicates of five fruit each. Fruit were evaluated for peel scalding 1, 3, and 7 weeks after treatment. After 4 and 7 weeks of storage, marked fruit were evaluated for weight loss. After 4 weeks of storage, peel color, TSS, TA, PPR, and percent juice were evaluated from another five fruit randomly selected from each replicate. After 6 weeks of storage, fruit stored at 5 C (90% RH) were transferred to 16 C (90%RH). After 7 weeks of storage, remaining fruit were evaluated for CI and decay. Chilling Injury Chilling injury severity was rated from 0 to 3 (0-none, 1-slight, 2-moderate and 3-severe). The number of fruit in each rating was multiplied by its corresponding rating number and the sum of these products was divided by the total number of fruit in the replicate to give the average CI severity for that replicate. Peel Scalding Peel scalding was evaluated as described in chapter 3. Weight Loss Weight loss was calculated as described in chapter 4.

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59 Peel Color Peel color was measured as described in chapter 3. Total Soluble Solids and Titratable Acidity Total soluble solids and titratable acidity were measured as described in chapter 4. Peel Puncture Resistance Peel puncture resistance was measured as described in chapter 4. Percent Juice Percent juice was calculated as described in chapter 4. Statistical Analysis Percentage data were transformed to arcsine values and analyzed by ANOVA using SAS (PROC GLM) for PC (SAS Institute Inc, Cary, N.C.). When differences were significant (P < 0.05), individual treatment means were separated using Duncans Multiple Range Test (P = 0.05). Means presented are untransformed values. Results and Discussion Hot water dip had a greater effect in reducing CI severity of inner canopy fruit than of outer canopy fruit. Compared to fruit dipped at 25 C, dipping fruit in 53, 56, or 59 C water for 30 s reduced CI severity by 3%, 6% or 10%, respectively, in outer canopy fruit stored at 5 C, but reduced CI severity by 11%, 18% or 32%, respectively, in inner canopy fruit (Figure 5-1). Purvis (1980) has reported that outer canopy fruit are more susceptible to CI than the inner canopy fruit, but our results indicated little effect of canopy position on non-heated fruit with both inner and outer canopy fruit severely affected by CI. So, heat treatment by itself had a major role in reducing the CI severity in inner canopy fruit. None of the fruit stored at 16 C developed CI.

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60 Fruit stored at chilling temperature (5 C) developed more decay than fruit stored at non-chilling temperature (16 C). After 7 weeks of storage, fruit stored at 16 C developed only 0%-4% decay whereas fruit stored at 5 C developed 27%-58% decay (Figure 5-2). Most decay was due to anthracnose (Colletotrichum gloeosporioides). High incidence of anthracnose was observed on fruit that developed CI. For fruit stored at 5 C, HW dipping at 53, 56 or 59 C for 30 s reduced decay by 25%, 21% or 44%, respectively, compared with dipping in 25 C water for 30 s. After 3 weeks of storage, 2% of fruit treated at 59 C developed visible peel scalding (data not shown). No scalding was observed on fruit dipped in 25, 53 or 56 C water. After 4 weeks of storage, TSS in fruit from outer canopy was significantly higher than in fruit from inner canopy and TA was significantly lower in outer canopy fruit than in inner canopy fruit. The percent juice was not affected by the canopy position. The percent juice and TA was significantly lower in fruit stored at 5 C than in fruit stored at 16 C. However, HW dipping did not affect the percent juice, TSS, and TA of the fruit (data not shown). After 4 weeks of storage, PPR was significantly greater in fruit treated at 59 C than in all other treatments (Table 5-1). At the end of the experiment, weight loss from fruit treated at 25 C was significantly greater than from the other three treatment temperatures (Table 5-2). The weight loss was higher in the fruit stored at 5 C than the fruit stored at 16 C. Higher weight loss at 5 C could be due to accelerated weight loss in fruit that developed CI. Purvis (1984) correlated higher weight loss during storage with CI development in citrus fruit. Cohen et al. (1994) used weight loss as an early indicator of CI.

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61 0.00.51.01.52.02.53.025 C53 C56 C59 CTreatment (30 s)CI Index Inner Canopy 5 C Outer Canopy 5 CLSD0.05 Figure 5-1. Chilling injury in Ruby Red grapefruit after 30 s dip treatments of inner and outer canopy fruit. Fruit were evaluated after 6 weeks of storage at 5 C plus 1 week at 16 C. Chilling injury was rated from 0 (none) to 3 (severe). Vertical bar represents the 5% LSD value. 010203040506070809010025 C53 C56 C59 CTreatment temperature (30 s)Total decay (%) Inner Canopy 5 C Inner Canopy 16 C Outer Canopy 5 C Outer Canopy 16 C LSD0.05 Figure 5-2. Total decay in Ruby Red grapefruit after 30 s dip treatments of inner and outer canopy fruit. Fruit were evaluated after 7 weeks of storage at 5 or 16 C. Vertical bar represents the 5% LSD value.

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62 Table 5-1. Peel puncture resistance in Newtons of Ruby Red grapefruit after 30 s dip treatments. Fruit were evaluated after 4 weeks of storage at 5 or 16 C. 5 C16 C5 C16 C25 C17.2414.3918.4713.6853 C18.1914.5318.1214.3756 C18.2914.7318.3413.5559 C18.2514.9120.2815.17Tmnt. Temp.Inner CanopyOuter Canopy LSD 0.05 = 1.66 Table 5-2. Weight loss (%) from Ruby Red grapefruit after 30 s dip treatments. Fruit were evaluated after 7 weeks of storage at 5 or 16 C. 5 C16 C5 C16 C25 C4.343.774.423.8453 C4.153.613.683.5556 C3.963.053.713.3759 C4.233.613.923.36Tmnt. Temp.Inner CanopyOuter Canopy LSD 0.05 = 0.61

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CHAPTER 6 CONCLUSIONS The research reported in this thesis has shown the effects on ethylene treatment of grapefruit and physiological responses of oranges due to hot water (HW) dip treatments and also the effects of HW dip treatment in reducing postharvest decay and chilling injury (CI) in Florida grapefruit. As a result of these studies, it was found that HW dips administered before or after grapefruit degreening did not affect subsequent peel injury, respiration rate, ethylene production, total soluble solids (TSS) or titratable acidity (TA). Under commercial conditions, the various uses of ethylene treatment would not appear to have any affect on the use of HW treatments on grapefruit. Significantly higher electrolyte leakage was found only in fruit dipped at temperatures greater than 66 C for 60 s. In Valencia oranges, a HW dip at 56 C for up to 120 s or 60 C for 60 s did not affect electrolyte leakage though it resulted in 67% or 100% incidence of peel scalding, respectively. None of these treatments affected electrolyte leakage than the non-treated fruit. Hence electrolyte leakage is not an early indicator of peel injury. Valencia oranges dipped at temperatures above 66 C for 60 s had lower L* values than non-treated fruit. Hot water dip at 70-90 C for 60 s did not result in significant difference in chroma value though fruit developed visible peel discoloration. But, in a subsequent experiment, fruit dipped at 66-70 C for 60 s had significantly lower chroma value. The hue value was not affected by HW dip. The peroxidase activity of heat-treated fruit (66 C for 60 s) was lower than that of non-treated fruit or fruit treated 63

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64 at 60 C for 60 s. Hot water dips did not affect the total phenolics or the total protein content. To study the effects of washing or shellac coating of fruit immediately after a HW dip, two experiments were conducted with Ruby Red grapefruit. After 4 weeks of storage, washing and shellac coating of fruit immediately after HW dip reduced the development of peel scalding by 45% or 37% in fruit dipped at 59 or 62 C, respectively, compared with fruit that were not washed and coated. Dipping the fruit in ambient water or washing through brush beds after HW treatment did not significantly reduce peel scalding. Since the fruit used in the first coating experiment were harvested in November, they were highly susceptible to CI with 50% CI incidence in control fruit dipped in 25 C water followed by no post-dip treatment and stored at 10 C. After 12 weeks of storage, fruit dipped in 56 or 59 C water followed by washing and shellac coating reduced CI to 2% or 1%, respectively. To study the effects of HW dip on CI, fruit were harvested separately from the inner and outer canopy and stored at 5 C for 6 weeks followed by 1 week at 16 C. Hot water dip at 53, 56 or 59 C reduced CI by 3%, 6% or 10%, respectively, in outer canopy fruit, but reduced CI by 11%, 18% or 32%, respectively, in inner canopy fruit. Scalding was not significant in fruit dipped at 56 C water for 30 s. HW dip at 56 or 59 C for 30 s significantly reduced decay in fruit harvested in November. The fruit harvested in February were less susceptible to decay and so the effects of HW dip could not be seen. In none of the experiments did HW dip affect TSS or TA of the fruit.

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65 Although HW treatments reduced significantly the development of postharvest decay, there was still a substantial amount of decay. So, HW treatment alone did not effectively control the decay development. Heated solutions of sodium carbonate and imazalil were used at lower concentrations than used commercially at ambient temperature. Unfortunately, solutions of 3% or 6% sodium carbonate enhanced the development of stem-end rot. Imazalil at 125 and 250 ppm did not effectively control decay compared with fruit that were dipped in water only. From the experiments conducted on Ruby Red grapefruit, HW dip at 56 C for 30 s followed by washing and shellac coating significantly reduced development of postharvest decay and CI without causing significant damage to the peel and without affecting the juice quality of the fruit. Hence, HW dip at 56 C for 30 s followed by washing and shellac coating was the best treatment for Ruby Red grapefruit. Future experiments should be conducted to increase the efficacy of HW dip by using heated solutions of compounds generally regarded as safe or heated solutions of fungicides at lower concentrations. In the current study, heated solutions of sodium carbonate or imazalil did not effectively control postharvest decay in late season fruit. However, others have reported the efficacy of these compounds in heated solutions, which suggests they can be effective under modified conditions. Hence more work should be conducted on early season fruit when they are more susceptible to natural decay and economic losses from such decay is often more extensive.

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APPENDIX A ANALYSIS OF VARIANCE FOR CHAPTER 2 Table A-1. Analysis of variance for respiration rates of Ruby Red grapefruit during the first six days in storage at 20 C. 1 day2 days3 days4 days5 days6 daysTreatment43.12*6.13**3.95**3.45*2.05*2.37Error100.650.810.120.720.184.34d.f.Sources of variationMean squares of respiration rate F values significant at 5% ** F values significant at 1% Table A-2. Analysis of variance for ethylene production of Ruby Red grapefruit during the first six days in storage at 20 C. 1 day2 days3 days4 days5 days6 daysTreatment40.00120.00040.00070.00050.0027$0.0080Error100.00120.00030.00040.00050.00170.0037Sources of variationd.f.Mean squares of ethylene production Table A-3. Analysis of variance for percent of fruit scalded, total soluble solids and titratable acidity of Ruby Red grapefruit after 10 d of storage at 20 C. ScaldTotal soluble solidsTitratable acidityTreatment40.5255**1.27230.0228*Error100.00902.01800.0041Sources of variationd.f.Mean squares F values significant at 5% ** F values significant at 1% 66

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APPENDIX B ANALYSIS OF VARIANCE FOR CHAPTER 3 Experiment 1 Table B-1. Analysis of variance for percent of fruit scalded after 12 d of storage at 10 C, electrolyte leakage of treated and untreated sides of Valencia oranges immediately after treatment. ScaldElectrolyte leakage (treated side)Electrolyte leakage (untreated side)Treatment50.4560**26.78957.141*Error120.000521.60014.659Sources of variationd.f.Mean squares F values significant at 5% ** F values significant at 1% 67

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68 Experiment 2 Table B-2. Analysis of variance for peel scalding of Valencia oranges after 14 d of storage at 10 C. Percent of fruit scaldedPercent of fruit surface scaldedTreatment50.5096**0.5952**Error120.00640.0007Mean squaresSources of variationd.f. ** F values significant at 1% Table B-3. Analysis of variance for electrolyte leakage of treated and untreated sides of Valencia oranges immediately after treatment and after 14 d of storage at 10 C. Electrolyte leakage (treated side)Electrolyte leakage (untreated side)Electrolyte leakage (treated side)Electrolyte leakage (untreated side)Treatment50.2803**0.00250.1390**0.0011Error120.00200.00270.00140.0009Mean squares0 day14 daysSources of variationd.f. ** F values significant at 1%

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69 Experiment 3 Table B-4. Analysis of variance for peel scalding of Valencia oranges after 8 d of storage at 10 C. Percent of fruit scaldedPercent of fruit surface scaldedTreatment61.8531**1.2430**Error140.00640.0084Sources of variationd.f.Mean squares ** F values significant at 1% Table B-5. Analysis of variance for electrolyte leakage of treated and untreated sides of Valencia oranges immediately after treatment and after 8 d of storage at 10 C. Electrolyte leakage (treated side)Electrolyte leakage (untreated side)Electrolyte leakage (treated side)Electrolyte leakage (untreated side)Treatment60.0744**0.00200.0360*0.0013Error140.00460.00170.00510.0123Sources of variationd.f.Mean squares0 day8 days F values significant at 5% ** F values significant at 1%

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70 Experiment 4 Table B-6. Analysis of variance for peel scalding of Valencia oranges after 4 and 7 d of storage at 10 C. Percent of fruit scaldedPercent of fruit surface scaldedPercent of fruit scaldedPercent of fruit surface scaldedTreatment20.6691**0.2513**2.0104**0.4761**Error60.00450.00150.04060.01547 daysMean squaresSources of variationd.f.4 days ** F values significant at 1% Table B-7. Analysis of variance for electrolyte leakage from flavedo of Valencia oranges immediately after treatment and after 2, 4 and 7 d of storage at 10 C. 0 day2 days4 days7 daysTreatment20.0176**0.0116*0.0073*0.0117**Error60.00060.00120.00090.0008Sources of variationd.f.Mean squares of electrolyte leakage F values significant at 5% ** F values significant at 1% Table B-8. Analysis of variance for peroxidase activity in flavedo of Valencia oranges immediately after treatment and after 2, 4 and 7 d of storage at 10 C. 0 day2 days4 days7 daysTreatment214.22*29.78*45.3136.82*Error63.405.4314.279.35Sources of variationd.f.Mean squares of peroxidase activity F values significant at 5%

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71 Table B-9. Analysis of variance for total phenolics in flavedo of Valencia oranges immediately after treatment and after 2, 4 and 7 d of storage at 10 C. 0 day2 days4 days7 daysTreatment20.38900.69160.26320.1522Error60.53170.61040.19570.3273Sources of variationd.f.Mean squares of total phenolics Table B-10. Analysis of variance for total protein in flavedo of Valencia oranges immediately after treatment and after 2, 4 and 7 d of storage at 10 C. 0 day2 days4 days7 daysTreatment20.09391.28562.74580.7453Error60.43460.34321.68532.8706Sources of variationd.f.Mean squares of total protein content

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APPENDIX C ANALYSIS OF VARIANCE FOR CHAPTER 4 Experiment 1 Table C-1. Analysis of variance for peel scalding, total decay and chilling injury of Ruby Red grapefruit. Peel scalding was evaluated after 4 weeks of storage at 10 C. Total decay and chilling injury were evaluated after 12 weeks of storage at 10 C. Percent of fruit scaldedPercent of fruit surface scaldedTotal decayChilling injuryWater temperature (WT)42.0818**0.8977**0.3174**0.4455**Post dip treatment (PDT)20.0453*0.00450.7405**1.0092**WT x PDT80.01950.0042*0.02250.0494Error450.01230.00170.02790.0242Mean squaresSources of variationd.f. F values significant at 5% ** F values significant at 1% Table C-2. Analysis of variance for quality of Ruby Red grapefruit after 4 weeks of storage at 10 C. Weight lossPeel puncture resistanceJuice contentTotal soluble solidsTitratable acidityWater temperature (WT)41.0734**8.6801**2.08190.23320.0136Post dip treatment (PDT)20.21763.7180*2.83950.09220.0275WT x PDT80.28890.89832.99770.28220.0092Error450.19620.81202.03960.30280.0085Sources of variationd.f.Mean squares F values significant at 5% ** F values significant at 1% 72

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73 Experiment 2 Table C-3. Analysis of variance for peel scalding and total decay of Ruby Red grapefruit. Peel scalding was evaluated after 4 weeks and total decay was evaluated after 12 weeks of storage at 16 C. Percent of fruit scaldedPercent of fruit surface scaldedTotal decayWater temperature (WT)242.36*311.67**29.08Post dip treatment (PDT)22.2687.3413.97WT x PDT46.9441.2942.61Error277.9942.14109.37Sources of variationd.f.Mean squares F values significant at 5% ** F values significant at 1%

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74 Experiment 3 Table C-4. Analysis of variance for imazalil residue of Ruby Red grapefruit immediately after treatment. Sources of variationd.f.Mean squares of imazalil residueWater temperature (WT)13.5190**Imazalil treatment (IT)20.0968**WT x IT20.0933*Error180.0157 F values significant at 5% ** F values significant at 1% Table C-5. Analysis of variance for peel scalding and stem-end rot of Ruby Red grapefruit. Peel scalding was evaluated after 4 weeks and stem-end rot was evaluated after 12 weeks of storage at 16 C. Percent of fruit scaldedPercent of fruit surface scaldedStem-end rotWater temperature (WT)10.520818.75300.25*Chemical treatment (CT)50.20838.75238.14**WT x CT50.20838.7578.62Error360.260410.4249.26Sources of variationd.f.Mean squares F values significant at 5% ** F values significant at 1%

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APPENDIX D ANALYSIS OF VARIANCE FOR CHAPTER 5 Table D-1. Analysis of variance for peel scalding, chilling injury index and total decay of Ruby Red grapefruit. Peel scalding was evaluated at 4 weeks of storage. Chilling injury and total decay were evaluated after 7 weeks of storage. Percent of fruit scaldedPercent of fruit surface scaldedChilling injury indexTotal decayWater temperature (WT)311.11*25.00**0.2359**0.0718*Canopy position (CP)10.706.250.5968**0.0016Storage temperature (ST)10.000.00101.9090**6.5993**WT x CP30.706.250.06300.0043WT x ST30.000.000.2359**0.0504CP x ST16.266.250.5968**0.0001WT x CP x ST36.266.250.06300.0320Error482.665.210.04510.0184Sources of variationd.f.Mean squares F values significant at 5% ** F values significant at 1% Table D-2. Analysis of variance for quality of Ruby Red grapefruit after 4 weeks of storage. Peel puncture resistanceJuice contentTotal soluble solidsTitratable acidityWater temperature (WT)34.3335*2.68880.12420.0026Canopy position (CP)10.52930.09231.0000**0.0225**Storage temperature (ST)1253.6853**289.1275**1.0000**0.0286**WT x CP32.11142.05800.20830.0004WT x ST30.26910.06130.22830.0003CP x ST16.3252*0.00830.02250.0084WT x CP x ST30.68427.23400.19750.0016Error481.36324.88380.07830.0023Mean squaresSources of variationd.f. F values significant at 5% ** F values significant at 1% 75

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76 Table D-3. Analysis of variance for weight loss in Ruby Red grapefruit after 4 and 7 weeks of storage. Weight loss (4th week)Weight loss (7th week)Water temperature (WT)30.4211**0.8772**Canopy position (CP)10.00540.1828Storage temperature (ST)10.03754.5263**WT x CP30.02930.1445WT x ST30.02450.0720CP x ST10.01470.2678WT x CP x ST30.00870.0749Error480.06720.1862Sources of variationd.f.Mean squares ** F values significant at 1%

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78 Brown, G.E. 1975. Factors affecting postharvest development of Colletotrichum gloeosporioides in citrus fruits. Phytopathol. 65:404-409. Brown, G.E. 1978. Hypersensitive response of orange-colored Robinson tangerines to Collectotrichum gloeosporioides after ethylene treatment. Phytopathol. 68: 700-706. Brown, G.E. and C.R. Barmore. 1983. Resistance of healed citrus exocarp to penetration by Penicillium digitatum. Phytopathol. 73:691-694. Cabras, P., M. Schirra, F.M. Pirisi, V.L. Garau, and A. Angioni. 1999. Factors affecting imazalil and thiabendazole uptake and persistence in oranges following dip treatments. J. Agr. Food Chem. 47:3352-3354. Chace, W.G., Jr., P.L. Harding, J.J. Smoot, and R.H. Cubbedge. 1966. Factors affecting the quality of grapefruit exported from Florida. U.S. Dept. Agr. Res. Rpt. 739. Cohen, E., B. Shapiro, Y. Shalom and J.D. Klein. 1994. Water loss: a nondestructive indicator of enhanced cell membrane permeability of chilling injured citrus fruit. J. Amer. Soc. Hort. Sci. 119:983-986. Couey, H.M. 1989. Heat treatment for control of postharvest diseases and insect pests of fruits. HortScience 24:198-202. Dhallewin, G. and M. Schirra. 2000. Structural changes of epicuticular wax and storage response of Marsh grapefruits after ethanol dips at 21 and 50C. Proc. 4 th Intl. Conf. on Postharvest:441-442. Del Rio, M.A., T. Cuquerella, and M.L. Ragone. 1992. Effects of postharvest curing at high temperature on decay and quality of Marsh grapefruits and navel oranges. Proc. Intl. Soc. Citricult. 3:1081-1083. Eckert, J.W. and G.E. Brown. 1986. Postharvest citrus diseases and their control, p. 315-360. In: W.F. Wardowski, S. Nagy, and W. Grierson. (eds.), Fresh Citrus Fruits. AVI Publishing, Westport, CT. Eckert, J.W. and I.L. Eaks. 1988. Postharvest disorders and diseases of citrus fruit, p. 179-260. In: Reuther, W., Calavan, E.C., Carman, G.E., (eds.), The Citrus Industry, University of California Press, Berkeley. Eckert, J.W. and J.M. Ogawa. 1988. The chemical control of postharvest diseases: deciduous fruits, berries, vegetables and root/tuber crops. Annu. Rev. Phytopathol. 26:433-469. Fallik, E., Y, Aharoni, O. Yekutieli, A. Wiseblum, R. Regev, H. Beres, and E. Bar-Lev. 1996. A method for simultaneously cleaning and disinfecting agricultural produce. Israel patent No. 116965.

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79 Fallik, E., S. Grinberg, S. Alkalai, O. Yekutieli, A. Wiseblum, R. Regev, H. Beres, and E. Bar-Lev. 1999. A unique rapid hot water treatment to improve storage quality of sweet pepper. Postharvest Biol. Technol. 15:25-32. Fawcett, H.S. 1922. Packing house control of brown rot. Citrograph 7:232-234. Florida Department of Citrus. 2004. Citrus Reference Book. Economic and Market Research Department, Florida Department of Citrus, Gainesville. 04 June 2004. < http://www.fred.ifas.ufl.edu/citrus/pubs/ref/CRB2004.pdf >. Grierson, W. 1986. Physiological disorders, p. 361-378. In: W.F. Wardowski, S. Nagy, and W. Grierson. (eds.) Fresh Citrus Fruits. AVI Publishing, Westport, CT. Grierson, W. and T.T. Hatton. 1977. Factors involved in storage of citrus fruits: A new evaluation. Proc. Intl. Soc. Citricult. 1:227. Hallman, G.J., J.J. Gaffney, and J.L. Sharp. 1990a. Vapor heat treatment for grapefruit infested with Caribbean fruit fly (Diptera: Tephritidae). J. Econ. Entomol. 83:1475-1478. Hallman, G.J., J.J. Gaffney, and J.L. Sharp. 1990b. Vapor heat research unit for insect quarantine treatments. J. Econ. Entomol. 83:1965-1971. Holland, N., H.C. Menezes, and M.T. Lafuente. 2002. Carbohydrates as related to the heat-induced chilling tolerance and respiratory rate of Fortune mandarin fruit harvested at different maturity stages. Postharvest Biol. Technol. 25:181-191. Jacobi, K.K. and L.S. Wong. 1992. Quality of Kensington mango (Mangifera indica Linn.) following hot water and vapor-heat treatments. Postharvest Biol. Technol. 1:349-359. Kader, A.A. 2002. Postharvest Technology of Horticultural Crops. 3 rd ed. University of California, Oakland, Calif. Kim, J.J., S. Ben-Yehoshua, B. Shapiro, Y. Henis, and S. Carmeli. 1991. Accumulation of scoparone in heat-treated lemon fruit inoculated with Penicillium digitatum Sacc. Plant Physiol. 97:880-885. Klein, J.D. and S. Lurie. 1991. Postharvest heat treatment and fruit quality. Postharvest News Inf. 2:15-19. Lanza, G., E. di Martino Aleppo, M.C. Strano, and G. Reforgiato Recupero. 2000. Evaluation of hot water treatments to control postharvest green mold in organic lemon fruit p1167-1168. In: Proc. Intl. Soc. Citricult. XI Congr., Orlando, 3-7 Dec. 2000.

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81 Porat, R., A. Daus, B. Weiss, L. Cohen, E. Fallik, and S. Droby. 2000a. Reduction of postharvest decay in organic citrus fruit by a short hot water brushing treatment. Postharvest Biol. Technol. 18:151-157. Porat, R., B. Weiss, L. Cohen, A. Daus, R. Goren, and S. Droby. 1999. Effects of ethylene and 1-methycyclopropene on the postharvest qualities of Shamouti oranges. Postharvest Biol. Technol. 15:155-163. Porat, R., B. Weiss, L. Cohen, V. Vinokur, D. Pavoncello, A. Daus, and S. Droby. 2000b. Induction of grapefruit resistance to Penicillium digitatum by chemical, physical and biological elicitors. Proc. Intl. Soc. Citricult. XI Congr.:1146-1148. Prusky, D., Y. Fuchs, I. Kobiler, I. Roth, A. Weksler, Y. Shalom, E. Fallik, G. Zauberman, E. Pesis, M. Akerman, O. Yekutieli, A. Weisblem, R. Regev, and L. Artes. 1999. Effect of hot water brushing, prochloraz treatment and waxing on the incidence of black spot decay caused by Alternaria alternate in mango fruits. Postharvest Biol. Technol. 15:165-174. Purvis, A.C. 1980. Influence of canopy depth on susceptibility of Marsh grapefruit to chilling injury. HortScience 15:731-733. Purvis, A.C. 1984. Importance of water loss in the chilling injury of grapefruit stored at low temperature. Sci. Hort. 23:261-267. Ritenour, M.A., K-J. John-Karuppiah, R.R. Pelosi, M.S. Burton, T.G. McCollum, J.K. Brecht, and E.A. Baldwin. 2003. Response of Florida grapefruit to short-duration heat treatments using vapor heat or hot water dips. Proc. Fla. State Hort. Soc. 116:405-409. Rodov, V., T. Agar, J. Peretz, B. Nafussi, J.J. Kim, and S. Ben-Yehoshua. 2000. Effect of combined application of heat treatments and plastic packaging on keeping quality of Oroblanco fruit (Citrus grandis L C. paradisi Macf.). Postharvest Biol. Technol. 20:287-294. Rodov, V., S. Ben-Yehoshua, R. Albagli, and D.Q. Fang. 1993. Postharvest heat treatments of citrus fruits: curing vs. hot water application. In: Yupera, E.P., Calero, F.A., Sanchez, J.A. (Eds.), 2nd Intl. Congr. Food Technol. and Dev., Murcia, Spain PPU Publ., Barcelona, Spain:176-203. Rodov, V., S. Ben-Yehoshua, R. Albagli, and D.Q. Fang. 1995a. Reducing chilling injury and decay of stored citrus fruit by hot water dips. Postharvest Biol. Technol. 5:119-127. Rodov, V., S. Ben-Yehoshua, D.Q. Fang, J.J. Kim, and R. Ashkenazi. 1995b. Preformed antifungal compounds of lemon fruit: Citral and its relation to disease resistance. J. Agr. Food Chem. 43:1057-1061.

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82 Rodov, V., J. Peretz, T. Agar, G. Dhallewin, and S. Ben-Yehoshua. 1996. Heat applications as complete or partial substitute of postharvest fungicide treatments of grapefruit and Oroblanco fruits. Proc. VIII Intl. Citrus Congr., 12-17 May 1996. Sun City Resort, South Africa. 2:1187-1191. Rodov, V., J. Peretz, S. Ben-Yehoshua, T. Agar, and G. Dhallewin. 1997. Heat applications as complete or partial substitute for postharvest fungicide treatments of grapefruit and oroblanco fruits. In: Manicom, B., (Ed.), 1996 Proc. Intl. Soc. Citricult. 2:1153-1157. Roy, S., W.S. Conway, A.E. Watada, C.I. Sams, E.F. Erbe, and W.P. Wergin. 1994. Heat treatment affects epicuticular wax structure and postharvest calcium uptake in Golden delicious apples. HortScience 29:1056-1058. Roy, S., W.S. Conway, A.E. Watada, C.I. Sams, E.F. Erbe, and W.P. Wergin. 1999. Changes in ultrastructure of the epicuticular wax structure and postharvest calcium uptake in apple. HortScience 34:121-124. Schirra, M., M. Agabbio, G. Dhallewin, M. Pala, and R. Ruggiu. 1997. Response of Tarocco oranges to picking date, postharvest hot water dips, and chilling storage temperature. J. Agr. Food Chem. 45:3216-3220. Schirra, M. and S. Ben-Yehoshua. 1999. Heat treatments: a possible new technology in citrus handling Challenges and prospects, p. 133-147. In: Schirra, M. (ed.), Advances in Postharvest Diseases and Disorders Control of Citrus Fruit. Research Signpost Publisher, Trivandrum, India. Schirra, M. and G. Dhallewin. 1996. Storage of Fortune mandarin following postharvest dips in hot water and coating with an edible sucrose polyester. Proc. VIII Intl. Citrus Congr., 12-17 May 1996. Sun City Resort, South Africa. 2:1209-1214. Schirra, M. and G. Dhallewin. 1997. Storage performance of Fortune mandarins following hot water dips. Postharvest Biol. Technol. 10:229-238. Schirra, M., G. Dhallewin, S. Ben-Yehoshua, and E. Fallik. 2000. Host-pathogen interactions modulated by heat treatment. Postharvest Biol. Technol. 21:71-85. Schirra, M., G. Dhallewin, P. Cabras, A. Angioni, S. Ben-Yehoshua and S. Lurie. 2000. Chilling injury and residue uptake in cold-stored Star Ruby grapefruit following thiabendazole and imazalil dip treatments at 20 and 50 C. Postharvest Biol. Technol. 20:91-98. Schirra, M., G. Dhallewin, P. Cabras, A. Angioni, and V.L. Garau. 1998. Seasonal susceptibility of Tarocco oranges to chilling injury as affected by hot water and thiabendazole postharvest dip treatments. J. Agr. Food Chem. 46:1177-1180. Schirra, M. and M. Mulas. 1995a. Improving storability of Tarocco oranges by postharvest hot-dip fungicide treatments. Postharvest Biol. Technol. 6:129-138.

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BIOGRAPHICAL SKETCH Karthik-Joseph John-Karuppiah was born on November 10, 1979, in India. In 2001, he obtained his Bachelor of Science degree in Horticulture from the Tamil Nadu Agricultural University in India. In 2002, he started his masters program in Horticulture at the University of Florida and successfully completed his degree in 2004. 85


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HEAT TREATMENTS FOR CONTROLLING POSTHARVEST DISEASES AND
CHILLING INJURY IN FLORIDA CITRUS
















By

KARTHIK-JOSEPH JOHN-KARUPPIAH


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2004

































Copyright 2004

by

Karthik-Joseph John-Karuppiah

































This document is dedicated to my parents Dr. J. John Karuppiah and Mrs. J. Geetha.















ACKNOWLEDGMENTS

I would like to express my sincere thanks and gratitude to Dr. Mark A. Ritenour,

my committee chair, for giving me the opportunity to work on postharvest physiology

and for his valuable advice and guidance throughout my master's program. I thank my

committee member Dr. Jeffrey K. Brecht who initially taught me to organize and conduct

experiments. I would like to extend my thanks and gratitude to Dr. T. Gregory

McCollum, my committee member, for teaching me numerous laboratory techniques and

for helping me to statistically analyze my results. I would like to thank Kim Cordasco and

Michael S. Burton for their valuable help in conducting my experiments.

I thank my parents Dr. J. John Karuppiah and Mrs. J. Geetha, my sister R. Denniz

Arthi and her family for their love and encouragement during my whole life. I thank my

friends S.K. Ashok Kumar, A. Nithya Devi, M. Janakiraman, R. Rajesh and other friends

in India and in USA for their friendship and support.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ................................................. ................... viii

LIST OF FIGURES ......... ......................... ...... ........ ............ xi

A B S T R A C T .......................................... ..................................................x iii

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ..............................................1

C u rin g .......................................................3
V a p o r H e at .................................................................................. 4
H ot W after D ipping ................. ................................ ........ ........ .............. .
H ot W after Brushing .................. ..... ............ ...... .............. .................... .7
Commodity Responses to Heat Treatments..................................................8
S c a ld ............................................................ ................ 8
Electrolyte Leakage ....................................... ......... .. ...... .. ........ ..
R espiration and Juice Q quality .................................................................... ..... 9
P eel C olor ................................................................ .. .... ......... 10
Flesh and Peel Firmness ........................................................ ... ............11
Structural Changes of Epicuticular Wax ................ ............................... 11
H ost-Pathogen Interaction ................................................. ............................. 12
Stem -E n d R ot ...............................................................13
Anthracnose .................. .. ......... ................. 14
G re e n M o ld .................................................................................................... 14
M inor Postharvest D iseases............ ............... ........................... ............... 15
Physiological D isorders................................................ ............ ............... 15
Objectives ..................................... .................................. ........... 17

2 EFFECTS OF ETHYLENE TREATMENT ON 'RUBY RED' GRAPEFRUIT
QUALITY AFTER SHORT-DURATION HOT WATER DIP TREATMENTS .....18

In tro d u ctio n ...................................... ................................................ 18
M materials and M methods ....................................................................... .................. 18
F ru it .................. ............. .......................................................1 8
H eat Treatm ent and D egreening...................................... ........................ 18


v









Respiration Rate and Ethylene Production.............................. ...............19
Peel Scalding ........................................................... .. ........ ........ 20
Total Soluble Solids and Titratable Acidity ................................................20
Statistical A analysis .......................... .......... ............... .... ....... 20
R results and D iscu ssion .............................. ........................ .. ...... .... ...... ...... 2 1

3 PHYSIOLOGICAL RESPONSES OF 'VALENCIA' ORANGES TO SHORT-
DURATION HOT WATER DIP TREATMENT ............................................... 26

Introdu action ...................................... ................................................. 2 6
M materials and M methods ....................................................................... ..................26
F ru it ..............................................................................2 6
E x p e rim e n t 1 ................................................................................................. 2 6
E x p e rim e n t 2 ................................................................................................. 2 7
E x p e rim e n t 3 ................................................................................................. 2 7
E x p e rim e n t 4 ................................................................................................. 2 7
P e e l S c a ld in g ................................................................................................. 2 8
P e e l C o lo r ...................................................................................................2 8
E lectrolyte L eakage ....................................................................................... 28
Preparation of Flavedo Samples for Protein, Phenolic and Peroxidase Assays..29
Lowry Assay for Protein .................................................30
E stim ation of Total Phenolics ....................................................... 30
Peroxidase Assay .......................................... .................. ... .......31
Statistical A analysis ............................................................3 1
Results and Discussion .................................................................. ... ......31
E x p e rim e n t 1 ................................................................................................. 3 1
E x p e rim e n t 2 ................................................................................................. 3 3
E x p e rim e n t 3 ................................................................................................. 3 6
E x p e rim e n t 4 ................................................................................................. 4 0

4 CONTROL OF POSTHARVEST DISEASES IN 'RUBY RED' GRAPEFRUIT
BY HOT WATER DIP TREATMENT ........................ ...............44

In tro d u ctio n .......................................................................................4 4
M materials an d M eth od s ......................................................................................... 44
F ru it ..............................................................................4 4
E x p e rim e n t 1 ................................................................................................. 4 5
E x p e rim e n t 2 ................................................................................................. 4 5
E x p e rim e n t 3 ................................................................................................. 4 6
P e e l S c a ld in g ................................................................................................. 4 7
R e sid u e A n aly sis ........................................................................................... 4 7
W eight L oss.......................................................... 47
Peel Color .............. ........... ...... .........................48
Total Soluble Solids and Titratable Acidity ................. ................ ..........48
P percent Juice .............................................................................................. ........48
Peel Puncture R resistance .................................. ....... ............... .... ...........48
Statistical A naly sis ...........................................................4 8









R results and D iscu ssion .............................. ......................... ... ........ .... ............49
E x p e rim e n t 1 ................................................................................................. 4 9
E x p e rim e n t 2 ................................................................................................. 5 2
E x p e rim e n t 3 ................................................................................................. 5 4

5 CONTROL OF CHILLING INJURY IN 'RUBY RED' GRAPEFRUIT BY HOT
WATER DIP TREATMENT .................................................57

In tro d u ctio n ........................................................................................5 7
M materials an d M eth od s ......................................................................................... 57
F ru it ..............................................................................5 7
Heat Treatment .................. ..... ......... .......... ........ 58
C h illin g Inju ry ............................................................................................... 5 8
P e e l S c a ld in g ................................................................................................. 5 8
W eight L oss.......................................................... 58
Peel Color .............. ........... ...... ............... .......... 59
Total Soluble Solids and Titratable Acidity ................. ................ ..........59
Peel Puncture R resistance .................................. ....... ............... .... ...........59
P percent Juice .............................................................................................. ........59
Statistical A naly sis ............................................................59
R esu lts an d D iscu ssion ......................................................................................... 59

6 C O N C L U SIO N S ................................................................63

APPENDIX

A ANALYSIS OF VARIANCE FOR CHAPTER 2 .......................................... 66

B ANALYSIS OF VARIANCE FOR CHAPTER 3 ..............................................67

E x p e rim e n t 1 ......................................................................................................... 6 7
E x p e rim e n t 2 ......................................................................................................... 6 8
E x p e rim e n t 3 ......................................................................................................... 6 9
E x p e rim e n t 4 ......................................................................................................... 7 0

C ANALYSIS OF VARIANCE FOR CHAPTER 4 .......................................... 72

E x p e rim e n t 1 ......................................................................................................... 7 2
E x p e rim e n t 2 ......................................................................................................... 7 3
E x p e rim e n t 3 ......................................................................................................... 7 4

D ANALYSIS OF VARIANCE FOR CHAPTER 5 .......................................... 75

L IST O F R E F E R E N C E S ............................................................................................. 77

B IO G R A PH IC A L SK E T C H ....................................................................................... 85















LIST OF TABLES


Table pge

3-1 Peel scalding of 'Valencia' oranges on the heat-treated side of the fruit after
12 d at 10 C ........................................................................ ... ...... 32

3-2 Peel hue, chroma and L* values of 'Valencia' oranges after 14 d of storage
a t 1 0 C ...................................... ................................................. 3 6

3-3 Peel hue, chroma and L* values of the heat-treated and untreated sides of
'Valencia' oranges after 8 d of storage at 10 C............................................. 39

3-4 Total phenolics from flavedo of 'Valencia' oranges immediately after hot water
dip and after 2, 4, and 7 d of storage at 10 C.............................................. ........... 42

3-5 Total protein content from flavedo of 'Valencia' oranges immediately after hot
water dip and after 2, 4, and 7 d of storage at 10 C.............................................43

4-1 Peel scalding (percent of fruit scalded) of 'Ruby Red' grapefruit after 30 s
HW dip treatments followed by post-dip treatments. Fruit were evaluated after
4 w eeks of storage at 10 C .............................................. ............................ 50

4-2 Peel scalding incidence (percent of fruit scalded) of 'Ruby Red' grapefruit after
30 s HW dip treatments followed by post-dip treatments. Fruit were evaluated
after 4 w eeks of storage at 16 C...................... ......... ................................. 53

4-3 Peel scalding severity (percent of fruit surface scalded) of 'Ruby Red' grapefruit
after 30 s HW dip treatments followed by post-dip treatments. Fruit were
evaluated after 4 weeks of storage at 16 C.. ....................................................54

5-1 Peel puncture resistance in Newtons of 'Ruby Red' grapefruit after 30 s dip
treatments. Fruit were evaluated after 4 weeks of storage at 5 or 16 OC ................62

5-2 Weight loss (%) from 'Ruby Red' grapefruit after 30 s dip treatments. Fruit were
evaluated after 7 weeks of storage at 5 or 16 C.............................................. 62

A-1 Analysis of variance for respiration rates of 'Ruby Red' grapefruit during the
first six days in storage at 20 C...................... ......... .................................. 66

A-2 Analysis of variance for ethylene production of 'Ruby Red' grapefruit during the
first six day s in storage at 20 C .................................................... .....................66









A-3 Analysis of variance for percent of fruit scalded, total soluble solids and titratable
acidity of 'Ruby Red' grapefruit after 10 d of storage at 20 C.............................66

B-l Analysis of variance for percent of fruit scalded after 12 d of storage at 10 OC,
electrolyte leakage of treated and untreated sides of 'Valencia' oranges
im m ediately after treatm ent. ............................................ ............................ 67

B-2 Analysis of variance for peel scalding of 'Valencia' oranges after 14 d of storage
at 10 oC ................ ......... ..................... ............. .. ............ 68

B-3 Analysis of variance for electrolyte leakage of treated and untreated sides of
'Valencia' oranges immediately after treatment and after 14 d of storage
at 10 oC........... ......... ..... ......... ................ 68

B-4 Analysis of variance for peel scalding of 'Valencia' oranges after 8 d of storage
at 10 oC..................... ............................... 69

B-5 Analysis of variance for electrolyte leakage of treated and untreated sides
of 'Valencia' oranges immediately after treatment and after 8 d of storage at
1 0 C ................................................................................ 6 9

B-6 Analysis of variance for peel scalding of 'Valencia' oranges after 4 and 7 d of
storage at 10 OC .................................................... ................. 70

B-7 Analysis of variance for electrolyte leakage from flavedo of 'Valencia' oranges
immediately after treatment and after 2, 4 and 7 d of storage at 10 OC.................70

B-8 Analysis of variance for peroxidase activity in flavedo of 'Valencia' oranges
immediately after treatment and after 2, 4 and 7 d of storage at 10 C.................70

B-9 Analysis of variance for total phenolics in flavedo of 'Valencia' oranges
immediately after treatment and after 2, 4 and 7 d of storage at 10 OC.................71

B-10 Analysis of variance for total protein in flavedo of 'Valencia' oranges
immediately after treatment and after 2, 4 and 7 d of storage at 10 C....................71

C-1 Analysis of variance for peel scalding, total decay and chilling injury of 'Ruby
Red' grapefruit. Peel scalding was evaluated after 4 weeks of storage at 10 OC.
Total decay and chilling injury were evaluated after 12 weeks of storage
a t 1 0 C ...................................... ............. .................. ................ 7 2

C-2 Analysis of variance for quality of 'Ruby Red' grapefruit after 4 weeks of
storage at 10 OC .................................................... ................. 72

C-3 Analysis of variance for peel scalding and total decay of 'Ruby Red' grapefruit.
Peel scalding was evaluated after 4 weeks and total decay was evaluated after
12 w eeks of storage at 16 C ....................................... ............... ............... 73









C-4 Analysis of variance for imazalil residue of 'Ruby Red' grapefruit immediately
after treatm ent. ..................................................... ................. 74

C-5 Analysis of variance for peel scalding and stem-end rot of 'Ruby Red' grapefruit.
Peel scalding was evaluated after 4 weeks and stem-end rot was evaluated after
12 w eeks of storage at 16 C................................. ................................... 74

D-1 Analysis of variance for peel scalding, chilling injury index and total decay of
'Ruby Red' grapefruit. Peel scalding was evaluated at 4 weeks of storage.
Chilling injury and total decay were evaluated after 7 weeks of storage ...............75

D-2 Analysis of variance for quality of 'Ruby Red' grapefruit after 4 weeks of
sto rag e ........................................................................... 7 5

D-3 Analysis of variance for weight loss in 'Ruby Red' grapefruit after 4 and 7
w weeks of storage. ................................................... ................. 76















LIST OF FIGURES


Figure pge

2-1 Rates of respiration of 'Ruby Red' grapefruit for the first six d after 62 C hot
water treatment for 60 s and stored at 20 OC. .................... ......................... 22

2-2 Peel scalding of 'Ruby Red' grapefruit after 10 d of storage at 20 OC. Fruit were
dipped in 62 C w ater for 60 s.......................................... ............................ 22

2-3 Peel scalding (% of fruit scalded) of 'Ruby Red' grapefruit due to 62 C hot
water dip for 60 s after 10 d of storage at 20 C. ........................... ..................23

2-4 Total soluble solids content in 'Ruby Red' grapefruit on the 7th and 10th d after
62 C hot w ater treatm ent for 60 s. ........................................ ....... ............... 24

2-5 Titratable acidity in 'Ruby Red' grapefruit on the 7th and 10th d after 62 C hot
w ater treatm ent for 60 s................................................. ............................... 25

3-1 Electrolyte leakage from flavedo of 'Valencia' oranges immediately after 56 C
hot w after treatm ent ......................................... ....... ........ .. ........ .... 32

3-2 Peel scalding (percent of fruit scalded) of 'Valencia' oranges on the heat-treated
side of the fruit after 14 d of storage at 10 OC. ............ ........................................34

3-3 Peel scalding (percent of fruit surface scalded) of 'Valencia' oranges on the
heat-treated side of the fruit after 14 d of storage at 10 OC.............. ................ 35

3-4 Electrolyte leakage from flavedo of the heat-treated sides of 'Valencia' oranges
immediately after treatment and after 14 d of storage at 10 OC. ...........................35

3-5 Peel scalding of 'Valencia' oranges after 8 d of storage at 10 OC. Fruit were
dipped in 66, 68, or 70 C water for 60 s. ..................................... ............... 37

3-6 Peel scalding (percent of fruit scalded) of 'Valencia' oranges on the heat treated
side of the fruit after 8 d of storage at 10 OC. Fruit were dipped in 66, 68, or
70 C water for 60 s..................... ................. ..... ............ 38

3-7 Peel scalding (percent of fruit surface scalded) of 'Valencia' oranges on the
heat-treated side of the fruit after 8 d of storage at 10 OC. ................................... 38









3-8 Electrolyte leakage from flavedo of the heat-treated sides of 'Valencia' oranges
immediately after treatment and after 8 d of storage at 10 OC. .............................39

3-9 Peel scalding (percent of fruit scalded) of 'Valencia' oranges after heat treatment
at 60 and 66 C for 60 s. Scald was evaluated after 4 and 7 d of storage at
1 0 OC ................................................................................ 4 1

3-10 Electrolyte leakage from flavedo of 'Valencia' oranges immediately after hot
water dip for 60 s and after 2, 4, and 7 d of storage at 10 C ..............................41

3-11 Peroxidase activity from flavedo of 'Valencia' oranges immediately after hot
water dip for 60 s and after 2, 4, and 7 d of storage at 10 C..............................42

4-1 Total decay of 'Ruby Red' grapefruit after 30 s HW dip treatments followed by
post-dip treatments. Fruit were evaluated after 12 weeks of storage at 10 C........51

4-2 Chilling injury of 'Ruby Red' grapefruit after 30 s HW dip treatments followed
by post-dip treatments. Fruit were evaluated after 12 weeks of storage at 10 C....51

4-3 Total decay of 'Ruby Red' grapefruit after 30 s HW dip treatments followed by
post-dip treatments. Fruit were evaluated after 12 weeks of storage at 16 C........53

4-4 Imazalil residue in 'Ruby Red' grapefruit immediately after the 30 s dip
treatm ents at 25 and 56 C ........................................................... .....................55

4-5 Stem-end rot for different treatment temperatures after 12 weeks of storage at
16 OC ................................................................................ 5 6

5-1 Chilling injury in 'Ruby Red' grapefruit after 30 s dip treatments of inner and
outer canopy fruit. Fruit were evaluated after 6 weeks of storage at 5 OC plus
1 week at 16 oC. Chilling injury was rated from 0 (none) to 3 (severe). ................61

5-2 Total decay in 'Ruby Red' grapefruit after 30 s dip treatments of inner and outer
canopy fruit. Fruit were evaluated after 7 weeks of storage at 5 or 16 OC...............61















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

HEAT TREATMENTS FOR CONTROLLING POSTHARVEST DISEASES AND
CHILLING INJURY IN FLORIDA CITRUS

By

Karthik-Joseph John-Karuppiah

August 2004

Chair: Mark A. Ritenour
Cochair: Jeffrey K. Brecht
Major Department: Horticultural Science

For years, heat treatment has been tested and used on various fresh horticultural

commodities as a non-chemical method of reducing postharvest diseases, insects, and

physiological disorders such as chilling injury (CI). In citrus, the increasing demand for

fruit with less or no synthetic fungicide residues has led to the development and increased

use of hot water (HW) treatments, especially in Mediterranean climates such as those

found in Israel and California. However, the efficacy of these treatments on citrus under

subtropical Florida conditions is unclear.

In evaluation of physiological effects of HW dip on Florida citrus, rind discs of

'Valencia' oranges dipped in 66, 68 or 70 C water for 60 s had higher electrolyte

leakage immediately after HW dip than non-treated fruit. In another experiment, HW-

dipped fruit (66 C for 60 s) had higher electrolyte leakage and lower peroxidase activity

than non-treated fruit. In all these treatments, 100% of fruit developed scalding. Dipping

'Valencia' oranges in 60 C water for 60 s caused about 20% scalding, but did not









increase electrolyte leakage. Higher electrolyte leakage and lower peroxidase activity

were only observed when peel injury was severe. Hence, these parameters cannot be used

as early indicators of heat injury.

Treating grapefruit with ethylene (2-3 ppm for 3 d) before or after HW dip at 62 C

for 60 s did not affect the response of grapefruit to HW dip. Washing and coating

grapefruit with shellac immediately after HW dip at 56 or 59 C reduced peel scalding by

45% or 37%, respectively, compared with fruit that were not washed and coated. Dipping

grapefruit at 56 or 59 C for 30 s followed by washing and shellac coating reduced

incidence of CI to 2% or 1%, respectively, compared with 50% CI for fruit dipped at 25

C followed by no post-dip treatment. In another experiment, HW dip at 56 or 59 C for

30 s developed 18% or 32%, respectively, less CI after storage at 5 OC for 6 weeks plus 1

week at 16 C compared with fruit dipped at 25 C. Hot water dip was more effective in

reducing CI of inner canopy fruit (32%) compared with outer canopy fruit (10%).

After 12 weeks of storage, November-harvested grapefruit dipped in water at 56 or

59 C for 30 s developed 25% or 18% decay, respectively, compared with 40% decay in

fruit dipped at 25 C water. In an experiment using February-harvested fruit, dipping in

heated solutions of 3% or 6% sodium carbonate increased the incidence of stem-end rot

by 50% compared with fruit dipped in water alone. Dipping fruit in heated solutions of

125 or 250 ppm imazalil did not have any effect in reducing decay after 12 weeks of

storage. This could have been due to low incidence of decay in February-harvested fruit.

Dipping grapefruit in 59 C water for 30 s induced significant peel scalding in all

the experiments whereas treatment at 56 C for 30 s did not induce peel scalding. Hot

water dip treatment at 56 C for 30 s followed by washing and shellac coating was

effective in reducing postharvest decay and CI without causing damage to the peel.














CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW

The origin of almost all citrus species was probably the southern slopes of the

Himalayas in northeastern India and adjacent Burma. The origin of trifoliate orange and

kumquat is eastern China. In Florida, sweet oranges could have been introduced in 1565

when the colony at St. Augustine was established. The important species of citrus are

Citrus sinensis (sweet orange), C. paradisi (grapefruit), C. reticulata (mandarin), C.

aurantifolia (sour lime) and C. grandis (pummelo). Citrus production in the United States

is second only to Brazil. However, the U.S. ranks first worldwide in grapefruit

production. Commercial citrus production in the U.S. is limited to the states of Florida,

California, Texas and Arizona. Florida accounts for 74% of the total U.S. citrus

production, California produces 23% and Texas and Arizona contribute the remaining 3%

(NASS, 2003).

Though Florida is the number one grapefruit producer in the world, less than 50%

of the grapefruit produced in Florida is suitable for the fresh market primarily due to

cosmetic defects (NASS, 2003). In 2002-03, 65% of fresh grapefruit was exported from

the U.S., of which 36% was exported to Japan (Fla. Dept. Citrus, 2004).

Because of the increased attention on the importance of fresh fruits and vegetables

as part of good human nutrition, consumption of fresh fruits and vegetables continues to

increase. Furthermore, worldwide marketing and shipment of produce has increased the

requirement for maintaining fruit and vegetable quality throughout extended shipping and

storage durations. For a 50% packout of fresh Florida grapefruit, meaning that 50% of the









fruit are suitable for fresh market, harvesting and postharvest handling accounts for 63%

of the total cost of production and postharvest handling (University of Florida, 2003;

University of Florida, 2004). Fresh grapefruit shipped from Florida to Japan must

maintain its quality throughout the almost 30-d transit, plus an additional 1 to 3 months in

storage at destination warehouses. During this time, as with most commodities, decay

represents one of the greatest sources of product and economic loss. Grapefruit exported

from the U.S. must meet U.S. grade standards of U.S. Fancy, U.S. #1 Bright, or U.S. #1.

The tolerance level for decay for these grades is only 3% (USDA, 1997). So, reducing

postharvest decay of citrus is of critical importance for maintaining Florida's

competitiveness, especially in international markets.

The susceptibility of fresh horticultural commodities to postharvest diseases

increases during prolonged storage as a result of physiological changes in the fruits and

vegetables that enable pathogens to develop (Eckert and Ogawa, 1988). Postharvest

chemical treatments are very effective in controlling decay and are widely used on citrus.

Recently there has been an increased demand for fresh horticultural commodities with

less or no chemical residues. A number of fungicides are no longer registered for use on

fresh citrus, including those that were used to effectively control postharvest diseases.

There are only three fungicides (imazalil, thiabendazole and sodium o-phenylphenate)

currently registered for postharvest use on citrus and there are problems like development

of resistant pathogenic strains and environmental concerns in disposing the chemicals. To

minimize pre- or postharvest treatments of fresh citrus with synthetic fungicides, research

efforts are currently focused on enhancement of host resistance to pathogens through









physical, chemical, or biological agents (Ben-Yehoshua et al., 1988, 1997; Wilson et al.,

1994).

Of the physical treatments, prestorage heat treatment appears to be a promising

method of decay control for fresh horticultural commodities in general (Couey, 1989;

Klein and Lurie, 1991; Schirra and Ben-Yehoshua, 1999). Many fresh horticultural

products can tolerate temperatures of 50 C to 60 OC for up to 10 min, but shorter

exposure at this temperature can control many postharvest diseases (Barkai-Golan and

Phillips, 1991). Heat treatments may be applied to fruits and vegetables via: 1) long-

duration (e.g., 3-7 d) curing at warm temperatures and high relative humidity (RH) (Kim

et al., 1991), 2) long- or short-duration hot water (HW) dips (Schirra and Ben-Yehoshua,

1999), 3) vapor heat (Hallman et al., 1990a; Paull, 1994), 4) hot dry air (Lurie, 1998a, b;

Schirra and Ben-Yehoshua, 1999) or 5) short-duration (< 1 min) HW sprays (Fallik et al.,

1996).

Curing

Curing involves heat treatment for relatively long periods (i.e., 3-7 d). The first

curing experiments on citrus fruit were conducted by Fawcett in 1922 to reduce

Phytophthora citrophthora. In this case, fruit were held for 1-3 d at 30-36 C in a water-

saturated atmosphere. Ben-Yehoshua et al. (1987 a, b) showed that curing of seal-

packaged citrus fruit at 36 C and saturated humidity for 3 d effectively reduced decay

without damage during subsequent storage at 17 OC for 35 d. Curing citrus fruits

increases their resistance to green mold development (Brown and Barmore, 1983; Kim et

al., 1991). Postharvest curing at 34-36 C for 48-72 h effectively controls citrus decay









and reduces chilling injury (CI) symptoms (Ben-Yehoshua et al., 1987b; Del Rio et al.,

1992).

Curing lemons for 3 d at 36 C reduced the decline of anti-fungal compounds (that

inhibit germ tube elongation) in the flavedo tissue, reduced the loss of citral, and thus

suppressed decay development (Ben-Yehoshua et al., 1995).

In spite of its beneficial effects on reducing decay and CI in different citrus fruits,

curing is not widely utilized on a commercial scale for citrus. Practical implementation of

curing is difficult because of the long treatment durations, which result in fruit damage,

and the high cost of heating large volumes of fruit for up to 3 d (Schirra et al., 2000).

Vapor Heat

At temperatures higher than what is normally used for curing, the major obstacle to

widespread use of heat to reduce postharvest disease or insect infestation is the sensitivity

of many fruits to the high temperatures required for effective treatment (Couey, 1989).

For example, navel oranges, lemons, and avocados grown in California were easily

damaged by the vapor heat treatment (Sinclair and Lindgren, 1955). Visible heat damage

was reported for grapefruit exposed to forced vapor at 46 'C for 3.75 h (Hallman et al.,

1990b). Flavor and appearance of grapefruit air-heated at 46 C for 3 h with controlled

atmosphere were inferior to those of non-heated fruit (Shellie et al., 1997). Vapor heat

treatment at 59 C for 180 s resulted in severe peel scalding of grapefruit (over 77% of

the fruit surface) (Ritenour et al., 2003).

Hot Water Dipping

Hot water dip treatments are applied for only a few seconds to minutes at

temperatures higher than those used for vapor heat or hot air. Hot water dips have been









used for many years as non-chemical methods to control postharvest decay in various

fruits and vegetables (Barkai-Golan and Phillips, 1991; Couey, 1989; Lurie, 1998b).

Fawcett (1922) conducted the first studies using HW treatments and first reported control

of decay in oranges. For citrus, HW dips (2-3 min at 50-53 C) were shown to be as

effective as curing for 72 h at 36 C in controlling postharvest decay and CI in various

citrus fruits and are much less expensive, mainly because of shorter treatment duration

(Rodov et al., 1993, 1995a).

Dipping grapefruit in water at 53 C for 3 min resulted in about 50% reduction in

decay (Rodov et al., 1995a). Ben-Yehoshua et al. (2000) reported that the effective

temperature range for 2 min grapefruit dip treatments is between 51 and 54 C;

temperatures above 54 C caused brown discoloration of the peel and temperatures below

51 C were not effective in reducing decay.

Hot water dips at 52 C for 3 min have been shown to reduce green mold in organic

lemon inoculated with the spores ofPenicillium digitatum (Lanza et al., 2000). In

'Bianchetto' and 'Verdello' lemons, there was only 1% and 0% decay, respectively, in

the HW dip-treated fruit, which was as effective as non-heated imazalil treatment (Ig

a.i./L), which had no decay. The untreated 'Bianchetto' and 'Verdello' fruit developed

86% and 75% decay, respectively.

Most work on HW treatments of citrus has evaluated their effectiveness in reducing

decay from Penicillium molds. While these represent the most important cause of citrus

postharvest decay worldwide (Eckert and Brown, 1986), stem-end rot (SER; primarily

from Lasiodiplodia theobromae) commonly is the primary cause of postharvest decay in

Florida. Ritenour et al. (2003) reported that grapefruit dipped in water at 56 C for 120 s









developed SER in only 8% of the fruit compared with 33% of the fruit with SER in fruit

dipped in ambient water. However, they found very few instances where water

temperatures and dipping times that reduced SER did not also injure the fruit.

Fungicides in heated solutions (50-60 C) are more effective at controlling decay

than non-heated solutions. Heated solutions leave higher fungicide residues on the fruit

than non-heated solutions and are therefore effective even at much lower concentrations

(Cabras et al., 1999). Heated solutions ofthiabendazole (TBZ) and imazalil are more

effective at controlling postharvest decay in citrus than non-heated solutions (McDonald

et al., 1991; Schirra and Mulas, 1995a, 1995b; Wild, 1993).

Heated solutions of compounds generally recognized as safe (GRAS) like sulfur

dioxide, ethanol, and sodium carbonate have been found to be efficient in controlling

green mold in citrus (Smilanick et al., 1995, 1997). For example, navel and 'Valencia'

oranges inoculated with Penicillium digitatum spores, and after 24 h immersed in 2%, 4%

or 6% sodium carbonate solutions for 1 or 2 min at 35.0, 40.6, 43.3, or 46.1 C, had 40%-

70% less green mold than in fruit dipped in water alone (Smilanick et al., 1997).

Treatments of 4% or 6% sodium carbonate at 40.6 or 43.3 C provided better decay

control than did 2% sodium carbonate at 35.0 or 46.1 C.

In contrast to the report of Smilanick et al. (1997), Palou et al. (2001) found that

temperature of the sodium carbonate solution had more effect than concentration of the

sodium carbonate. Treating oranges in 3% or 4% sodium carbonate solution for 150 s at

45 C reduced the incidence of green mold and blue mold to 1% and 14%, respectively,

whereas the hot water without sodium carbonate reduced the incidence of green mold and

blue mold to 12% and 27%, respectively. The untreated fruit had 100% decay incidence.









Palou et al. (2002) evaluated the effects of dipping 'Clementine' mandarins in 0%,

2% or 3% sodium carbonate solutions at 20, 45 or 50 C for 60 or 150 s. Fruit were

artificially inoculated with Penicillium digitatum 2 h prior to treatment. Treatment with

45 or 50 C water alone did not effectively control the decay. Adding sodium carbonate

to the solution enhanced decay control compared with water alone at all temperatures and

immersion periods. Dipping the fruit in 3% sodium carbonate solution at 50 OC for 150 s

completely controlled decay development. At lower temperatures, sodium carbonate

solution was more effective than water alone and reduced the risk of peel damage by

heat.

Hot Water Brushing

Recently, interest has been focused on short-duration HW rinsing and brushing of

fresh fruits and vegetables (Fallik et al., 1996). In this method, HW is sprayed over the

produce as it moves along a set of brush rollers, thus, simultaneously cleaning and

disinfecting the produce (Porat et al., 2000a). Hot water brushing (10-30 s at 55-64 C;

Israeli patent 116965) has been commercially used in Israel with bell peppers (Fallik et

al., 1999), mangoes (Prusky et al., 1999), kumquat (Ben-Yehoshua et al., 1998), citrus

(Porat et al., 2000a), and several other crops to reduce postharvest decay. Hot water

brushing of grapefruit for 20 s at 56, 59 or 62 C, reduced decay by 80%, 95%, and 99%,

respectively, compared with brushing in ambient water (Porat et al., 2000a).

In separate experiments, 'Oroblanco' citrus fruit (a pummelo-grapefruit hybrid)

were either dipped in water for 2 min at 52 C or HW brushed (10 s at 52, 56 or 60 C)

(Rodov et al., 2000). After air-drying and waxing, dipped fruit developed significantly

less decay than did the non-treated fruit. Decay development was less after HW brushing









for 10 s at 56 or 60 C than after HW brushing at 52 C, but was higher than HW-dipped

fruit.

For grapefruit, HW brushing at 62 C for 20 s significantly reduced green mold

even when the fruit were inoculated with Penicillium digitatum; only 22% or 32% of the

inoculated wounds were infected with green mold if inoculated 1 or 3 d after HW

treatment, respectively, compared with 50% or 59% infection if inoculated on the HW

treatment day or 7 d later, respectively (Pavoncello, 2001). For grapefruit brushed and

rinsed for 20 s with 20, 53, 56, 59, or 62 C water and then inoculated with Penicillium

digitatum 24 h later, treatments at 53 C did not significantly reduce decay, whereas

treatments at 56, 59, or 62 C reduced postharvest decay by 20%, 52%, or 69%,

respectively, compared with the untreated fruit (Porat et al., 2000b). Since inoculation

occurred after HW brushing in these experiments, they demonstrate that HW treatment

reduce decay by enhancing grapefruit resistance to the decay organisms.

Commodity Responses to Heat Treatments

When fruit are exposed to high temperatures, there is potential risk of injury.

Symptoms of heat injury can be external (i.e., peel scalding, pitting, etc.) or internal (i.e.,

softening, discoloration, tissue disintegration, off-flavors, etc.) (Lurie, 1998a). For

example, Miller et al. (1988) dipped grapefruit in 43.5 C water for 4 h and observed

resulting peel discoloration after 3 weeks.

Scald

Peel scalding is a brown discoloration of the flavedo that can be caused by heat

treatment. Schirra and D'hallewin (1997) dipped 'Fortune' mandarins in 50, 54, 56, or

58 C water for 3 min and then stored at 6 OC for 30 d followed by 3 d at 20 OC. After









storage, 10%, 70% or 100% of the fruit developed peel scalding after HW dip in 54, 56,

or 58 C water, respectively.

Dipping 'Tarocco' oranges in 53 C water for 3 min caused little peel scald in fruit

harvested in February and none in fruit harvested in March (Schirra et al., 1997). Fruit

harvest before February developed 20-30% peel scald, whereas 70% of fruit harvested in

April developed peel scald.

In preliminary experiments, HW brushing at 60 OC damaged 'Shamouti' oranges.

In contrast, HW brushing treatment at 56 C for 20 s is non-damaging in all tested

cultivars of citrus (Porat et al., 2000a).

Ritenour et al. (2003) showed a time x temperature interaction for peel scalding in

grapefruit. Fruit dipped in water at 56 C for 120 s, 59 C for 20-120 s or 62 C for 10-

120 s developed significant peel scalding after 33 d in storage at 10 OC. Hot water dips at

62 C for 60 or 120 s resulted in 100% peel scalding.

Electrolyte Leakage

'Fortune' mandarins dipped in 50, 52, 54, 56 or 58 C water for 3 min were stored

at 6 C for 30 d followed by 3 d at 20 OC (Schirra and D'hallewin, 1997). Immediately

after treatment, there was no significant difference in electrolyte leakage, while at the end

of storage period, fruit dipped in water at 56 or 58 C had greater electrolyte leakage than

did fruit dipped in water at 50, 52 or 54 C. However, Schirra et al. (1997) reported no

change in electrolyte leakage due to HW dips of 'Tarocco' oranges at 53 C for 3 min.

Respiration and Juice Quality

Respiration is the process of breakdown of organic materials to simple end

products, providing energy required for various metabolic processes and results in the

loss of food reserves (Kader, 2002). Increased respiration allows enhanced metabolic









rates to occur in the commodity and thus supports more rapid senescence. Temperature

affects the respiration rate of fruits and vegetables by increasing the demand for energy to

drive metabolic reactions. The respiration rate thus increases with increase in temperature

of the product.

After 33 d in storage, the respiration rate was higher in 'Fortune' mandarins

previously dipped in 56 or 58 C water (Schirra and D'hallewin, 1997). The rate of

ethylene production was also higher in fruit treated at 58 C. Total soluble solids (TSS)

and titratable acidity (TA), however, did not show much difference between the untreated

and the heat-treated (50, 54, 56 and 58 C for 3 min) fruit.

Dipping 'Tarocco' oranges in 53 C water for 3 min did not influence the

respiration rate, ethylene production, TSS and TA (Schirra et al., 1997). Hot water

brushing at 56 C for 20 s did not affect juice TSS and TA in 'Minneola' tangerines,

'Shamouti' oranges and 'Star Ruby' red grapefruit (Porat et al., 2000a).

In general, respiration rate was higher immediately after heat treatment, but later

decreased to levels like that of the non-treated fruit. Juice TSS and TA were not affected

by heat treatments.

Peel Color

No significant difference could be found in rind color between untreated 'Fortune'

mandarin fruit and those treated in water at 50 or 54 C for 3 min (Schirra and

D'hallewin, 1997). L* values were lower for mandarins treated at 58 C, a* values were

lower for fruit treated at 54, 56 and 58 C, and b* values were lower for fruit treated at 56

and 58 C. Hot water brushing at 60 OC for 10 s followed by waxing slowed the

yellowing process in 'Oroblanco' (Rodov et al., 2000). There was a delay of about 2

weeks in the change of rind color as compared with the non-treated fruit.









Flesh and Peel Firmness

Firmness of citrus fruit depends mainly on turgidity and weight loss (Rodov et al.,

1997). Heat treatment causes the redistribution of natural epicuticular wax on the fruit

surface and closes many microscopic cuticular cracks (Rodov et al., 1996). This could be

the reason for better maintenance of firmness in heat-treated citrus fruit. However, in

further experiments, maintenance of citrus fruit firmness by heat treatment was not

accompanied by reduction in weight loss (Rodov et al., 2000). Heat treatment could have

inhibited enzymatic action involved in softening or enhanced cell wall strengthening

processes like lignification. Hot water dips for 2 min at 52 C maintained firmness by

inhibiting fruit softening 'Oroblanco'. Hot water brushing at 60 OC for 10 s also

maintained fruit firmness but brushing at 52 or 56 C did not.

Structural Changes of Epicuticular Wax

A number of deep surface cracks that form an interconnected network on peel

surface are observed on the epicuticular wax of non-heated apples (Roy et al., 1999).

Changes in the structure of the epicuticular wax are quite similar following different

types of heat treatment (Schirra et al., 2000). After heat treatment at 38 C for 4 d, the

cuticular cracks on apples disappeared, probably due to the melting of wax platelets (Roy

et al., 1994). Changes in epicuticular wax structure have been noticed in several types of

produce when heat-treated, such as after a 2-min water dip at 52 C of 'Oroblanco'

(Rodov et al., 1996), a 2-min water dip at 50-54 C of 'Fortune' mandarins (Schirra and

D'hallewin, 1996, 1997), and a 20 s HW brushing at 56-62 C of grapefruit (Porat et al.,

2000a).









Heat treated 'Marsh' grapefruit showed that during long term storage the cuticular

cracks became wider and deeper (D'hallewin and Schirra, 2000). Also during long term

storage, severe alterations occurred in the outer stomatal chambers, thus becoming

important invasion sites for wound pathogens (Eckert and Eaks, 1988).

Host-Pathogen Interaction

Inoculum levels are directly related to decay potential of a particular commodity

(Trapero-Casas and Kaiser, 1992; Yao and Tuite, 1989). The effect of heat on decay-

causing organisms can be influenced by factors like moisture content of spores, age of the

inoculum, and inoculum concentration (Barkai-Golan and Phillips, 1991), as well as

factors inherent within the host (i.e., physiological maturity and stress) (Klein and Lurie,

1991). Schirra et al. (2000) reported that heat treatments have a direct effect on fungal

pathogens by slowing germ tube elongation or by inactivating or killing the germinating

spores.

Heat treatments may also have an indirect effect on decay development by inducing

anti-fungal substances within the commodity that inhibit fungal development or by

promoting the healing of wounds on the commodity (Schirra et al., 2000). Oil glands of

citrus flavedo contain compounds, such as citral in lemons that have anti-fungal activity

(Ben-Yehoshua et al., 1992; Kim et al., 1991; Rodov et al., 1995b). Heat treatments

enhance wound healing by promoting the synthesis of lignin-like compounds and these

compounds act as physical barriers to the penetration of pathogens (Schirra et al., 2000).

High concentration of scoparone was found in heat-treated citrus and this could have

anti-fungal properties (Kim et al., 1991).

Heat treatments may also influence decay susceptibility by altering surface features

of the commodity. For example, HW brushing of 'Minneola' tangerines at 56 C for 20 s









smoothed the fruit epicuticular waxes, which covered and sealed the stomata and

microscopic cracks on the fruit (Porat et al., 2000a). This could reduce the entry of

pathogens and thus reduce decay development.

Heat treatments can also damage tissues and thus make them more susceptible to

invasion by pathogens. For example, HW and vapor heat treatments used to control

Caribbean fruit fly resulted in increased decay compared with the non-treated fruit

(Hallman et al., 1990a; Miller et al., 1988). The long duration of treatment caused

damage to the tissue and this led to increased decay (Jacobi and Wong, 1992).

Water dips for 4.5 h at 43.5 C increased decay of 'Marsh' grapefruit compared

with fruit dipped in ambient water and the non-treated fruit (Miller et al., 1988). Heat-

treated fruit developed 45% decay in 2 weeks whereas fruit dipped in ambient water and

untreated fruit developed only 6% and 1% decay, respectively. Grapefruit dipped in

62 C water for 30 s developed only 5% SER after 82 d in storage, whereas increasing

the treatment duration to 120 s caused significant peel scalding (100%) and increased

incidence of SER (23%) (Ritenour et al., 2003).

Stem-End Rot

Stem-end rot of citrus fruits is caused by either Lasiodiplodia theobromae

(previously known as Diplodia natalensis and commonly called Diplodia stem-end rot)

or Phomopsis citri. The pathogen infects the calyx of immature citrus fruit and remains

quiescent (Eckert and Brown, 1986). After harvest, decay develops when the fungus

grows from the calyx into the fruit. The fungus does not spread from fruit to fruit in

packed containers. Stem-end rot may also begin in wounds in the rind and at the stylar

end of the fruit.









Diplodia SER is the most serious postharvest disease in Florida citrus. The warm

and humid conditions in Florida favor the development of SER. Development of

Diplodia SER is hastened during degreening with ethylene and it is more serious in early

season degreened fruits (Brooks, 1944; McCornack, 1972). Phomopsis SER is more

prevalent later in the season when degreening is not necessary.

Stem-end rots appear as a leathery, pliable area encircling the button or stem-end of

citrus fruit. The affected area is buff colored to brown. Diplodia proceeds more rapidly

through the core than the rind and the decay often appears at both ends of the fruit. It

usually develops unevenly in the rind and forms finger-like projections. Phomopsis SER

spreads through the core and in a nearly even rind pattern from the stem-end without

finger-like projections.

Anthracnose

Anthracnose of citrus fruits is caused by Colletotrichum gloeosporioides. This

fungus also infects immature fruit before harvest and remains quiescent (Eckert and

Brown, 1986). Degreening with ethylene enhances anthracnose development and so it is

more severe in early season fruit (Brown, 1975, 1978). The lesions initially appear silvery

gray and leathery. The rind becomes brown to grayish black and softens as the rot

progresses. In humid conditions, pink masses of spores may form on the lesions.

Infections do not spread to adjacent healthy fruit.

Green Mold

Green mold of citrus fruits is caused by Penicillium digitatum. The fungus enters

the fruit only through injuries in the skin (Eckert and Brown, 1986). Initially the decay

appears as soft, water-soaked spots. Later the white fungal mycelium appears on the

surface and produces a mass of powdery olive green spores. As the disease advances, the









colored sporulating area is surrounded by a white margin. The fungus produces enzymes

that break down the cell walls and macerate the tissues as the mycelium grows. Finally

the decayed fruit becomes soft, shrunken, and shriveled and is entirely covered with

green spores. Green mold is the major decay causing organism in citrus fruits worldwide.

But in Florida this disease is less severe than SER and anthracnose.

Minor Postharvest Diseases

Some of the other citrus postharvest diseases that are of less importance are black

rot (Alternaria citri), blue mold (Penicillium italicum) and sour rot (Galactomyces citri-

aurantii).

Physiological Disorders

Physiological disorders are caused by nutritional imbalances, improper harvesting

and handling practices or storage at undesirable temperatures (Grierson, 1986). Some of

the common physiological disorders in citrus fruits are CI, stem-end rind breakdown,

postharvest pitting, blossom-end clearing, and oleocellosis.

Chilling injury occurs mainly in tropical and subtropical commodities when held

below a critical threshold temperature, but above their freezing point (Kader, 2002).

Grapefruit develops CI when stored at temperatures below 10-12 C (Chace et al., 1966).

Symptoms of CI become more noticeable when the fruit are transferred to non-chilling

temperatures. Grapefruit harvested early and late in the season are more susceptible to CI

than are fruit harvested in mid-season (Grierson and Hatton, 1977). Fruit from the outer-

canopy are more susceptible to CI than are fruit from the inner-canopy (Purvis, 1980).

Common symptoms of CI are surface pitting, discoloration of the skin or water-soaked

area of the rind (Grierson, 1986).









Chilling injury in many horticultural commodities has been successfully reduced by

high temperature prestorage conditioning or by curing treatments (Wang, 1990). Changes

in respiratory rate, soluble carbohydrates, and starch content in 'Fortune' mandarin due to

conditioning (3 d at 37 C) were evaluated by Holland et al. (2002). They found that

changes in carbohydrate concentration were mainly due to the consumption of

carbohydrates for respiration, which was highest in the heat-treated fruit. There was no

relation between CI and changes in carbohydrates. The sucrose levels in untreated fruit

were 57-79% lower than the heat-treated fruit. But there were losses in glucose, fructose,

and starch in heat-treated fruit. So sucrose could be involved in heat-induced chilling

tolerance of citrus fruit.

Rodov et al. (1995a) studied the effect of curing and HW dip treatments on CI

development of grapefruit. Curing (36 C for 72 h), HW dip (53 C for 3 min) or hot

imazalil dip (53 C for 3 min) each reduced CI by about 40%. When grapefruit were

sealed in plastic film (D-950 film, Cryovac) after HW dip treatment, CI was reduced by

about 60%. But curing and sealing the fruit did not provide better decay control than

curing alone.

'Tarocco' oranges, harvested monthly from November to April and then dipped in

53 C water for 3 min, were stored at 3 OC for 10 weeks followed by 1 week at 20 C

(Schirra et al., 1997). The fruit harvested between November and January were more

susceptible to CI than fruit harvested in February, March or April. Heat treatment

reduced the development of CI, especially in fruit that were more susceptible to CI.

'Tarocco' oranges harvested monthly from December to April were dipped in water

or TBZ solution (200 ppm) at 50 OC for 3 min (Schirra et al., 1998). Dipping fruit in HW









alone reduced CI only in fruit that were harvested during January or April. But, dipping

the fruit in heated TBZ solution significantly reduced CI throughout the season.

'Valencia' oranges dipped in water, benomyl (500 mg/L) or TBZ (1000 mg/L) for

2 min at ambient temperature or 53 C developed less CI after 15 weeks of storage at

1 C when dipped at 53 C (Wild and Hood, 1989). In another experiment, grapefruit

dipped in water, TBZ (1000mg/L) or benomyl (500 mg/L) for 2 min at 14 OC or 50 C

developed significantly less CI after 8 weeks storage at 1 OC when dipped at the higher

temperature (Wild, 1993).

Objectives

* Determine the effects of ethylene treatment of grapefruit on response to hot water
dip treatment

* Determine the physiological responses of oranges to hot water dip treatment

* Determine if postharvest diseases in Florida grapefruit can be reduced by hot water
dip treatment

* Determine if hot water dip treatment can reduce chilling injury in Florida grapefruit














CHAPTER 2
EFFECTS OF ETHYLENE TREATMENT ON 'RUBY RED' GRAPEFRUIT
QUALITY AFTER SHORT-DURATION HOT WATER DIP TREATMENTS

Introduction

Degreening with ethylene is a common practice with early season grapefruit that

enhances rind color without affecting the fruit's internal quality. But ethylene treatment

can have adverse effects like making the fruit susceptible to Diplodia stem-end rot

(Brooks, 1944; McCornack, 1972). The objective of this experiment was to determine if

ethylene treatment affects the response of grapefruit to hot water (HW) dip treatments.

Materials and Methods

Fruit

Commercially mature (TSS:TA > 7:1) and healthy 'Ruby Red' grapefruit were

harvested randomly from 1-1.5 m above ground level on healthy trees, evenly spaced

around the tree and distributed through the inner and outer canopy on 22 Nov. 2002 at the

Indian River Research and Education Center research grove in Fort Pierce, Fla. The trees

received standard commercial care. The fruit were transported to Gainesville, Fla. on the

day of harvest and stored at 10 OC overnight before receiving their respective treatments

the following day.

Heat Treatment and Degreening

The fruit were exposed to one of the following treatments.

1. Control (no HW dip or degreening)

2. Degreening only

3. Hot water dip only









4. Hot water dip followed by degreening

5. Degreening followed by HW dip

Each treatment had three replicates of 20 fruit each. Hot water dips were done at

62 C for 60 s. Hot water dip was administered using a laboratory scale fruit heating

system (Model HWH-2, Gaffney Engineering, Gainesville, Fla.) capable of maintaining

water temperatures up to 65 C, to a stability within +2.0 C of the initial water

temperature for the first 4-5 min after submerging up to 32 kg of fruit with an initial fruit

temperature of 20 C. Following treatment, the fruit were air dried and stored at 20 OC.

Degreening was accomplished by treating fruit with 2-3 ppm ethylene for 3 d at 29 C

and 95% relative humidity (RH). Fruit that were not degreened were held at 20 OC and

95% RH while fruit from the other treatments were being degreened. Initial total soluble

solids (TSS) and titratable acidity (TA) were measured from three replicates of 10 fruit

each. Following treatments, fruit were stored at 20 OC, with 95% RH. Rates of respiration

and ethylene production were measured for 6 d. On the 7th d of storage, half the fruit were

evaluated for peel scalding, TSS and TA, and on the 10th d, the remaining 10 fruit per

replication were evaluated.

Respiration Rate and Ethylene Production

Rates of respiration and ethylene production were measured by the static system.

Three fruit per replicate were weighed and sealed together in a 3 L container for 2 h. Gas

samples (0.5 mL) were withdrawn through a rubber septum using a syringe and the

percentage of carbon dioxide determined using a Gow-Mac gas chromatograph (Series

580, Bridgewater, N.J.) equipped with a thermal conductivity detector. The respiration

rate was calculated using the following formula:









Respiration rate (mL CO2-kg1-h-1) = % CO2 void volume (mL)
Sample weight (kg) sealed time (h) 100

Ethylene production was measured by injecting a 1 mL gas sample into a HP 5890

gas chromatograph (Hewlett Packard, Avondale, Pa.) equipped with a flame ionization

detector. The rate of ethylene production was calculated using the following formula:

iL C2H4.kg-lh-1 = ppm C2H4 void volume (mL)
Sample weight (kg) sealed time (h) 1000

Peel Scalding

Peel scalding was evaluated on each fruit and the percentage of fruit showing any

peel scalding was calculated.

Total Soluble Solids and Titratable Acidity

A 13 mm thick cross sectional slice was removed from the equatorial region of

each fruit and the peel removed. The samples were macerated in a blender and then

centrifuged at 4,000 gn for 20 min. The supernatant solution was used for measuring TSS

and TA. Total soluble solids were measured using a Mark II refractometer (Model 10480,

Reichert-Jung, Depew, N.Y.) and the values expressed as Brix. Titratable acidity was

measured with an automatic titrimeter (Fisher Titrimeter II, No. 9-313-10, Pittsburg, Pa.)

using 6.00 g of juice that was diluted with 50 mL of distilled water and titrated with 0.1 N

NaOH to an endpoint of pH 8.2. Titratable acidity was expressed as percentage citric

acid. The TA was calculated as follows:

TA (% citric acid) = mL NaOH Normality of NaOH 0.064 100
6 g of juice

Statistical Analysis

Percentage data were transformed to arcsine values and analyzed by ANOVA using

SAS (PROC GLM) for PC (SAS Institute Inc, Cary, N.C.). When differences were









significant (P < 0.05), individual treatment means were separated using Duncan's

Multiple Range Test (P = 0.05). Means presented are untransformed values.

Results and Discussion

Dipping grapefruit in 62 C water for 60 s before or after degreening did not result

in consistent differences in the respiration rate (Figure 2-1). Respiration increased

dramatically in fruit from all treatments on the 6th d of storage, but there were no

significant differences between treatments. There were no decay of fruit and hence the

sudden increase can be due to mistake in calibration of the gas chromatograph. Ethylene

production was low (<0.1 L-kg-lhrf1) and not significantly different among treatments

throughout the entire 6-d monitoring period (data not shown). Schirra and D'hallewin

(1997) have shown that ethylene production from 'Fortune' mandarins was stimulated by

treatment at 58 C for 3 min. But, Schirra et al. (1997) reported that a heat treatment at

53 C for 3 min did not affect the respiration rate and ethylene production of 'Tarocco'

oranges.

Peel scalding (Figire 2-2) did not develop on fruit that were not dipped in heated

water, regardless if they were degreened or not (Figure 2-3). However, 50% of the fruit

receiving the HW dip treatments developed peel scalding after 10 d in storage. Scalding

was not significantly affected by the degreening treatments. There were no consistent

differences in TSS (Figure 2-4) or TA (Figure 2-5). Porat et al. (1999) reported that

ethylene degreening does not affect the juice TSS and TA of 'Shamouti' oranges. Neither

did HW dips at 53 C for 3 min have any effect on the TSS and TA of 'Tarocco' oranges

(Schirra et al., 1997). Heat treating grapefruit before or after degreening did not have any

effect on the incidence of peel scalding, rates of respiration and ethylene production, or










16

14
LSDo,,,
12 12



--. --- --*---*DW HT
10

8


o- HTWD

2 -. HTBD

0 -----------------------------
1 2 3 4 5 6
Days after treatment

Figure 2-1. Rates of respiration of 'Ruby Red' grapefruit for the first six d after 62 C hot
water treatment for 60 s and stored at 20 C. Vertical bar represents the 5%
LSD value. Ctrl Control (no HW dip or degreening), DWHT Degreening
without heat treatment, HTWD Heat treatment without degreening, HTBD -
Heat treatment before degreening and HTAD Heat treatment after
degreening.


Figure 2-2. Peel scalding of 'Ruby Red' grapefruit after 10 d of storage at 20 OC. Fruit
were dipped in 62 C water for 60 s.











on juice quality (TSS and TA). From these results it may be concluded that degreening


grapefruit with ethylene had no effect on the response to HW dip treatment.


DWHT


HTWD
Treatment (62 OC for 60 s)


HTBD


HTAD


Figure 2-3. Peel scalding (% of fruit scalded) of 'Ruby Red' grapefruit due to 62 C hot
water dip for 60 s after 10 d of storage at 20 OC. Bars with different letters are
significantly different by Duncan's multiple range test at P < 0.05. Ctrl -
Control (no HW dip or degreening), DWHT Degreening without heat
treatment, HTWD Heat treatment without degreening, HTBD Heat
treatment before degreening and HTAD Heat treatment after degreening.


60


50


S40
&

330
B3U

20


10


0


a a















b b











12


10a a
10
b a .1 --

8--
OCtrl
o IEDWHT
S6 OHTWD

-- HTBD
S HTAD
4 --






0
2 -




7th Day 10th Day

Days after treatment
Figure 2-4. Total soluble solids content in 'Ruby Red' grapefruit on the 7th and 10th d
after 62 C hot water treatment for 60 s. Bars within each day with different
letters are significantly different by Duncan's multiple range test at P < 0.05.
Ctrl Control (no HW dip or degreening), DWHT Degreening without heat
treatment, HTWD Heat treatment without degreening, HTBD Heat
treatment before degreening and HTAD Heat treatment after degreening.










1.4

ab a
1.2- 0 a
:,b ,b ab
S1.0 bc bc

Ctrl
S0-.8 3MDWHT

O HTWD
0.6 -- HTBD
Z E HTAD
S0.4


0.2


0.0
7th Day 10th Day
Days after treatment

Figure 2-5. Titratable acidity in 'Ruby Red' grapefruit on the 7th and 10th d after 62 C
hot water treatment for 60 s. Bars within each day with different letters are
significantly different by Duncan's multiple range test at P < 0.05. Ctrl -
Control (no HW dip or degreening), DWHT Degreening without heat
treatment, HTWD Heat treatment without degreening, HTBD Heat
treatment before degreening and HTAD Heat treatment after degreening.














CHAPTER 3
PHYSIOLOGICAL RESPONSES OF 'VALENCIA' ORANGES TO SHORT-
DURATION HOT WATER DIP TREATMENT

Introduction

When fruit are dipped in hot water (HW), there is a potential risk of damaging the

tissues. Tissue damage may be associated with changes in peel electrolyte leakage,

peroxidase activity, total phenolics or total protein content. If so, such changes could

even serve as early, quantitative measures of heat injury. The following experiments were

conducted to evaluate the physiological responses of 'Valencia' oranges to HW dips.

Materials and Methods

Fruit

'Valencia' oranges were harvested at the Indian River Research and Education

Center research grove in Fort Pierce, Fla. between June and August 2003. Inner-canopy

fruit harvested from 1-1.5 m above ground level were used in all experiments. The fruit

were harvested in the morning and were treated later the same day.

Experiment 1

Harvested fruit were dipped in water at 56 C for 10, 20, 30, 60 or 120 s. Hot water

dips were conducted in a temperature controlled water bath (Optima series immersion

circulators, Boekel Scientific, Feasterville, Pa.). Fruit were dipped such that half the

surface of each fruit along the meridian was immersed in the water. Control fruit were not

dipped. Each treatment had three replicates of three fruit each. Electrolyte leakage from

flavedo tissue was measured using the procedure described below on the treated and









untreated sides of each fruit immediately after treatment. After taking discs of peel for

electrolyte leakage measurement, the fruit were stored at 10 OC (90% RH). This resulted

in decay development on the 12th d after treatment at which time scald was evaluated and

the experiment terminated.

Experiment 2

Because treatments in the previous experiment did not result in measurable

differences, fruit were dipped in water at 50, 60, 70, 80 or 90 OC for 60 s. Dipping was

conducted as described in experiment 1. Each treatment had three replicates of three fruit

each and these were duplicated so that one set of three replicates was used to measure

electrolyte leakage immediately after treatment, and the other set of three replicates was

stored at 10 OC (90% RH). After 14 d of storage, fruit were evaluated for scald, peel

color, and electrolyte leakage.

Experiment 3

Based on results from the second experiment and to focus on the temperature range

where changes in electrolyte leakage occurred, harvested fruit were dipped in water at 60,

62, 64, 66, 68 or 70 oC for 60 s. Hot water dip was administered as described in

experiment 1. Each treatment had three replicates of five fruit each and these were

duplicated so that one set of three replicates was used to measure electrolyte leakage

immediately after treatment, and the other set of three replicates was stored at 10 C

(90% RH). After 8 d of storage, fruit were evaluated for scald, peel color, and electrolyte

leakage.

Experiment 4

Because peel electrolyte leakage and scalding changed significantly between 60

and 66 OC, harvested fruit were dipped in water at 60 or 66 OC for 60 s and peel color,









total phenolics, protein content, and peroxidase activity were evaluated in addition to

electrolyte leakage and peel scalding over time. Dipping was conducted as described in

experiment 1 except that the whole fruit were completely submerged. Each treatment had

three replicates of five fruit each and these were duplicated four times so that one set was

evaluated at each of four sampling times: immediately after treatment, and after storage at

10 C (90% RH) for 2, 4, or 7 d.

Peel Scalding

Peel scalding was evaluated on each fruit and expressed on a 0 to 10 scale with 0

representing no scalding and 10 representing scald on 100% of the fruit surface. The

percentage of fruit with any peel scalding (rated as a 1 or above) was also calculated.

Peel Color

Peel color was measured using a Minolta Chroma Meter (CR-300 series, Minolta

Co. Ltd., Japan) at three equidistant locations on each fruit along the equator of the fruit

and expressed as L*, a* and b* values. The hue and chroma values were calculated from

a* and b* values using the following formula:

Hue = arc tangent (b*-a*-1)

Chroma = (a*2 + b*2)1/2

Electrolyte Leakage

Electrolyte leakage of flavedo tissue was determined following the procedure of

McCollum and McDonald (1991). Discs of peel tissue were taken from the equator of

each fruit using a 13-mm cork borer. Two discs were taken per fruit during the first three

experiments, and only one during the fourth experiment. The albedo was removed using a

razor blade and the flavedo discs were incubated in 25 mL of 0.4 M mannitol solution for

4 h at room temperature with constant shaking. After 4 h, the initial conductivity was









measured using a conductivity meter (D-24, Horiba Ltd., Japan). The samples were then

frozen overnight. Following thawing and warming to room temperature total conductivity

was measured. Electrolyte leakage was calculated using the following formula:

Electrolyte leakage (%) = Initial conductivity (S-m-1) 100
Total conductivity (S-m-1)



Preparation of Flavedo Samples for Protein, Phenolic and Peroxidase Assays

Flavedo was peeled from one meridian half of each fruit using a fruit peeler, frozen

in liquid nitrogen, crushed into small pieces with a mortar and pestle, and then freeze

dried. After drying, the samples were ground into a fine powder and stored at -20 OC.

Acetone-washed flavedo powder was prepared by shaking 1 g of freeze dried

flavedo in 25 mL of acetone for 1 h. The samples were filtered through Whatman No. 1

filter paper under vacuum and washed with 25 mL acetone before being collected in

beakers and dried overnight.

Protein was extracted by adding 25 mL borate buffer (0.1 M, pH 8.8) to 0.3 g of the

acetone-washed powder and shaking at 5 OC for 1 h. Extracts were then filtered through

4-6 layers of cheesecloth and the volume brought up to 25 mL with the borate buffer

before centrifugation at 25,000 gn for 10 min. Supernatants were decanted, brought to

70% saturation with solid ammonium sulfate and shaken overnight at 5 OC. Samples were

centrifuged at 25,000 gn for 10 min the following day and the supernatant discarded. The

pellets were dissolved in 4 mL borate buffer. Desalting was done by dialysis using

dialysis tubes with molecular weight cut-off = 6,000-8,000.









Lowry Assay for Protein

For protein measurement, standards for the Lowry assay (Lowry et al., 1951) were

prepared from 0, 4, 8, 12, 16, and 20 [g of bovine serum albumin. Samples were

prepared by adding 190 [L of water to 10 [L of protein extract. Working solution of

copper-tartrate-carbonate (CTC) was prepared by mixing 5 mL of CTC, 5 mL of NaOH

(0.8 M), and 10 mL of water. The CTC working solution (200 IL) was added to the

protein solution, and the mixture was vortexed and allowed to stand for 10 min. Working

solution of Folin-Ciocalteu was prepared by mixing one volume of Folin-Ciocalteu

phenol reagent with five volumes of water. Then 100 [L of Folin-Ciocalteu working

solution was added to the protein and CTC solution, and the mixture was vortexed and

allowed to stand for 30 min. Absorbance was read at 750 nm using a microplate reader

(Ultramark, BioRad, Hercules, Calif.). The results were expressed as mg of protein per g

dry weight of the tissue.

Estimation of Total Phenolics

For phenolics estimation (Swain and Hillis, 1959), methanol (10 mL) was added to

200 mg of freeze dried flavedo acetone powder and kept on a shaker overnight. The

extracts were filtered through Whatman No. 1 filter paper under vacuum. To 0.2 mL of

filtered extract, Folin-Ciocalteu reagent (0.4 mL) and 0.5 M ethanolamine (0.9 mL) were

added and the mixture vortexed. After 20 min, 200 [L aliquot aliquots were applied to a

microplate reader (Ultramark, Bio-Rad Laboratories, Hercules, Calif.) and absorbance

was measured at 630 nm. Gallic acid solutions in methanol at 0, 50, 100, 150, and

200 ppm were used as standards. The results were expressed as mg of total phenolics per

g dry weight of the tissue.









Peroxidase Assay

For peroxidase assay (Worthington, 1972), protein extract (100 pL) was added to

1.4 mL of 0.0025 M 4-aminoantipyrine and 1.5 mL of 0.0017 M H202 and mixed well by

shaking. The absorbance was read in a UV-visible recording spectrophotometer

(UV160U, Shimadzu, Japan) at 510 nm over a period of time. The spectrophotometer

was set under kinetic program to have a lag time of 30 s, rate time of 180 s and interval

time of 30 s. The results were expressed as peroxidase activity per minute per g dry

weight of the tissue.

Statistical Analysis

Percentage data were transformed to arcsine values and analyzed by ANOVA using

SAS (PROC GLM) for PC (SAS Institute Inc, Cary, N.C.). When differences were

significant (P < 0.05), individual treatment means were separated using Duncan's

Multiple Range Test (P = 0.05). Means presented are untransformed values.

Results and Discussion

Experiment 1

Electrolyte leakage on the heat-treated sides of fruit from each treatment averaged

between 33% and 42%, and was not significantly different from each other (Figure 3-1).

Schirra and D'hallewin (1997) reported that HW dip at 50, 52, 54, 56 or 58 C for 3 min

of 'Fortune' mandarin did not affect the electrolyte leakage immediately after treatment.

However, they observed higher electrolyte leakage after 30 d of storage in fruit dipped at

56 or 58 C. After 12 d of storage, only fruit dipped in 56 C water for 120 s developed

peel scalding (Table 3-1). Of these fruit, about 67% were scalded and an average of 89%

of the fruit surface was scalded, but this did not result in a significant change in










electrolyte leakage. Therefore, HW dip at 56 C for 10-120 s did not result in significant

changes in electrolyte leakage.


Table 3-1. Peel scalding of'Valencia'
12 d at 10 oC.


oranges on the heat-treated side of the fruit after


Treatment Scalding Scalding
(56 C) (% of fruit surface) (% of fruit)

0s 0.00 bz 0.00 b

10 s 0.00 b 0.00 b

20 s 0.00 b 0.00 b

30 s 0.00 b 0.00 b

60 s 0.00 b 0.00 b

120 s 89.00 a 66.67 a

Significance *
z Values within each column followed by different letters are significantly different by
Duncan's multiple range test at P < 0.05.
* Significant at P < 0.05.


50


. 40


S30
20

1 20


10


0


4 Treated side
-A *Untreated side


Os 10 s


20 s 30 s
Treatment duration (56 OC)


Figure 3-1. Electrolyte leakage from flavedo of 'Valencia' oranges immediately after
56 OC hot water treatment. Vertical bar represents the 5% LSD value.


120 s


LSDo 05









Experiment 2

To further evaluate the effect of HW dip on electrolyte leakage, the range of

treatment temperatures was increased to between 50 and 90 C, all for 60 s. Hot water dip

at 60, 70, 80 or 90 C for 60 s resulted in all fruit developing peel scalding (Figure 3-2).

An average of 64% of the fruit surface was scalded in fruit dipped at 60 OC, which was

significantly less than the 100% of the fruit surface scalded in fruit dipped at 70, 80 or

90 C (Figure 3-3). Scalding incidence was still high (56%) on fruit dipped at 50 OC for

60 s, but was significantly lower than on fruit dipped at higher temperatures (Figure 3-2);

20% of the fruit surface was scalded when dipped at 50 OC (Figure 3-3). The untreated

half of the fruit did not develop peel scalding in any of the treatment temperatures (data

not shown).

Electrolyte leakage from fruit dipped at 50 or 60 OC was not significantly different

from the non-treated fruit when evaluated immediately after the treatments, or after 14 d

of storage (Figure 3-4). Fruit dipped in 70, 80 or 90 C water had higher electrolyte

leakage (79-97%) compared with the non-treated fruit (35%), 50 C (35%), or 60 C

(39%) treated fruit. Electrolyte leakage from 50 and 60 C-dipped fruit was not

significantly different from the non-treated fruit, although the 60 C HW dip resulted in

significant peel scalding (Figures 3-2 and Figure 3-3). Schirra and D'hallewin (1997)

reported that HW dip at 54, 56 or 58 C for 3 min did not result in significant difference

in electrolyte leakage immediately after treatment though the scalding incidence was

10%, 70%, or 100%, respectively, after 33 d in storage. Thus, flavedo electrolyte leakage

is not an early indicator of peel scalding. Electrolyte leakage of flavedo from the

untreated sides of fruit from all treatments did not result in significant differences from

the non-treated fruit throughout the 14 d experiment (data not shown).










120



100



S 80



60
o 60

.
c 40
jI


20


n


Ctrl 50 C 60 C 70 C 80 C 90 C

Treatment temperature (60 s)

Figure 3-2. Peel scalding (percent of fruit scalded) of 'Valencia' oranges on the heat-
treated side of the fruit after 14 d of storage at 10 OC. Bars with different
letters are significantly different by Duncan's multiple range test at P < 0.05.
Ctrl Control (no HW dip).

Peel hue and chroma values did not show consistent treatment differences

(Table 3-2). The L* values from fruit treated at 70, 80 or 90 OC, which had scalding on

100% of the peel surface, were significantly lower than the non-treated fruit. Though not

significantly lower, L* values also tended to decline at dipping temperatures above 50 or

60 oC treatment. Schirra and D'hallewin (1997) reported that 'Fortune' mandarins dipped

at 58 OC for 3 min had lower L* values after 33 d in storage. Differences in treatment

duration and orange cultivar studied likely explain the slight differences in results.


a a a a















- --- -


c
































50 C


60 C


70 C


80 C


90 C


Treatment temperature (60 s)


Figure 3-3. Peel scalding (percent of fruit surface scalded) of 'Valencia' oranges on the
heat-treated side of the fruit after 14 d of storage at 10 OC. Bars with different
letters are significantly different by Duncan's multiple range test at P < 0.05.
Ctrl Control (no HW dip).


Ctrl 50 C 60 C 70 C 80 C 90 C
Treatment temperature (60 s)


Figure 3-4. Electrolyte leakage from flavedo of the heat-treated sides of 'Valencia'
oranges immediately after treatment and after 14 d of storage at 10 OC.
Vertical bar represents the 5% LSD value. Ctrl Control (no HW dip).


a a a





b







d

d---- -










Table 3-2. Peel hue, chroma and L* values of 'Valencia' oranges after 14 d of storage at
10 C.
Hue Chroma L*
Treatment (60 s) Treated side Untreated side Treated side Untreated side Treated side Untreated side
Ctrl 75.109 b c 75.109 55.607 55.607 56.090 a 56.090
50 C 77.630 ab 77.141 53.671 54.707 55.240 ab 56.097
60 C 79.236 a 77.600 51.146 54.563 54.09 abc 55.783
70 C 73.128 cd 77.230 49.837 57.930 51.047 cd 56.644
80 C 70.28 d 71.949 49.054 57.498 50.197 d 54.791
90 C 73.619 cd 75.297 51.438 58.128 52.373 bcd 57.596
Significance ns ns ns ns
z Values within each column followed by different letters are significantly different by Duncan's multiple
range test at P < 0.05.
* Significant at P <0.05.
ns Not significant at P < 0.05.
Ctrl Control (no HW dip).

Experiment 3

Because flavedo electrolyte leakage increased markedly following dip treatments

between 60 and 70 oC, experiments were conducted further evaluating this range of

temperatures. There was a distinct increase in peel scalding at treatment temperatures of

64 OC and above (Figure 3-5, Figure 3-6); fruit dipped at 60 or 62 oC developed no

scalding, whereas all fruit dipped at 66, 68 or 70 oC developed scalding after 8 d of

storage. Parallel increases in the percentage of fruit surface scalded at higher dip

temperatures were also observed (Figure 3-7).

Electrolyte leakage immediately after the treatments also increased significantly at

treatment temperatures of 64 OC and above (Figure 3-8); values for fruit dipped in 66, 68,

or 70 oC water were 45%, 53%, and 78%, respectively, higher than non-treated fruit.

After 8 d of storage, electrolyte leakage was significantly higher only in fruit dipped at 68

or 70 oC, which were 23% and 36% higher, respectively, than the non-treated fruit.

Electrolyte leakage on the untreated side of fruit did not vary significantly immediately

after treatments or after 8 d of storage (data not shown). While scalding developed in fruit









dipped at 64 C or higher, increased electrolyte leakage was only detected in tissue

exposed to 66 C or higher immediately after treatment, and at 68 C or higher after 8 d

storage. Thus, increased electrolyte leakage was only observed if severe scalding

developed, and is not suitable as an early indicator of peel scalding.

After 8 d of storage, there was no significant change in hue angle between the

treated and untreated tissue (Table 3-3). However, chroma and L* values for fruit dipped

at 66, 68 or 70 C were significantly lower than for fruit not dipped. These results are

similar to the previous experiment where HW dips at higher temperatures resulted in

lower L* values. But the chroma values in the previous experiment were not affected by

HW dips. The untreated sides of the fruit did not have any significant changes in the

chroma and L* values.

























Figure 3-5. Peel scalding of 'Valencia' oranges after 8 d of storage at 10 OC. Fruit were
dipped in 66, 68, or 70 'C water for 60 s.



































Figure 3-6







120


100


S80


60


.,


20


a a a





b










Ctrl 60 OC 62 C 64 C 66 C 68 C 70 C

Treatment temperature (60 s)


. Peel scalding (percent of fruit scalded) of 'Valencia' oranges on the heat
treated side of the fruit after 8 d of storage at 10 OC. Fruit were dipped in 66,
68, or 70 oC water for 60 s. Bars with different letters are significantly
different by Duncan's multiple range test at P < 0.05. Ctrl Control (no HW
dip).


Ctrl 60 C


62 C


64 C


66 C


68 C


70 C


Treatment temperature (60 s)


Figure 3-7. Peel scalding (percent of fruit surface scalded) of 'Valencia' oranges on the
heat-treated side of the fruit after 8 d of storage at 10 OC. Bars with different
letters are significantly different by Duncan's multiple range test at P < 0.05.
Ctrl Control (no HW dip).


a

b b







C




d d d















LSDo o5
80 _

70 -

3 60 ,0

W 50

2 40
"" Initial
3 30
-X 8th day
20

10

0 -
Ctrl 60 C 62 C 64 C 66 C 68 C 70 C
Treatment Temperature (60 s)

Figure 3-8. Electrolyte leakage from flavedo of the heat-treated sides of 'Valencia'
oranges immediately after treatment and after 8 d of storage at 10 OC. Vertical
bar represents the 5% LSD value. Ctrl Control (no HW dip).

Table 3-3. Peel hue, chroma and L* values of the heat-treated and untreated sides of


'Valencia' oranges after 8 d of storage at 10 OC. Ctrl


Control (no HW dip).


Hue Chroma L*
Treatment (60 s) Treated side Untreated side Treated side Untreated side Treated side Untreated side
Ctrl 75.877 75.877 70.252 abz 70.252 63.913 a 63.913
60 C 66.157 65.508 71.247 a 71.917 63.880 a 64.809
62 C 77.358 77.746 70.859 ab 69.186 63.895 a 63.826
64 C 77.576 77.407 66.957 be 64.884 62.011 a 61.531
66 C 75.559 79.141 64.957 c 69.835 59.047 b 63.44
68 C 77.465 78.958 63.320 c 68.322 59.529 b 62.933
70 C 74.348 75.686 63.844 c 71.066 58.529 b 63.317
Sinificance ns ns ns ns
z Values within each column followed by different letters are significantly different by
Duncan's multiple range test at P < 0.05.
* Significant at P < 0.05.
ns Not significant at P < 0.05.


I









Experiment 4

In testing for other potential physiological changes in heat-treated 'Valencia'

oranges, all fruit dipped in 66 C water for 60 s developed peel scalding within 7 d,

whereas only 20% of the fruit developed scalding when dipped in 60 C water

(Figure 3-9). In experiment 2, all the fruit dipped at 60 OC for 60 s were scalded after 14 d

of storage and in experiment 3, none of the fruit dipped at 60 OC for 60 s were scalded

after 8 d of storage. The differences in sensitivity to heat injury could be due to the

differences in the time of harvest which was between 1st week of June and 1st week of

August. Again, only severely scalded fruit from the 66 C water treatment developed

significantly higher electrolyte leakage throughout the 7 d storage period (Figure 3-10).

Electrolyte leakage averaged 19% higher in 66 C-treated fruit than in untreated fruit.

These results are consistent with the previous experiments.

Peroxidase activity of fruit treated at 66 C was lower than the non-treated fruit and

60 C treatment (Figure 3-11). Higher electrolyte leakage and lower peroxidase activity

were observed only on fruit that were dipped at temperatures of 66 C or greater, which

resulted in significant peel scalding. The HW dip treatments did not significantly affect

total phenolics or total protein contents in the peel (Table 3-4 and Table 3-5).

Peel browning is generally caused by the oxidation of phenols mainly by the

enzymes polyphenol oxidase (PPO) and peroxidase (Lattanzio et al., 1994). The total

phenolics did not change with heat treatment and there was lower peroxidase activity. So

the flavedo browning was likely due to oxidation by PPO. Martinez-Tellez and Lafuente

(1993) have reported that chilling-induced browning had no correlation with PPO and

peroxidase activities. There are also non-enzymatic browning reactions in which colored

complexes are formed by the interactions between phenolics and heavy metals (Lattanzio














100


80


60


4 days


7 days


Days after Treatment


Figure 3-9. Peel scalding (percent of fruit scalded) of 'Valencia' oranges after heat
treatment at 60 and 66 C for 60 s. Scald was evaluated after 4 and 7 d of
storage at 10 OC. Bars within each day with different letters are significantly
different by Duncan's multiple range test at P < 0.05. Ctrl Control (no HW
dip).

70
9 Ctrl
a 60 C
60


50 -


40


30


20


10


0 day


2 days


4 days


7 days


Days after Treatment

Figure 3-10. Electrolyte leakage from flavedo of 'Valencia' oranges immediately after
hot water dip for 60 s and after 2, 4, and 7 d of storage at 10 OC. Bars within
each day with different letters are significantly different by Duncan's multiple
range test at P < 0.05. Ctrl Control (no HW dip).


R Ctrl
a
-n .n c














b b







42


et al., 1994). These could have also contributed to the peel browning caused by HW

treatment.


S20



15


*a
^ 10



5
53


Today


2 days


4 days


7 days


Days after Treatment


Figure 3-11. Peroxidase activity from flavedo of 'Valencia' oranges immediately after
hot water dip for 60 s and after 2, 4, and 7 d of storage at 10 OC. Vertical bar
represents the 5% LSD value. Ctrl Control (no HW dip).


Table 3-4. Total phenolics from flavedo of 'Valencia' oranges immediately after hot
water dip and after 2, 4, and 7 d of storage at 10 OC. Ctrl Control (no HW
dip).

Total phenolics (mg/g dry wt.)

Treatment (60 s) 0 day 2 days 4 days 7 days

Ctrl 3.101 3.499 3.510 3.746

60 C 3.336 4.071 3.900 3.333

66 C 3.808 3.117 3.320 3.695

Significance ns ns ns ns
ns Not significant at P < 0.05.


LSDo 05
LSD,,,,



x.-



-A ---- --A
- .


Ctrl
*60C
66C






43


Table 3-5. Total protein content from flavedo of 'Valencia' oranges immediately after hot
water dip and after 2, 4, and 7 d of storage at 10 OC. Ctrl Control (no HW
dip).
Total protein (mg/g dry wt.)
Treatment (60 s) 0 day 2 days 4 days 7 days
Ctrl 18.969 21.241 25.569 23.732
60 C 20.468 19.096 23.543 23.897
66 C 18.906 14.812 16.459 19.501
Significance ns ns ns ns
ns Not significant at P < 0.05.














CHAPTER 4
CONTROL OF POSTHARVEST DISEASES IN 'RUBY RED' GRAPEFRUIT BY
HOT WATER DIP TREATMENT

Introduction

Short duration, hot water (HW) dip treatments appear to be a promising method to

control postharvest decay in citrus (Rodov et al., 1995a). Heated solutions of sodium

carbonate have been more effective in controlling decay than non-heated solutions (Palou

et al., 2002). The following experiments were conducted to evaluate the effectiveness of

HW dip, hot sodium carbonate dip, and hot imazalil dip for decay control in Florida

grapefruit. The effect of washing or coating the fruit immediately after HW dip was also

studied.

Materials and Methods

Fruit

Three experiments were conducted between November 2003 and May 2004. For

the first experiment, fruit were harvested during November 2003 and for the last two

experiments, fruit were harvested during February 2004 at the Indian River Research and

Education Center research grove in Fort Pierce, Fla. Commercially mature (TSS:TA >

7:1) and healthy 'Ruby Red' grapefruit were harvested randomly from 1-1.5 m above

ground level on healthy trees, evenly spaced around the tree and distributed through the

inner and outer canopy. Harvested fruit were stored at room temperature overnight and

heat treatment was done on the next day.









Experiment 1

Harvested fruit were dipped in 25, 53, 56, 59 or 62 C water for 30 s. There were

three post-dip treatments:

1. Hot water dip only

2. Hot water dip, followed immediately by a 1 min dip in water at ambient (-25 C)
temperature

3. Hot water dip, followed immediately by washing and coating (simulated
commercial packinghouse treatment)

The HW dip treatments were conducted using stainless steel tanks holding 95 L

of rapidly stirred water. Heating was accomplished using a large gas burner with the

temperature varying by -+1 C during each treatment. Fruit were treated by placing them

into perforated plastic crates that allowed water circulation past the fruit. Each treatment

had four replicates of 40 fruit each. Fruit were washed over a brush bed and then coated

with shellac (FMC Foodtech., Lakeland, Fla.) to simulate commercial handling.

Fungicides were not used. Following treatment, the fruit were stored at 10 OC (90% RH).

Ten fruit from each replicate were randomly selected, marked and weighed to follow

weight loss during storage. Initial analyses of peel color, total soluble solids (TSS),

titratable acidity (TA), peel puncture resistance (PPR), and percent juice were done using

four replications of 10 fruit each. Fruit were evaluated for peel scalding 1 and 4 weeks

after treatment. After 4 weeks of storage, marked fruit were weighed, and peel color,

TSS, TA, PPR, and percent juice were determined. Decay was evaluated after 4, 8, and

12 weeks in storage.

Experiment 2

Harvested fruit were dipped in water at 25, 56 or 59 C for 30 s. There were three

post-dip treatments:









1. Hot water dip only

2. Hot water dip, followed immediately by washing (-25 C water) through a
commercial packing line

3. Hot water dip, followed immediately by washing (-25 C water) and coating
(simulated commercial packinghouse treatment)

The HW dips were administered as described in experiment 1. Each treatment had

four replicates of 40 fruit each. Fungicides were not used. Following treatment, the fruit

were stored at 16 C (90% RH). Ten fruit from each replicate were randomly selected,

marked, and weighed to follow weight loss measurement during storage. Fruit were

evaluated for peel scalding 1, 2, and 4 weeks after treatment. After 4 weeks of storage,

marked fruit were weighed. Decay was evaluated after 4, 8, and 12 weeks in storage.

Experiment 3

There were two dip temperatures and six chemical treatments:

1. Water alone

2. 3% sodium carbonate solution

3. 6% sodium carbonate solution

4. 125 ppm imazalil solution

5. 250 ppm imazalil solution

6. 3% sodium carbonate + 125 ppm imazalil solution

Harvested fruit were dipped at 25 or 56 C for 30 s for each of the above

treatments. The HW dips were administered as described in experiment 1. Each treatment

had four replicates of 40 fruit each. There were five additional fruit in each replication for

the imazalil treatments, which were taken for residue analysis after coating the fruit with

shellac. Fruit were washed and shellac coated immediately after HW dip treatment.

Following treatment, fruit were stored at 16 OC (90% RH). Ten fruit from each replicate









were randomly selected, marked, and weighed to follow weight loss during storage.

Initial analyses of peel color, TSS, TA, PPR and percent juice were done using four

replications of 10 fruit each. Fruit were evaluated for peel scalding 1 and 4 weeks after

treatment. After 4 weeks of storage, marked fruit were weighed and peel color, TSS, TA,

PPR, and percent juice were determined. Decay was evaluated after 4, 8, and 12 weeks in

storage.

Peel Scalding

Peel scalding was evaluated as described in chapter 3.

Residue Analysis

Residue analysis was done by Decco, Ceraxagri, Inc., Fort Pierce, Fla. Fruit were

cut into quarters and one section from each fruit was used. The samples were weighed

and then blended. To the blended samples, DI water (100 mL), 5N NaOH (10 mL) and

NaCl (50 g) were added and the homogenate was weighed. After homogenizing for

3 min, 50 g of the sample was taken in centrifuge tubes and 10 mL of iso-octane was

added. After centrifuging at 1800 gn for 10 min, 5 mL of supernatant solution was

decanted into a 25 mL beaker. The samples were dried with anhydrous sodium sulfate. A

2 [iL sample of the extract was injected into the gas chromatograph with electron capture

(EC) or nitrogen phosphorus (NP) detector. The residue level was calculated from the

following formula:

Imazalil (ppm) = Chromatogram reading 10 Total weight
Weight of blended fruit taken Initial weight

Weight Loss

Fruit were weighed on the 1st d after treatment and then again on the 4th week after

treatment. Weight loss was calculated as follows:









Weight loss (%) = Initial weight (g) Final weight (g) 100
Initial weight (g)

Peel Color

Peel color was measured as described in chapter 3.

Total Soluble Solids and Titratable Acidity

The fruit were cut into halves along the equator and juice was extracted using a test

juice extractor (Model 2700, Brown Citrus Systems Inc., Winter Haven, Fla.). Juice TSS

(Brix) was measured using a refractometer (Abbe-3L, Spectronic Instruments Inc.,

Rochester, N.Y.) and the juice TA (% citric acid) was measured by titrating 40 mL of

juice samples to pH 8.3 with 0.3125 N NaOH using an automatic titrimeter (DL 12,

Mettler-Toledo Inc., Columbus, Ohio).

Percent Juice

Percent juice was calculated from the total weight of fruit and total weight of juice.

Percent juice = Juice weight (g) 100
Fruit weight (g)

Peel Puncture Resistance

Peel puncture resistance was measured at two equidistant spots along the equator of

each fruit using a texture analyzer (Model TAXT2i, Stable Micro Systems, Godalming,

England) with a 2 mm diameter, flat-tipped, cylindrical probe. The analyzer was set such

that the probe traveled at a speed of 2 mm-s-1 and the maximum force exerted to puncture

the peel was recorded. Peel puncture resistance was expressed in Newtons.

Statistical Analysis

Percentage data were transformed to arcsine values and analyzed by ANOVA using

SAS (PROC GLM) for PC (SAS Institute Inc, Cary, N.C.). When differences were









significant (P < 0.05), individual treatment means were separated using Duncan's

Multiple Range Test (P = 0.05). Means presented are untransformed values.

Results and Discussion

Experiment 1

Dipping fruit in 25, 53, or 56 C water for 30 s did not cause any peel scalding after

4 weeks of storage at 10 OC (Table 4-1). Fruit dipped in 59 C water for 30 s developed

17%-31% peel scalding and fruit dipped in 62 C water for 30 s developed 46%-72%

peel scalding depending on the post-dip treatment (Table 4-1). Schirra and D'hallewin

(1997) reported peel scalding in 'Fortune' mandarins after HW dips at 56 or 58 C for

3 min. Washing and shellac coating of fruit immediately after HW dip reduced the

development of peel scalding by 45% or 37% in fruit dipped at 59 or 62 C, respectively,

compared with fruit that were not washed and coated. Dipping fruit in ambient water

immediately after HW dip reduced the development of scalding by only 6% or 5% in

fruit dipped at 59 or 62 C, respectively, compared with fruit that were not dipped in

ambient water after HW dips. So, washing and coating fruit immediately after HW dip

can significantly reduce the heat damage.

Grapefruit dipped in 56 or 59 C water followed by washing and shellac coating

showed the best result in reducing decay to 25% or 18%, respectively, after 12 weeks of

storage at 10 OC (Figure 4-1). Fruit dipped in 25 C water followed by no post-dip

treatment developed 75% decay. Decay development in fruit dipped in ambient water

after HW dip at 62 C did not differ from fruit that received no post-dip treatment after

HW dip at 62 C. But fruit treated at other temperatures followed by dipping in ambient

water developed higher decay than fruit that received no post-dip treatment. Fruit dipped

in 62 C water were injured by the treatment, which negated the beneficial effect of









reducing decay. Hot water (Miller et al., 1988) and vapor heat (Hallman et al., 1990)

treatments of grapefruit that caused scalding were reported to also result in increased

decay, which was suggested to be a result of the damaged tissue being more susceptible

to pathogen invasion.

Though others have reported the development of chilling injury (CI) of Florida

grapefruit stored at 10 OC (Grierson and Hatton, 1977), it is fairly uncommon

commercially because of the almost universal use of wax coatings that restrict gas

exchange to varying degrees and reduce chilling sensitivity. Early season fruit

(September-November) are more susceptible to CI than fruit harvested during the middle

of the season (December-February) (Grierson and Hatton, 1977; Schirra et al., 2000). The

fruit utilized for the current studies were still very chilling sensitive and developed CI

during storage at 10 OC. Dipping fruit in 56 or 59 C water followed by washing and

Table 4-1. Peel scalding (percent of fruit scalded) of 'Ruby Red' grapefruit after 30 s HW
dip treatments followed by post-dip treatments. Fruit were evaluated after 4
weeks of storage at 10 OC. Dip Fruit were dipped in ambient water for 1 min
and Shellac Fruit were washed over a brush bed and coated with shellac.
Post-dip treatment
Tmnt. temp. None Dip Shellac
25 oC 0.00 0.00 0.00
53 OC 0.00 0.00 0.00
56 C 0.00 0.00 0.00
59 OC 30.63 28.75 16.88
62 C 71.88 68.13 45.63
LSDo.05 = 14.37

shellac coating reduced CI to 2% or 1%, respectively, after 12 weeks of storage at 10 C

(Figure 4-2). Fruit dipped in 25 OC water followed by no post-dip treatment developed

50% CI. Hot water dip did not affect the TSS, TA or the amount of juice (data not


















---None
- > Dip
-A Shellac


25 C 53 C 56 C 59 C 62 C
Treatment temperature (30 s)


Figure 4-1. Total decay of 'Ruby Red' grapefruit after 30 s HW dip treatments followed
by post-dip treatments. Fruit were evaluated after 12 weeks of storage at
10 C. Vertical bar represents the 5% LSD value. Dip Fruit were dipped in
ambient water for 1 min and Shellac Fruit were washed over a brush bed
and coated with shellac.

100

90 LSD 05
90

80 LSDo a0

70



'. 50 -- Dip
"* Shellac


25 C 53 C 56 C 59 C 62 C
Treatment temperature (30 s)


Figure 4-2. Chilling injury of 'Ruby Red' grapefruit after 30 s HW dip treatments
followed by post-dip treatments. Fruit were evaluated after 12 weeks of
storage at 10 OC. Vertical bar represents the 5% LSD value. Dip Fruit were
dipped in ambient water for 1 min and Shellac Fruit were washed over a
brush bed and coated with shellac.









shown). There were no consistent differences in weight loss or peel puncture resistance

(data not shown).

Experiment 2

Fruit dipped in 59 OC water for 30 s had significantly more scalding than the fruit

that were dipped in 25 OC water after 1, 2, and 4 weeks of storage at 16 OC. After 4 weeks

in storage, the incidence of peel scalding for fruit dipped in 59 OC water for 30 s was 3%-

5% depending on the post-dip treatment (Table 4-2) and peel scalding severity ranged

from 8%-14% of the fruit surface (Table 4-3). In the previous experiment using

November-harvested fruit, HW dips at 59 OC resulted in 17%-31% peel scalding

incidence after 4 weeks of storage. So, the late season (February-harvested) fruit used in

the second experiment may have been more resistant to heat damage. Hot water dips at

56 OC water for 30 s followed by no post-dip treatment resulted in only 2% incidence of

peel scalding whereas fruit that were shellac coated after HW dipping at 56 oC did not

develop any peel scalding. Washing alone or washing and coating the fruit immediately

after HW dip did not have a significant effect on the development of peel scalding. Since

the level of scalding was very low compared with the previous experiment, the effect of

coating the fruit on heat damage could not be seen.

None of the HW or post-dip treatments were effective in controlling decay (Figure

4-3). Heat treatment and washing alone or washing and coating the fruit did not result in

significant reduction in postharvest decay. The incidence of total decay was very low in

this season compared with the previous experiment. This could be a reason why

significant effect of treatments could not be seen.







53


Table 4-2. Peel scalding incidence (percent of fruit scalded) of 'Ruby Red' grapefruit
after 30 s HW dip treatments followed by post-dip treatments. Fruit were
evaluated after 4 weeks of storage at 16 OC. Wash Fruit were washed over a
brush bed and Shellac Fruit were washed over a brush bed and coated with
shellac.

Post-dip treatment
Tmnt. temp. None Wash Shellac

25 OC 0.00 0.00 0.00

56 C 1.88 3.13 0.00

59 C 2.50 3.75 5.00
LSDo.05 = 2.05


25 C


56 C


- None
- X Wash
-A Shellac


59 C


Treatment temperature (30 s)


Figure 4-3. Total decay of 'Ruby Red' grapefruit after 30 s HW dip treatments followed
by post-dip treatments. Fruit were evaluated after 12 weeks of storage at
16 oC. Vertical bar represents the 5% LSD value. Wash Fruit were washed
over a brush bed and Shellac Fruit were washed over a brush bed and coated
with shellac.


LSDo os



^. A ___









Table 4-3. Peel scalding severity (percent of fruit surface scalded) of 'Ruby Red'
grapefruit after 30 s HW dip treatments followed by post-dip treatments. Fruit
were evaluated after 4 weeks of storage at 16 OC. Wash Fruit were washed
over a brush bed and Shellac Fruit were washed over a brush bed and coated
with shellac.
Post-dip treatment
Tmnt. temp. None Wash Shellac
25 OC 0.00 0.00 0.00
56 C 6.67 11.25 0.00
59 OC 7.50 13.75 9.17
LSDo.05 = 2.05

Experiment 3

Fruit dipped in heated solutions of imazalil had greater residue levels immediately

after dipping than fruit dipped in non-heated solutions of imazalil (Figure 4-4). Fruit

dipped in heated solutions of imazalil had 0.6-1 ppm of residue whereas fruit dipped in

ambient temperature solution had only 0.1 ppm of residue. 'Salustiana' oranges dipped in

imazalil solution at 50 oC had 8-fold greater residue than when dipping was done at

20 oC (Cabras et al., 1999). Hence, lower concentrations of imazalil could be more

effective when the application occurs at higher temperatures.

After 4 weeks of storage, no significant scalding was observed in fruit dipped at

56 OC for 30 s. Only 0.2% of the fruit were scalded. Most decay was due to stem-end rot

(SER). After 12 weeks of storage at 16 OC, HW dipping at 56 OC for 30 s resulted in

lower SER than the non-heated solutions for all the imazalil and sodium carbonate

solutions except for the water dip treatment (Figure 4-5). Ritenour et al. (2003) reported

that a HW dip at 56 OC for 30 s decreased SER to 20% after 12 weeks in storage

compared with 33% SER in fruit dipped in ambient water. Treatments with 3% or 6%

sodium carbonate solutions at 25 OC increased the incidence of SER to 27% or 23%,










respectively, compared with water dip treatment at 25 C, which developed 6% SER.

Treatments with 3% or 6% sodium carbonate solutions at 56 C also increased the

incidence of SER (to 14% or 19%, respectively) compared with the water dip treatment at

56 C, which developed 11% SER. This is in contrast to the effect of hot sodium

carbonate solutions on green and blue molds. Smilanick et al. (1997) and Palou et al.

(2001, 2002) have shown that sodium carbonate solutions, especially heated solutions,

significantly reduce the development of green and blue molds in citrus. The SER may be

enhanced by the high pH (10-11) of the sodium carbonate solutions. Treating the fruit

with imazalil solution did not result in significant reduction in SER though the fruit

dipped in heated imazalil solutions had higher residue levels. Since the incidence of

natural decay was low in fruit harvested during February, the effect of imazalil could not

be observed.

1.2


1.0
I NO
0.8

S25 'C
0.6
0 56C

0.4


0.2


0.0 -- --
125 ppm 125 ppm + 3% SC 250 ppm
Imazalil concentration

Figure 4-4. Imazalil residue in 'Ruby Red' grapefruit immediately after the 30 s dip
treatments at 25 and 56 C. Vertical bar represents the 5% LSD value. SC -
Sodium carbonate.







56



100

90
LSDoo5
80

70

60
50
E 56 'C
40

30

20 -

10


0% SC 3% SC 6% SC 125 ppm 125 ppm 250 ppm
imazalil imazalil + imazalil
3% SC
Treatment (30 s)

Figure 4-5. Stem-end rot for different treatment temperatures after 12 weeks of storage at
16 C. Bars with different letters are significantly different by Duncan's
multiple range test at P < 0.05. SC Sodium carbonate.














CHAPTER 5
CONTROL OF CHILLING INJURY IN 'RUBY RED' GRAPEFRUIT BY HOT
WATER DIP TREATMENT

Introduction

Grapefruit develops chilling injury (CI) when stored at temperatures below

10-12 C (Chace et al., 1966). Grapefruit harvested early and late in the season are more

susceptible to CI than are fruit harvested in mid-season (Grierson and Hatton, 1977).

Fruit from the outer-canopy are more susceptible to CI than are fruit from the inner-

canopy (Purvis, 1980). Heat treatments have been successful in reducing CI in many

citrus varieties (Rodov et al., 1995a; Schirra et al., 1997). The following experiment was

conducted to study the effect of short duration, hot water (HW) dip treatments in

controlling CI in Florida grapefruit.

Materials and Methods

Fruit

Commercially mature (TSS:TA > 7:1) and healthy 'Ruby Red' grapefruit were

harvested randomly from 1-1.5 m above ground level on healthy trees, evenly spaced

around the tree on 3 Nov. 2003 at the Indian River Research and Education Center

research grove in Fort Pierce, Fla. Fruit were harvested separately from the inner and

outer canopies of the tree. The harvested fruit were stored at room temperature overnight

and heat treatment was done on the next day.









Heat Treatment

Fruit were dipped in water at 25, 53, 56 or 59 C for 30 s. Hot water dips were

administered as described in chapter 4. Each treatment had four replicates of 30 fruit

each. After the HW dip, half of the fruit from each treatment and canopy position were

stored at 5 C (90% RH) and the other half stored at 16 OC (90% RH). Five fruit from

each replicate were randomly selected, marked, and weighed to follow weight loss during

storage. Initial analyses of peel color, total soluble solids (TSS), titratable acidity (TA),

peel puncture resistance (PPR) and percent juice were done using four replicates of five

fruit each. Fruit were evaluated for peel scalding 1, 3, and 7 weeks after treatment. After

4 and 7 weeks of storage, marked fruit were evaluated for weight loss. After 4 weeks of

storage, peel color, TSS, TA, PPR, and percent juice were evaluated from another five

fruit randomly selected from each replicate. After 6 weeks of storage, fruit stored at 5 C

(90% RH) were transferred to 16 OC (90%RH). After 7 weeks of storage, remaining fruit

were evaluated for CI and decay.

Chilling Injury

Chilling injury severity was rated from 0 to 3 (0-none, 1-slight, 2-moderate and

3-severe). The number of fruit in each rating was multiplied by its corresponding rating

number and the sum of these products was divided by the total number of fruit in the

replicate to give the average CI severity for that replicate.

Peel Scalding

Peel scalding was evaluated as described in chapter 3.

Weight Loss

Weight loss was calculated as described in chapter 4.









Peel Color

Peel color was measured as described in chapter 3.

Total Soluble Solids and Titratable Acidity

Total soluble solids and titratable acidity were measured as described in chapter 4.

Peel Puncture Resistance

Peel puncture resistance was measured as described in chapter 4.

Percent Juice

Percent juice was calculated as described in chapter 4.

Statistical Analysis

Percentage data were transformed to arcsine values and analyzed by ANOVA using

SAS (PROC GLM) for PC (SAS Institute Inc, Cary, N.C.). When differences were

significant (P < 0.05), individual treatment means were separated using Duncan's

Multiple Range Test (P = 0.05). Means presented are untransformed values.

Results and Discussion

Hot water dip had a greater effect in reducing CI severity of inner canopy fruit than

of outer canopy fruit. Compared to fruit dipped at 25 C, dipping fruit in 53, 56, or 59 C

water for 30 s reduced CI severity by 3%, 6% or 10%, respectively, in outer canopy fruit

stored at 5 C, but reduced CI severity by 11%, 18% or 32%, respectively, in inner

canopy fruit (Figure 5-1). Purvis (1980) has reported that outer canopy fruit are more

susceptible to CI than the inner canopy fruit, but our results indicated little effect of

canopy position on non-heated fruit with both inner and outer canopy fruit severely

affected by CI. So, heat treatment by itself had a major role in reducing the CI severity in

inner canopy fruit. None of the fruit stored at 16 OC developed CI.









Fruit stored at chilling temperature (5 C) developed more decay than fruit stored at

non-chilling temperature (16 C). After 7 weeks of storage, fruit stored at 16 C

developed only 0%-4% decay whereas fruit stored at 5 OC developed 27%-58% decay

(Figure 5-2). Most decay was due to anthracnose (Colletotrichum gloeosporioides). High

incidence of anthracnose was observed on fruit that developed CI. For fruit stored at

5 C, HW dipping at 53, 56 or 59 C for 30 s reduced decay by 25%, 21% or 44%,

respectively, compared with dipping in 25 C water for 30 s.

After 3 weeks of storage, 2% of fruit treated at 59 OC developed visible peel

scalding (data not shown). No scalding was observed on fruit dipped in 25, 53 or 56 C

water. After 4 weeks of storage, TSS in fruit from outer canopy was significantly higher

than in fruit from inner canopy and TA was significantly lower in outer canopy fruit than

in inner canopy fruit. The percent juice was not affected by the canopy position. The

percent juice and TA was significantly lower in fruit stored at 5 OC than in fruit stored at

16 C. However, HW dipping did not affect the percent juice, TSS, and TA of the fruit

(data not shown). After 4 weeks of storage, PPR was significantly greater in fruit treated

at 59 C than in all other treatments (Table 5-1). At the end of the experiment, weight

loss from fruit treated at 25 C was significantly greater than from the other three

treatment temperatures (Table 5-2). The weight loss was higher in the fruit stored at 5 C

than the fruit stored at 16 C. Higher weight loss at 5 OC could be due to accelerated

weight loss in fruit that developed CI. Purvis (1984) correlated higher weight loss during

storage with CI development in citrus fruit. Cohen et al. (1994) used weight loss as an

early indicator of CI.






























25 C


53 C


56 C


59 C


Treatment (30 s)

Figure 5-1. Chilling injury in 'Ruby Red' grapefruit after 30 s dip treatments of inner and
outer canopy fruit. Fruit were evaluated after 6 weeks of storage at 5 OC plus 1
week at 16 C. Chilling injury was rated from 0 (none) to 3 (severe). Vertical
bar represents the 5% LSD value.


25 C


53 C


56 C


59 C


- Inner Canopy 5 oC
SOuter Canopy 5 C


Treatment temperature (30 s)
-A Inner Canopy 16 C
)"- Outer Canopy 16 C


Figure 5-2. Total decay in 'Ruby Red' grapefruit after 30 s dip treatments of inner and
outer canopy fruit. Fruit were evaluated after 7 weeks of storage at 5 or 16 OC.
Vertical bar represents the 5% LSD value.


LSDo 05



Inner Canopy 5 C
-A Outer Canopy 5 C


LSDO 0,










--............ --.-.-.-A









Table 5-1. Peel puncture resistance in Newtons of 'Ruby Red' grapefruit after 30 s dip
treatments. Fruit were evaluated after 4 weeks of storage at 5 or 16 OC.
Inner Canopy Outer Canopy
Tmnt. Temp. 5 C 16 C 5 C 16 C
25 oC 17.24 14.39 18.47 13.68
53 OC 18.19 14.53 18.12 14.37
56 OC 18.29 14.73 18.34 13.55
59 C 18.25 14.91 20.28 15.17
LSDoo5 = 1.66

Table 5-2. Weight loss (%) from 'Ruby Red' grapefruit after 30 s dip treatments. Fruit
were evaluated after 7 weeks of storage at 5 or 16 OC.
Inner Canopy Outer Canopy
Tmnt. Temp. 5 C 16 C 5 C 16 C
25 oC 4.34 3.77 4.42 3.84
53 OC 4.15 3.61 3.68 3.55
56 C 3.96 3.05 3.71 3.37
59 C 4.23 3.61 3.92 3.36
LSDo s = 0.61














CHAPTER 6
CONCLUSIONS

The research reported in this thesis has shown the effects on ethylene treatment of

grapefruit and physiological responses of oranges due to hot water (HW) dip treatments

and also the effects of HW dip treatment in reducing postharvest decay and chilling

injury (CI) in Florida grapefruit. As a result of these studies, it was found that HW dips

administered before or after grapefruit degreening did not affect subsequent peel injury,

respiration rate, ethylene production, total soluble solids (TSS) or titratable acidity (TA).

Under commercial conditions, the various uses of ethylene treatment would not appear to

have any affect on the use of HW treatments on grapefruit.

Significantly higher electrolyte leakage was found only in fruit dipped at

temperatures greater than 66 C for 60 s. In 'Valencia' oranges, a HW dip at 56 C for up

to 120 s or 60 C for 60 s did not affect electrolyte leakage though it resulted in 67% or

100% incidence of peel scalding, respectively. None of these treatments affected

electrolyte leakage than the non-treated fruit. Hence electrolyte leakage is not an early

indicator of peel injury.

'Valencia' oranges dipped at temperatures above 66 C for 60 s had lower L*

values than non-treated fruit. Hot water dip at 70-90 C for 60 s did not result in

significant difference in chroma value though fruit developed visible peel discoloration.

But, in a subsequent experiment, fruit dipped at 66-70 C for 60 s had significantly lower

chroma value. The hue value was not affected by HW dip. The peroxidase activity of

heat-treated fruit (66 C for 60 s) was lower than that of non-treated fruit or fruit treated









at 60 C for 60 s. Hot water dips did not affect the total phenolics or the total protein

content.

To study the effects of washing or shellac coating of fruit immediately after a HW

dip, two experiments were conducted with 'Ruby Red' grapefruit. After 4 weeks of

storage, washing and shellac coating of fruit immediately after HW dip reduced the

development of peel scalding by 45% or 37% in fruit dipped at 59 or 62 C, respectively,

compared with fruit that were not washed and coated. Dipping the fruit in ambient water

or washing through brush beds after HW treatment did not significantly reduce peel

scalding.

Since the fruit used in the first coating experiment were harvested in November,

they were highly susceptible to CI with 50% CI incidence in control fruit dipped in 25 C

water followed by no post-dip treatment and stored at 10 OC. After 12 weeks of storage,

fruit dipped in 56 or 59 C water followed by washing and shellac coating reduced CI to

2% or 1%, respectively. To study the effects of HW dip on CI, fruit were harvested

separately from the inner and outer canopy and stored at 5 OC for 6 weeks followed by 1

week at 16 C. Hot water dip at 53, 56 or 59 C reduced CI by 3%, 6% or 10%,

respectively, in outer canopy fruit, but reduced CI by 11%, 18% or 32%, respectively, in

inner canopy fruit.

Scalding was not significant in fruit dipped at 56 C water for 30 s. HW dip at 56 or

59 C for 30 s significantly reduced decay in fruit harvested in November. The fruit

harvested in February were less susceptible to decay and so the effects of HW dip could

not be seen. In none of the experiments did HW dip affect TSS or TA of the fruit.









Although HW treatments reduced significantly the development of postharvest

decay, there was still a substantial amount of decay. So, HW treatment alone did not

effectively control the decay development. Heated solutions of sodium carbonate and

imazalil were used at lower concentrations than used commercially at ambient

temperature. Unfortunately, solutions of 3% or 6% sodium carbonate enhanced the

development of stem-end rot. Imazalil at 125 and 250 ppm did not effectively control

decay compared with fruit that were dipped in water only.

From the experiments conducted on 'Ruby Red' grapefruit, HW dip at 56 C for

30 s followed by washing and shellac coating significantly reduced development of

postharvest decay and CI without causing significant damage to the peel and without

affecting the juice quality of the fruit. Hence, HW dip at 56 C for 30 s followed by

washing and shellac coating was the best treatment for 'Ruby Red' grapefruit.

Future experiments should be conducted to increase the efficacy of HW dip by

using heated solutions of compounds generally regarded as safe or heated solutions of

fungicides at lower concentrations. In the current study, heated solutions of sodium

carbonate or imazalil did not effectively control postharvest decay in late season fruit.

However, others have reported the efficacy of these compounds in heated solutions,

which suggests they can be effective under modified conditions. Hence more work

should be conducted on early season fruit when they are more susceptible to natural

decay and economic losses from such decay is often more extensive.















APPENDIX A
ANALYSIS OF VARIANCE FOR CHAPTER 2


Table A-1. Analysis of variance for respiration rates of 'Ruby Red' grapefruit during the
first six days in storage at 20 C.

Sources of Mean squares of respiration rate
variation d.f. 1 day 2 days 3 days 4 days 5 days 6 days
Treatment 4 3.12* 6.13** 3.95** 3.45* 2.05* 2.37
Error 10 0.65 0.81 0.12 0.72 0.18 4.34
F values significant at 5%
** F values significant at 1%


Table A-2. Analysis of variance for ethylene production of 'Ruby Red' grapefruit during
the first six days in storage at 20 C.

Sources of Mean squares of ethylene production
variation d.f. 1 day 2 days 3 days 4 days 5 days 6 days
Treatment 4 0.0012 0.0004 0.0007 0.0005 0.0027 $0.0080
Error 10 0.0012 0.0003 0.0004 0.0005 0.0017 0.0037


Table A-3. Analysis of variance for percent of fruit scalded, total soluble solids and
titratable acidity of 'Ruby Red' grapefruit after 10 d of storage at 20 OC.


Mean squares
Sources of Total soluble Titratable
variation d.f. Scald solids acidity
Treatment 4 0.5255** 1.2723 0.0228*

Error 10 0.0090 2.0180 0.0041


* F values significant at 5%
** F values significant at 1%


v














APPENDIX B
ANALYSIS OF VARIANCE FOR CHAPTER 3

Experiment 1


Table B-1. Analysis of variance for percent of fruit scalded after 12 d of storage at 10 OC,
electrolyte leakage of treated and untreated sides of 'Valencia' oranges
immediately after treatment.
Mean squares
Sources of Electrolyte leakage Electrolyte leakage
variation d.f. Scald (treated side) (untreated side)
Treatment 5 0.4560** 26.789 57.141*
Error 12 0.0005 21.600 14.659
F values significant at 5%
** F values significant at 1%










Experiment 2


Table B-2. Analysis of variance for peel scalding of 'Valencia' oranges after 14 d of
storage at 10 OC.
Mean squares
Sources of Percent of fruit Percent of fruit
variation d.f. scalded surface scalded
Treatment 5 0.5096** 0.5952**

Error 12 0.0064 0.0007
** F values significant at 1%


Table B-3. Analysis of variance for electrolyte leakage of treated and untreated sides of
'Valencia' oranges immediately after treatment and after 14 d of storage at 10
oC.
Mean squares
0 day 14 days
Sources of Electrolyte leakage Electrolyte leakage Electrolyte leakage Electrolyte leakage
variation d.f. (treated side) (untreated side) (treated side) (untreated side)
Treatment 5 0.2803** 0.0025 0.1390** 0.0011
Error 12 0.0020 0.0027 0.0014 0.0009
** F values significant at 1%










Experiment 3


Table B-4. Analysis of variance for peel scalding of 'Valencia' oranges after 8 d of
storage at 10 OC.
Mean squares
Sources of Percent of fruit Percent of fruit
variation d.f. scalded surface scalded
Treatment 6 1.8531** 1.2430**

Error 14 0.0064 0.0084
** F values significant at 1%


Table B-5. Analysis of variance for electrolyte leakage of treated and untreated sides of
'Valencia' oranges immediately after treatment and after 8 d of storage at 10
oC.
Mean squares
0 day 8 days
Sources of Electrolyte leakage Electrolyte leakage Electrolyte leakage Electrolyte leakage
variation d.f. (treated side) (untreated side) (treated side) (untreated side)
Treatment 6 0.0744** 0.0020 0.0360* 0.0013
Error 14 0.0046 0.0017 0.0051 0.0123
F values significant at 5%
** F values significant at 1%









Experiment 4


Table B-6. Analysis of variance for peel scalding of 'Valencia' oranges after 4 and 7 d of
storage at 10 OC.
Mean squares
4 days 7 days
Sources of Percent of Percent of fruit Percent of Percent of fruit
variation d.f. fruit scalded surface scalded fruit scalded surface scalded
Treatment 2 0.6691** 0.2513** 2.0104** 0.4761**
Error 6 0.0045 0.0015 0.0406 0.0154
** F values significant at 1%


Table B-7. Analysis of variance for electrolyte leakage from flavedo of 'Valencia'
oranges immediately after treatment and after 2, 4 and 7 d of storage at 10 C.

Sources of Mean squares of electrolyte leakage
variation d.f. 0 day 2 days 4 days 7 days
Treatment 2 0.0176** 0.0116* 0.0073* 0.0117**
Error 6 0.0006 0.0012 0.0009 0.0008
F values significant at 5%
** F values significant at 1%


Table B-8. Analysis of variance for peroxidase activity in flavedo of 'Valencia' oranges
immediately after treatment and after 2, 4 and 7 d of storage at 10 0C.

Sources of Mean squares of peroxidase activity
variation d.f. 0 day 2 days 4 days 7 days
Treatment 2 14.22* 29.78* 45.31 36.82*
Error 6 3.40 5.43 14.27 9.35
F values significant at 5%









Table B-9. Analysis of variance for total phenolics in flavedo of 'Valencia' oranges
immediately after treatment and after 2, 4 and 7 d of storage at 10 0C.

Sources of Mean squares of total phenolics
variation d.f. 0 day 2 days 4 days 7 days
Treatment 2 0.3890 0.6916 0.2632 0.1522
Error 6 0.5317 0.6104 0.1957 0.3273


Table B-10. Analysis of variance for total protein in flavedo of 'Valencia' oranges
immediately after treatment and after 2, 4 and 7 d of storage at 10 0C.

Sources of Mean squares of total protein content
variation d.f. 0 day 2 days 4 days 7 days
Treatment 2 0.0939 1.2856 2.7458 0.7453
Error 6 0.4346 0.3432 1.6853 2.8706
















APPENDIX C
ANALYSIS OF VARIANCE FOR CHAPTER 4

Experiment 1

Table C-1. Analysis of variance for peel scalding, total decay and chilling injury of 'Ruby
Red' grapefruit. Peel scalding was evaluated after 4 weeks of storage at 10 OC.
Total decay and chilling injury were evaluated after 12 weeks of storage at 10
0C.
Mean squares
Percent of fruit Percent of fruit
Sources of variation d.f. scalded surface scalded Total decay Chilling injury
Water temperature (WT) 4 2.0818** 0.8977** 0.3174** 0.4455**
Post dip treatment (PDT) 2 0.0453* 0.0045 0.7405** 1.0092**
WTxPDT 8 0.0195 0.0042* 0.0225 0.0494
Error 45 0.0123 0.0017 0.0279 0.0242
* F values significant at 5%
** F values significant at 1%


Table C-2. Analysis of variance for quality of 'Ruby Red' grapefruit after 4 weeks of
storage at 10 OC.
Mean squares
Peel puncture Juice Total soluble Titratable
Sources of variation d.f. Weight loss resistance content solids acidity
Water temperature (WT) 4 1.0734** 8.6801** 2.0819 0.2332 0.0136
Post dip treatment (PDT) 2 0.2176 3.7180* 2.8395 0.0922 0.0275
WT xPDT 8 0.2889 0.8983 2.9977 0.2822 0.0092
Error 45 0.1962 0.8120 2.0396 0.3028 0.0085
* F values significant at 5%
** F values significant at 1%









Experiment 2


Table C-3. Analysis of variance for peel scalding and total decay of 'Ruby Red'
grapefruit. Peel scalding was evaluated after 4 weeks and total decay was
evaluated after 12 weeks of storage at 16 OC.
Mean squares
Percent of Percent of fruit
Sources of variation d.f. fruit scalded surface scalded Total decay
Water temperature (WT) 2 42.36* 311.67** 29.08
Post dip treatment (PDT) 2 2.26 87.34 13.97
WT x PDT 4 6.94 41.29 42.61
Error 27 7.99 42.14 109.37
F values significant at 5%
** F values significant at 1%









Experiment 3

Table C-4. Analysis of variance for imazalil residue of 'Ruby Red' grapefruit
immediately after treatment.


Sources of variation
Water temperature (WT)
Imazalil treatment (IT)
WT x IT
Error
* F values significant at 5%
** F values significant at 1%


Mean squares of
imazalil residue
3.5190**
0.0968**
0.0933*
0.0157


Table C-5. Analysis of variance for peel scalding and stem-end rot of 'Ruby Red'
grapefruit. Peel scalding was evaluated after 4 weeks and stem-end rot was
evaluated after 12 weeks of storage at 16 OC.


Sources of variation
Water temperature (WT)
Chemical treatment (CT)
WT x CT
Error
* F values significant at 5%
** F values significant at 1%


Mean squares
Percent of fruit Percent of fruit
scalded surface scalded Stem-end rot
0.5208 18.75 300.25*
0.2083 8.75 238.14**
0.2083 8.75 78.62
0.2604 10.42 49.26















APPENDIX D
ANALYSIS OF VARIANCE FOR CHAPTER 5


Table D-1. Analysis of variance for peel scalding, chilling injury index and total decay of
'Ruby Red' grapefruit. Peel scalding was evaluated at 4 weeks of storage.
Chilling injury and total decay were evaluated after 7 weeks of storage.
Mean squares
Percent of Percent of fruit Chilling
Sources of variation d.f fruit scalded surface scalded injury index Total decay
Water temperature (WT) 3 11.11* 25.00** 0.2359** 0.0718*
Canopy position (CP) 1 0.70 6.25 0.5968** 0.0016
Storage temperature (ST) 1 0.00 0.00 101.9090** 6.5993**
WT x CP 3 0.70 6.25 0.0630 0.0043
WT x ST 3 0.00 0.00 0.2359** 0.0504
CP x ST 1 6.26 6.25 0.5968** 0.0001
WT x CP x ST 3 6.26 6.25 0.0630 0.0320
Error 48 2.66 5.21 0.0451 0.0184
F values significant at 5%
** F values significant at 1%


Table D-2. Analysis of variance for quality of 'Ruby Red' grapefruit after 4 weeks of
storage.
Mean squares
Peel puncture Juice Total soluble Titratable
Sources of variation d.f. resistance content solids acidity
Water temperature (WT) 3 4.3335* 2.6888 0.1242 0.0026
Canopy position (CP) 1 0.5293 0.0923 1.0000** 0.0225**
Storage temperature (ST) 1 253.6853** 289.1275** 1.0000** 0.0286**
WT xCP 3 2.1114 2.0580 0.2083 0.0004
WT x ST 3 0.2691 0.0613 0.2283 0.0003
CP x ST 1 6.3252* 0.0083 0.0225 0.0084
WT x CP x ST 3 0.6842 7.2340 0.1975 0.0016
Error 48 1.3632 4.8838 0.0783 0.0023
F values significant at 5%
** F values significant at 1%









Table D-3. Analysis of variance for weight loss in 'Ruby Red' grapefruit after 4 and 7
weeks of storage.


Sources of variation
Water temperature (WT)
Canopy position (CP)
Storage temperature (ST)
WT x CP
WT x ST
CP x ST
WT x CP x ST
Error
** F values significant at 1%


Mean squares
Weight loss Weight loss
(4th week) (7th week)
0.4211** 0.8772**
0.0054 0.1828
0.0375 4.5263**
0.0293 0.1445
0.0245 0.0720
0.0147 0.2678
0.0087 0.0749
0.0672 0.1862
















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BIOGRAPHICAL SKETCH

Karthik-Joseph John-Karuppiah was born on November 10, 1979, in India. In 2001,

he obtained his Bachelor of Science degree in Horticulture from the Tamil Nadu

Agricultural University in India. In 2002, he started his master's program in Horticulture

at the University of Florida and successfully completed his degree in 2004.