Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. POSTHARVEST ENVIRONMENTAL FACTORS AFFECTING INFECTION OF KIWIFRUIT BY BOTRYTIS CINEREA. A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Phi losophy in Plant Science at Massey University Palmerston North New Zealand. Silvia Bautista Banos May 1 995 -------c---- � To my husband J . Raul : With my deepest gratitude and love, forever. i i i ABSTRACT In vitro germination of B. cinerea isolates from diseased blueberry, camell ia, grapes, kiwifruit and strawberry were compared at Massey University (Palmerston North) and pathogenicity on kiwifruit at both Massey University and at the New Zealand Institute for Crop and Food Research (Levin) . An average of 74.4% of spores germinated on agar when the concentration was 5.9 x 1 04 but only 62.8% when it was 1 .5 x 1 06. Percentage spore germination on agar did not indicate relative pathogenicity on kiwifruit and there was no significant difference in disease incidence of fruit inoculated with d ifferent isolates. The effect of culture medium and of age of culture from which conidial inoculum was prepared were evaluated by growing B. cinerea on malt agar, potato dextrose agar and autoclaved kiwifruit leaves for seven, 1 8 or 28 days. Each fruit was inoculated with one drop of a 0.05% Tween 20 suspension containing 1 ,000, 5,000, 25,000 or (Levin only) 1 25,000 spores per drop. Disease incidence was proportional to inoculum concentration . There was no significant effect of colony age. The highest disease incidence at Massey University was with inoculum produced on malt agar whereas at Crop and Food Research it was with inoculum produced on autoclaved kiwifruit leaves. All further inoculation work was carried out using the K3 isolate from Massey University grown on Malt agar for 1 0-1 4 days. The ability of B. cinerea conidia to survive temperature/humidity regimes that could be used for curing kiwifruit was tested by exposing conidia on glass slides to combinations of 0, 1 0, 1 5, 25 or 30°C with low «50%) , medium-low (64-80%), medium-high (80-90%) or high (>90%) relative humidities for two, four, six or eight days. Both the percentage germination and the speed of germination decreased at the higher temperatures and with longer exposure times. iv The effect of temperature during curing on subsequent infection levels was investigated in 1 992, of humidity in 1 993 and of both temperature and humidity in 1 994. After harvest, each fruit was inoculated with 1 25,000 spores ( 1 992) or 25,000 spores (1 993 and 1 994) . In 1 994 dry conidial appl ication using a paintbrush was also included. The greatest curing effect was obtained at 1 0°C. Disease incidence increased at O°C and the curing effect diminished at temperatures above 1 0°C. Fruit cured at 20°C and at 30°C softened rapidly and developed a high incidence of disease. In 1 994 a three day curing period was used and 1 0°C again gave the lowest subsequent disease incidence. After twelve weeks coolstorage ( 1 993) there was less disease in fruit cured at 89-95% relative humidity than at lower humidities. In 1 994 comparable resu lts were obtained. The effect of curing regimes on fruit physiology showed that ethylene production increased and rate of respiration decreased with higher curing temperature but both increased with incubation time. There was no consistent pattem of treatment effect on ethylene production or on rate of respiration during subsequent coolstorage. Fruit firmness decreased with h igher curing temperatures and as the curing period was extended. Firmness fluctuated with harvest and in general decreased with storage although a satisfactory firmness was maintained throughout coolstorage from all treatments. There was no consistent relationship between temperature/time of incubation and total soluble solids content during curing and during storage. As the period of storage increased glucose and fructose content of fruit increased. pH remained constant in fruit from all treatments and there was no consistent relationship between acid buffering capacity measured as citric acid equ ivalent and curing temperature/incubation times during subsequent coolstorage. For all experiments, weight loss increased with increased curing temperature or with decreased relative humidity. Kiwifruit stem scars consisted of two main tissue systems: Ground and v vascular. Parenchyma, collenchyma and idioblasts containing raphides were the main components of the ground tissue. The vascular system consisted of xylem vessels, phloem and cambium. There was no evidence of anatomical structures blocking the xylem vessels in Botrytis infected fruit cured at O°C or at 1 0°C. Samples from both showed some evidence of thicken ing of the parenchyma cell walls in contact with conidial hyphae. Positive reactions to lignin, suberin and reducing compounds were observed in all treatments. Suberin development in xylem and parenchyma scar tissue was found at 1 0°C but not at 0, 20 or 30°C. In itial relative humidity ranges of 34-80%, 75-90% and 1 00% were tested during coolstorage at O°C in 1 992 and 40-59%, 65-80% and 92-97% in 1 994. Inoculum levels appl ied to the stem scar were 5000 and 25000 spores/ml respectively and infection levels were evaluated after 1 2 weeks coolstorage. There was no definite pattem in ethylene production and rate of respi ration during the incubation period . In both, 1 992 and 1 994 experiments weight loss increased as relative humidity decreased. TSS increased during incubation for all treatments. Firmness decreased with incubation time and after three months coolstorage for all treatments. In the second experiment of 1 994 there was a more marked effect of relative humidity on firmness. Fruit firmness decreased with harvest maturity. In the 1 992 experiment fruit disease decreased as incubation time increased and in 1 994, infection levels decreased as relative humidity increased. vi ACKNOWLEDGEMENTS I appreciate the assistance of Dr. Peter G. Long for valuable advice on planning for the experiments and writing of this thesis and Dr. S. Ganesh for his statistical advice. I appreciate the support of Hugh Neilson for all the laboratory work. Special thanks to The National Council of Science and Technology (Mexico) for providing financial assistance. My gratitude to my brothers: Alfredo, Sergio and Armando and my dear friend Charles R. Ensor for their continuous encouragement throughout my studies in this country. vii TABLE OF CONTENTS ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i i ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi i LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv LIST OF FIGURES xvi i i LIST OF PLATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxii i CHAPTER ONE GENERAL INTRODUCTION . . . . . . . . . . . . . . . . 1 World market and economic importance . . . . . . . . . . . . . . . . 1 Commercial orchards in New Zealand . . . . . . . . . . . . . . . . . 1 Cultural, Management and Harvesting Practices for kiwifruit in New Zealand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 :::::�: and. sorti�9 : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : (h'1 Storage and Marketing . . . . . . . . . . . . . . . . . . . . . . . . . . . . � Diseases of kiwifruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 BOTRYTIS CINEREA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Taxonomy, morphology and general l ife cycle . . . . . . . 9 Pathogenicity of B. cinerea on different commodities . . 1 2 The infection process by Botrytis . . . . . . . . . . . . . . . . 1 2 Effect of environment during the infection process by Botrytis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 Kiwifruit and B. cinerea . . . . . . . . . . . . . . . . . . . . . . . 1 4 CONTROL MEASURES . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 vii i Alternatives for control of B. cinerea . . . . . . . . . . . . . . 1 5 Chemical control . . . . . . . . . . . . . . . . . . . . . . . 1 5 Non-chemical control methods . . . . . . . . . . . . . 1 6 Definition of curing . . . . . . . . . . . . . . . . . . . . . . 1 7 Impact of curing to control postharvest d iseases. . . . . . . . . . . . . . . . . . . . . . . . 1 8 In tubers, bulbs and roots . . . . . . . . . . . . 1 8 I n vegetables . . . . . . . . . . . . . . . . . . . . . 2 1 In leafy vegetables . . . . . . . . . . . . . . . . . 2 1 In tropical and subtropical fruits . . . . . . . 2 1 In temperate fruits . . . . . . . . . . . . . . . . . 22 Curing as an alternative for control of B. cinerea during kiwifruit storage . . . . . . . . . . . . . . . . . . . . . . . . 22 Long-term storage conditions to reduce B. cinerea on kiwifruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 GENERAL OBJECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 CHAPTER TWO GENERAL MATERIALS AND METHODS . . . . . . 25 Fruit harvesting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Preparation of inoculum . . . . . . . . . . . . . . . . . . . . . . . 25 Quality measurements . . . . . . . . . . . . . . . . . . . . . . . . 26 Defined relative humidity . . . . . . . . . . . . . . . . . . . . . . 26 Measurement of ethylene and carbon dioxide production . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Assessment of infection . . . . . . . . . . . . . . . . . . . . . . . 29 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 CHAPTER THREE INOCULUM VARIABLES AFFECTING PATHOGENICITY OF BOTRYTIS CINEREA INFECTION OF KIWIFRUIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 ix INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 OBJECTIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . 33 Experiment No. 1 Title: Effect of B. cinerea isolates, culture age and inoculum concentration on in vitro conidial germination . . . . . . . . . . . . . . . . . . 33 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Experiment No.2 Title: Pathogenicity of B. cinerea isolates from a variety of sources on kiwifruit. . . 34 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Experiment No.3 Title: Effect of growth media, culture age and inoculum level on pathogenicity of B. cinerea to kiwifruit. . . . . . . . . . . . . . . . . . . . . . . 35 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Experiment No.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Experiment No.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Experiment No.3 . . . . . . . . . . . . . . . . . . . . . . . . . . .. 44 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 CHAPTER FOUR EFFECT OF RELATIVE HUMIDITY AND TIME OF EXPOSURE AT DIFFERENT TEMPERATURES ON SURVIVAL OF CONIDIA OF BOTRYTIS CINEREA. . . . . . . . 54 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 OBJECTIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . 55 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 64 x CHAPTER FIVE CURING OF KIWIFRUIT TO CONTROL BOTRYTIS CINEREA DURING STORAGE. . . . . . . . . . . . . . 71 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Physiological considerations of curing . . . . . . . . . . . . . 71 Control of postharvest d iseases by curing . . . . . . . . . . 76 OBJECTIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . 77 Experiment No.1 Title: Curing temperatures, physiological changes and incidence of B. cinerea stem-end rot during subsequent coolstorage in 1 992 . . . . . . . . . . . . . . . . . . . .. 77 Fruit harvesting . . . . . . . . . . . . . . . . . . . . . . . . 77 Inoculum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Preparation of treatments . . . . . . . . . . . . . . . . . 78 Assessments . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Assessment period . . . . . . . . . . . . . . . . . . . . . 79 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . 80 Experiment No.2 Title: Curing temperature and incidence of B. cinerea stem-end rot of kiwifruit during subsequent coolstorage in 1 994. . . . . . . 80 Fruit harvesting . . . . . . . . . . . . . . . . . . . . . . . . 80 Inoculum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Preparation of treatments . . . . . . . . . . . . . . . . . 80 Assessments . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . 81 Experiment No. 3 Title: Relative humidity during curing and B. cinerea stem-end rot incidence in kiwifruit during subsequent coolstorage in 1 993. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Fruit harvesting . . . . . . . . . . . . . . . . . . . . . . . . 81 Inoculum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 xi Treatment preparation . . . . . . . . . . . . . . . . . . . 81 Assessments . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . 82 Experiment No. 4 Title: Relative humidity, type of inoculum and harvest maturity on incidence of B. cinerea stem-end rot in kiwifruit during subsequent coolstorage in 1 994. . . . . . . . . . . . 82 Fruit harvesting . . . . . . . . . . . . . . . . . . . . . . .. 84 Inoculum . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 84 Treatments and experimental procedure . . . . .. 84 Statistical analysis . . . . . . . . . . . . . . . . . . . . .. 84 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Experiment No. 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Physiological changes of fruit during curing and coolstorage in 1 992 . . . . . . . . . . . . . . . . 85 Chemical composition of fruit . . . . . . . . . . . . . . 90 Infection levels during coolstorage . . . . . . . . . . 92 Experime�No. 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Fruit quality after the curing and coolstorage in 1 994 . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Infection levels during fruit coolstorage . . . . . . . 98 Experiment No.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 02 Relative humidity behaviour during curing period in 1 993 . . . . . . . . . . . . . . . . . . . . . . . . . 1 02 Fruit quality after the curing period . . . . . . . . . . 1 02 Infection levels during coolstorage . . . . . . . . . . 1 02 Experiment NO. 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 0 Relative humidity behaviour during curing in 1 994 . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 0 Fruit quality after the curing period . . . . . . . . . . 1 1 0 Infection levels during coolstorage . . . . . . . . . . 1 1 3 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 9 xii Physiological response during curing and coolstorage periods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 9 Chemical composition of fruit . . . . . . . . . . . . . . . . . . . 1 24 Infection levels during coolstorage . . . . . . . . . . . . . . . 1 26 CHAPTER SIX ANATOMICAL AND HISTOCHEMICAL STUDY OF INOCULATED KIWIFRUIT STEM SCARS DURING CURING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 31 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 31 Curing and wound healing . . . . . . . . . . . . . . . . . . . . . 1 31 Anatomical and/or histochemical responses during wound healing . . . . . . . . . . . . . . . . . . . . . . . . . 1 32 Factors affecting response to wound healing . . . . . . . . 1 33 OBJECTIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 34 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . 1 34 Fruit samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 34 Tissue preparation for anatomical and histochemical study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 35 Anatomical and Histochemical staining . . . . . . . . . . . . 1 35 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 37 Anatomical components of stem scar tissues . . . . . . . 1 37 Histochemical tests . . . . . . . . . . . . . . . . . . . . . . . . . . 1 42 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 47 CHAPTER SEVEN IN ITIAL COOLSTORAGE RELATIVE HUMIDITY OF KIWIFRUIT AND INFECTION BY BOTRYTIS CINEREA . 1 53 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 53 Storage temperature and relative humidity . . . . . . . . . 1 54 OBJECTIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 55 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . 1 55 Experiment No. 1 Title: Relative humidity at DOC, physiological changes and incidence of B. cinerea stem-end rot during coolstorage in xii i 1 992 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 55 Fruit harvesting . . . . . . . . . . . . . . . . . . . . . . . . 1 55 Inoculum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 56 Preparation of treatments . . . . . . . . . . . . . . . . . 1 56 Assessments . . . . . . . . . . . . . . . . . . . . . . . . . . 1 56 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . 1 56 Experiment No.2 Title: Effect of initial coolstorage relative humidity and fruit maturity on fruit quality and infection of kiwifruit by B. cinerea in 1 993 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 58 Fruit Harvesting . . . . . . . . . . . . . . . . . . . . . . . . 1 58 Inoculum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 58 Preparation of treatments . . . . . . . . . . . . . . . . . 1 58 Assessments . . . . . . . . . . . . . . . . . . . . . . . . . . 1 58 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . 1 59 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 59 Experiment No. 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 59 Relative humidity behaviour during treatment . . . 1 59 Fruit quality and physiological changes during inCUbation and storage periods. . . . . . . . 1 59 Infection levels during coolstorage . . . . . . . . . . 1 62 Experiment No.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 68 Relative humidity behaviour . . . . . . . . . . . . . . . 1 68 Fruit qual ity after initial relative humidity coolstorage period . . . . . . . . . . . . . . . . . 1 68 Infection levels during coolstorage . . . . . . . . . . 1 78 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 78 Fruit quality and physiological changes during incubation time . . . . . . . . . . . . . . . . . . . . . . . . 1 78 xiv Infection levels during coolstorage . . . . . . . . . . . . . . . 1 84 CHAPTER EIGHT GENERAL DISCUSSION AND FUTURE RESEARCH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 87 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 95 APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 PUBLICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 TABLE 1 -1 3-1 3-2 3-3 3-4 3-5 4-1 LIST OF TABLES Diseases of kiwifruit. Summary of in vitro germination of B. cinerea conidia after 1 2h incubation at 20°C on MA. Summary of effect of B. cinerea isolates and spore concentrations on percentage infection of kiwifruit after storage at O°C at Massey University Fruit Crops Orchard. Summary of the effect of different B. cinerea isolates and spore concentrations on percentage infection of kiwifruit after storage at O°C at Crops and Food Research Levin. Summary of the effects of media, culture ages and inoculum levels on incidence of stem end rot caused by B. cinerea at Massey University Fruit Crops Orchard after storage at O°C. Summary of the effects of media, culture ages and inoculum levels on incidence of stem end rot caused by B. cinerea at Crops and Food Research Levin after storage at O°C. Summary of B. cinerea conidial survival at different temperatures and humidities as assessed by subsequent germination on MA. xv PAGE 1 0 37 42 43 46 47 60 4-2 5-1 5-2 5-3 5-4 5-5 5-6 5-7 5-8 5-9 Survival of B. cinerea conidia as indicated by germination on MA after 8h or 24h following incubation at various combination of relative humidities and temperatures for d ifferent period of time (P < 0.001). 1 992: Percentage weight loss (Mean ± SE) of kiwifruit cured at four temperatures for up to six days. 1 992: Firmness (Mean ± SE) of kiwifruit cured at four temperatures for up to six days. 1 992: Firmness (Lsmean ± SE) of kiwifruit cured for up to six days then stored at O°C for up to six months. 1 992: Ethylene production (Mean ± SE) of kiwifruit cured at four temperatures for up to six days. 1 992: Ethylene production (Lsmean ± SE) of kiwifruit cured for up to six days then stored at O°C for up to six months. 1 992: Rate of respiration (Mean ± SE) of kiwifruit cured at four temperatures for up to six days. 1 992: Rate of respiration (Lsmean ± SE) of kiwifruit cured for up to six days then stored at O°C for up to six months. 1 992: Total soluble sol ids (Mean ± SE) of kiwifruit cured at four temperatures for up to six days. 1 992: Total soluble sol ids (Lsmean ± SE) of kiwifruit cured for up to six days then stored at O°C for up to six months. xvi 61 85 86 87 87 88 89 90 91 91 5-1 0 5-1 1 5-1 2 5-1 3 5-1 4 5-1 5 5-1 6 5-1 7 6-1 1 992: Sugar content of cured kiwifruit during subsequent storage at O°C. 1 992: Acidity of cured kiwifruit during subsequent storage at O°C. 1 992: Percentage infection (Mean ± SE) of cured kiwifruit after 1 2 weeks storage. 1 994: Daily percentage weight loss of inoculated kiwifruit from three harvests cured at five temperatures. 1 994: Cumulative percentage weight loss of inoculated kiwifruit from three harvest after curing and coolstorage at O°C. 1 994: Firmness of inoculated kiwifruit from three harvests after curing and coolstorage at O°C. 1 994: Weight loss, firmness and total soluble solids of inoculated kiwifruit harvested at d ifferent maturities and cured for three days at 1 0°C and one of three relative humidities. 1 994: Infection levels of B. cinerea developed during coolstorage of kiwifruit inoculated with spore suspension or dry conidia before curing at 1 0°C and a range of relative humidities. Histochemical tests of kiwifruit stem scars inoculated with B. cinerea and incubated at various temperatures for up to six days. xvi i 93 94 95 96 97 99 1 1 2 1 1 5 1 46 FIG. 1 - 1 1 -2 1 -3 1 -4 1 -5 3-1 3-2 3-3 3-4 3-5 4-1 4-2 LIST OF FIGURES Kiwifruit growing areas in New Zealand T-bar support system for kiwifruit. a) Standard T-bar and b) Winged T-bar. Pergola support system for kiwifruit. Kiwifruit pruning methods. Kiwifruit handling system. Interaction between isolates and spore concentration during in vitro conidial germination on MA. Vertical bar indicates overall standard error of the mean (SEM). Interaction between isolates and culture age during in vitro conidial germination on MA. Vertical bar indicates overall standard error of the mean (SEM). Interaction between culture age and spore concentration during in vitro conidial germination on MA. Vertical bar indicates overall standard error of the mean (SEM). Interaction between isolates and spore concentration on storage rot incidence at MUFCO. Vertical bar indicates overall standard error of the mean (SEM). Interaction between media and B. cinerea culture age on storage rot incidence at MUFCO. a) 6 weeks, b) 1 2 weeks of storage. Vertical bars indicate overall standard error of the mean (SEM). Relative humidity system. Actual relative humidities attained at six temperatures. xvi i i PAGE 2 4 5 6 8 38 40 41 45 48 57 58 5-1 5 -2 5-3 5-4 5-5 5-6 5-7 5-8 Relative humidity system. 1 994: Percentage infection of kiwifruit from three harvests after curing at a range of temperatures and coolstorage for six weeks. Letters a, b, c, d & e refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. 1 994: Percentage infection of kiwifruit from three harvests after curing at a range of temperatures and coolstorage for 1 2 weeks. Letters a, b, c, d & e refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. 1 993: First harvest relative humidity during a three day incubation time at 1 0°C (Mean ± SE). 1 993: Second harvest relative humidity during a three day incubation time at 1 0°C (Mean ± SE) . 1 993: Weight loss of inoculated kiwifruit after curing for three days at 1 0°C and one of three relative humidities. Letters a, b & c refer to Duncan's test (P < 0.05) . Vertical bars indicate SEM. 1 993: Firmness of inoculated kiwifruit after curing for three days at 1 0°C and one of three relative humidities. Letters a & b refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. 1 993: TSS of inoculated kiwifruit after curing for three days at 1 0°C and one of three relative humidities. Letter a refers to Duncan's test (P < 0.05). Vertical bars indicate SEM. xix 83 1 00 1 01 1 03 1 04 1 05 1 06 1 07 5-9 1 993: Effect of curing at 1 0°C on incidence of B. cinerea infection of kiwifruit after six weeks of coolstorage. Letters a, b & c refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. xx 1 08 5-1 0 1 993: Effect of curing at 1 0°C on incidence of B. 1 09 5-1 1 cinerea infection of kiwifruit after 1 2 weeks of coolstorage. Letters a, b & c refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. 1 994: Range of relative humidities attained during three days incubation at 1 0°C for each of four harvests. 1 1 1 5-1 2 1 994: Interaction between maturity and relative 1 1 4 humidity on firmness and total soluble sol ids of inoculated kiwifruit cured at one of three rh's and four harvest maturities. Vertical bars indicate overall standard error of the mean (SEM). 5- 1 3 1 994: I nteraction between matu rity and relative 1 1 6 humidity on B. cinerea storage rot incidence of kiwifruit cured at 1 0°C. Vertical bars indicate overall standard error of the mean (SEM) . 5-1 4 1 994: Interaction between maturity and type of 1 1 7 inoculum on B. cinerea storage rot incidence of kiwifruit cured at 1 0°C. Vertical bars indicate overall standard error of the mean (SEM) . 5-1 5 1 994: Interaction between relative humidity and type 1 1 8 of inoculum on B. cinerea storage rot incidence of kiwifruit cured at 1 0°C. Vertical bars indicate overall standard error of the mean (SEM). 7-1 Relative humidity system. 1 57 7-2 7-3 7-4 7-5 7-6 7-7 7-8 7-9 1 992: Input and output relative humidity over a six week period at O°C. 1 992: Percentage weight loss of inoculated kiwifruit over a six week period at O°C and one of three relative humidities. Letters a, b, c & d refer to Duncan's test (P < 0.05) . Vertical bars indicate SEM. 1 992: Firmness of inoculated kiwifruit over a six week period at O°C and one of three relative humidities. Letters a & b refer to Duncan's test (P < 0.05) . Vertical bars indicate SEM. Firmness at harvest 9.9. 1 992: Mean (± SE) firmness of inoculated kiwifruit stored at O°C after incubation at d ifferent relative humidities for up to six weeks. (Overall P < 0.001) . 1 992: Total soluble solids of inoculated kiwifruit over a six week period at O°C and one of three relative humidities. Letters a & b refer to Duncan's test (P < 0.05) . Vertical bars indicate SEM. TSS at harvest 6.9%. 1 992: Ethylene production of inoculated kiwifruit over a six week period at O°C and one of three relative humidities. Letters a, b & c refer to Duncan's test (P < 0.05) . Vertical bars indicate SEM. 1 992: Rate of respiration of inoculated kiwifruit over a six week period at O°C and one of three relative humidities. Letters a, b & c refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. 1 992: Mean percentage infection of inoculated kiwifruit over a six week period at O°C and one of three relative humidities after 1 2 weeks coolstorage. xxi 1 60 1 61 1 63 1 64 1 65 1 66 1 67 1 69 xxii 7-1 0 1 993: Input and output relative humidity for fruit from 1 70 the first harvest over a seven day period at O°C. 7-1 1 1 993: Input and output relative humidity for fruit from the second harvest over a seven day period at O°C. 1 71 7-1 2 1 993: Input and output relative humidity for fruit from 1 72 the third harvest over a seven day period at O°C. 7 - 1 3 1 993: Percentage weight loss of inoculated kiwifruit 1 73 incubated for seven days at O°C and one of three relative humidities. Letters a, b & c refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. 7-1 4 1 993: Firmness of inoculated kiwifruit incubated for 1 74 seven days at O°C and one of three relative humidities. Letters a & b refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. 7-1 5 1 993: Total soluble solids of inoculated kiwifruit 1 75 incubated for seven days at O°C and one of three relative humidities. Letters a & b refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. 7 -1 6 1 993: Percentage infection of inoculated kiwifruit 1 76 incubated for seven days at O°C and one of three relative humidities after six weeks coolstorage. Letters a & b refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. 7-1 7 1 993: Percentage infection of inoculated kiwifruit 1 77 incubated for seven days at O°C and one of three relative humidities after 1 2 weeks coolstorage. Letters a & b refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. PLATE 6-1 6-2 6-3 6-4 6-5 6-6 LIST OF PLATES Longitudinal section of a fresh kiwifruit (Plate 6-2 stained with Phloroglucinol-HCLmg. x1 .8). Longitudinal section of a fresh kiwifruit with pedicel attached stained with Phloroglucinol-HCL(mg. xO.7) . Positions of the cross-section shown in Plates 6-4 - 6-7 are indicated with horizontal lines. Section A 1 (Plate 6-4) , section A2 (Plate 6-5), section A3 (Plate 6-6), section A4 (Plate 6-7) and section A5 (Plate 6- 7) . Cross section (A 1 ) of a fresh kiwifruit stem scar stained with Phloroglucinol-HCL(mg. x4.0). At the union between pedicel and fruit, there is a circular arrangement of three vascular bundles (v) surrounded by, parenchyma and the suberized tissue from the fruit shoulder. Cross section (A2) of a fresh kiwifruit stem scar stained with Phloroglucinol-HCI.(mg. x1 .8). The original three vascular bundles (v) have divided to form five. The upper most point of the sclerified plug (p) can be seen in the centre. Cross section (A3) of a fresh kiwifruit stem scar stained with Phloroglucinol-HCL(mg. xO.7) , showing radial development of sclerified tissue (s) on the upper surface of the plug and vascular bundles (v) . xxi i i PAGE 1 36 1 38 1 39 1 39 1 40 6-7 6-8 6-9 6- 1 0 6-1 1 6-1 2 Cross section (A4) of a fresh kiwifruit stem scar stained with Phloroglucinol-HCI. (mg. xO.7) . The sclerified plug (p) is wel l defined and vascular bundles (v) are diverging between the inner and outer pericarp. Cross section (AS) of a fresh kiwifruit stem scar stained with Phloroglucinol-HCI.(mg. xO.7) . The lower, compacted region of the sclerified plug (p) is surrounded by wel l defined vascular bundles (v) and seeds (s). Longitudinal section of a kiwifruit stem scar stained with safranin-fast green showing parenchyma (p) , sclereids (s) and collenchyma (q) cells. Bar indicates 0.2 mm. Longitudinal section of a kiwifruit stem scar stained with methyl violet eosin showing idioblast containing calcium oxalate crystals (raphides) (r) and parenchyma (p) cells. Bar indicates 0.2 mm. Longitudinal section of a kiwifruit stem scar stained with methyl violet eosin showing xylem vessels (x) with helicoidal secondary wall thickening and parenchyma (p) cells. Bar indicates 0.02 mm. Longitudinal section of a kiwifruit stem scar stained with methyl violet eosin after two days curing at 20 or 30°C. Most of the spores (s) scattered on the surface of the xylem vessels (x) have germinated. Bar indicates 0.2 mm. xxiv 1 40 1 4 1 1 41 1 43 1 43 1 44 6- 1 3 6-1 4 Longitudinal section of a kiwifruit stem scar stained with methyl violet eosin after two days curing at 1 DoC. Most of the spores (s) scattered on the surface of the xylem vessels (x) have not germinated. Bar indicates 0.2 mm. Longitudinal section of a kiwifruit stem scar cured at either 0 or 1 DoC for two days stained with methyl violet eosin. Parenchyma cell walls (p) in contact with hyphae (h) are thicker than normal . Bar indicates 0.2 mm. xxv 1 44 1 45 CHAPTER ONE GENERAL INTRODUCTION World market and economic importance Worldwide, kiwifruit (Actinidia deliciosa (A. Chev.) C .F. Liang et A.R. Ferguson var. deliciosa) production has become one of the fastest­ developing horticultural industries of the last 1 0- 15 years. In New Zealand, the kiwifruit industry has developed as one of the most important horticultural crops. In 1 992 world production of kiwifruit was 793,000 tonnes with Italy and New Zealand each producing about 270,000 tonnes (Costa et al. 1 991 ) . At present, New Zealand's share of the international kiwifruit market returns NZ $500 mill ion per year. The main international importers are Japan, The United States and several European countries (Anonymous 1 995). Commercial orchards in New Zealand I n New Zealand, kiwifruit have traditionally been grown in citrus growing areas of the Auckland province, but commercial plantings can now be found from Northland to Nelson and even in some areas of Marlborough and Canterbury. There are some commercial orchards of kiwifruit in the Wanganui and Horowhenua regions and more recently, in Hawkes Bay. However the largest area of kiwifruit plantings are located in the various districts of the Bay of Plenty (eg. Te Puke, Opotiki, Tauranga) where almost 90% of New Zealand commercial kiwifruit plantings are to be found (Fig . 1 -1 ) (Ferguson & Bollard 1 990). Cultural, Management and Harvesting Practices for kiwifruit in New Zealand In earl ier years, varieties such as "Bruno" , "Abbot/Allison", "Monty", "Hayward", "Gracie", "Brodie", "Wilkins super", were selected and propagated throughout New Zealand, but in recent years the storage qual ities of cv "Hayward" made this selection the most important variety. At present it is almost (98%) the sole variety of commerce both in New Northland Auckland Nelson Coromandel Bay of Plenty North Island Marlborough South Island Hawke's Bay Wairarapa Canterbury Wanganui Manawatu Horowhenua Figure 1-1. Kiwifruit growing areas in New Zealand (compiled from various sources) . Chapter One 3 Zealand and overseas (Sale 1 990) . There is concern about an industry based on one cultivar only and Crown Research Institutes such as the Horticultural Research Institute of New Zealand are proceeding with the selection and development of new varieties. For comparison, the apple industry is of a simi lar size but is based on a number of varieties which include early, mid-season and late varieties and red, yellow or green fruit coloration. Since kiwifruit can be successfully stored for many months there is less of a market requirement for an extension of season, both earlier and later, although this could reduce peak season labour requirements for harvesting . The current research interest in the development of new varieties is for cultivars that are hairless, and for varieties with a different coloured flesh (Pringle et al. 1 991 ; ) . Among the wide range of cultural and management practices of kiwifruit, training and pruning have become the most important aspects of vine management (Sale 1 990). The development of training and pruning methods to control the vigorous vegetative growth of kiwifruit has increased fruit yields throughout the years. After extensive experimentation, the most popular commercial kiwifruit training methods reported are T -bar (Fig . 1 -2) and Pergolas (Fig. 1 -3) . In 1 980 more than 60% of the plantings in the Bay of Plenty were on T-bars (Sale & Lyford 1 990) . Clark & Gravett ( 1 992) found in a one year survey that those losses caused by B. cinerea from orchards util ising pergola support structures were greater than those with T-bars. Summer and winter prunings are important aspects of vine management. Winter pruning aims to retain an optimum number of evenly distributed one­ year old replacement canes, while summer pruning is essential to maintain order in the vine structure, proper spacing of laterals and canes and maximum light penetration into the canopy (Fig. 1 -4) . The most important objectives of this practice are to al low air movement and penetration of light around the vines to minimize the conditions which favour fungal diseases Chapter One 4 a) • • , \. � • • ;'1 ••• , <# b) Figure 1-2. T-bar support system for kiwifruit. a) Standard T-bar and b) Winged T-bar (Alexander 1 985; Sale and Lyford 1 990) . Chapter One 5 0' 0> 0> � "0 � o ->-...J "0 C ro Q) ro CJ) - � o - E Q) -en >­en t o 0-0-::J en ro (5 e> Q) Cl. M I T"" Chapter One leader --------------------------------- next season's fruiting arm unpruned in summer buds winter pruning cut summer .... � - pruned (}t fruiting laterals .. � Figure 1-4. Kiwifruit pruning methods (Sale 1 990) . 6 Chapter One 7 such as Bofrytis and to increase fruit yields (Sale & Lyford 1 990) . Commercially kiwifruit are harvested when the soluble solids content (TSS) of the fruit has reached 6.2% (Sale 1 990; Hopkirk 1 992) . Harvesting starts at the end of ApriVbeginning of May and continues until the middle/end of June. Fruit is hand harvested, by snapping off the fruit at the stalk. During harvest, workers wear cotton gloves to avoid bruises or blemishes to fruit and to protect hands from scratches and sap. In general , two pickings are normally made, with the larger fruits harvested at the first picking and the remainder during the subsequent harvest (Sale 1 990) . During harvest, fruit are placed into an apron bag which , when ful l is emptied into a bin. Bins are generally made of wood and hold 250 kg of fruit. Although bins do not have any special size or shape a maximum depth of 40 cm has been recommended to minimize crushing of the fruit in the bottom layers by the weight of fruit above them (McDonald 1 990) . Transportation of the fruit from orchard to packinghouse is by either trailer or truck. Bins are emptied and returned to the orchard for refil l ing (Harvey ef al. 1 983; McDonald 1 990; Lenting 1 99 1 ) . Grading and sorting Once the fruit reaches the packinghouse (Fig . 1 -5) , the main activities before packing are inspection and grading. The first activity, is carried out by trained operators to remove damaged or blemished fruit. The subsequent grading for size is done automatically by machine, each fruit being weighed separately. Some grading machines are of the orbital type in which the fruit is thrown away through the air, the distance projected depending on weight and fruit are caught in various canvas chutes (McDonald 1 990; Sale 1 990). Packing Fruit for export is packaged as specified by The New Zealand Kiwifruit Marketing Board. The more common packages are: a single-layer tray of Chapter One O h d ;- Packhouse rc ar ---- Tray materials ---- Tray Assembly Bin-tipper l Inspection __ Re'ect table J l Brushing j Singulator l Grader � . � t Tray filling l Palleting l Strapping l Precooler _ l Cool-store - � -t � Export Figure 1-5. Kiwifruit handling system. (Modified from McDonald 1 990) . 8 / Chapter One 9 approximately 3.5 Kg of fruit; tripack, containing 1 0.2- 1 1 .0 Kg fruit and bulkbins equivalent to 1 5 or 31 single layer trays. Although the material of fruit containers has been continuously modified in the last few years to meet the environmental requirements from European countries, in general fruit is placed into the preformed plastic pocket tray-packs, wrapped in polyethylene l iners and packed in cardboard trays (Sale & Lyford 1 990) . Storage and Marketing Once the fruit are packed into the appropriate container, they are often immediately precooled (Fig . 1 .5). The most common precool ing technique is forced air where cool air is forced between the fruit to remove heat and bring it more rapidly (8h from ambient to 2°C) to near coolstore temperature. After precooling, fruit are stored in cool rooms at 0.5-1 °C and a high relative humidity (at least 95%) to be kept for long term storage of 4-6 months (McDonald 1 990) . With the exception of fruit going to Australia, kiwifruit can only be exported by The New Zealand Kiwifruit Marketing Board (Earp 1 990) . Diseases of kiwifruit Major diseases of kiwifruit and a brief explanation of symptoms are given in Table 1 - 1 . BOTRYTIS CINEREA Taxonomy, morphology and general life cycle B. cinerea is the asexual stage of Botryotinia fuckeliana (de Bary) Whetzel . Botrytis is classified in the true fungi Eumycota, subdivision . Deuteromycotina, Class Hyphomycetes and Order Hyphales (Agrios 1 988) . B. cinerea produces abundant grey mycelium and long branched conidiophores with rounded apical cells bearing clusters of colourless or grey, one-celled, ovoid conidia which contain 4-1 8 nuclei. (Jarvis 1 980; Agrios 1 980). The fungus overwinters in the soil as mycelium growing on Table 1-1. DISEASES OF KIWIFRUIT Diseases Storage Botrytis storage rot (Botrytis cinerea) Ripe rot or Doth iorela rot (Botryosphaeria dothidea) Field Phytophthora root rot (Phytophthora cactorum, Phytophthora cinnamom/) Honey fungus (root rot) (Armillaria spp) Symptoms Extreme softness localized at the stem end. The fungus fi rst visible on the fru it surface as a white fluffs spreading alongside the fruit. Mycelium turns grey with sclerotia formation (Brook 1 990b). Fru it rotting after coolstorage. A portion of the fruit col lapses (Sommer et al. 1 992) . Drought and starvation symptoms on the leaves, plants become weak and susceptible to attack by other pathogens. Necrotic brown lesions on roots (Agrios 1 988) . Plant growth reduction, smaller yellowish leaves, dieback of twigs and branches and gradual or sudden death of the tree. Development of white mycelial mats on roots (Agrios 1 988) . ...... o Table 1-1. (Cont.) Sclerotinia fruit rot (Sc/erotinia sc/erotiorum) Leaf spot (Alternaria a/ternata) (G/omerella spp. , Colletotrichum spp.) Bacterial blossom rot (Pseudomonas spp.) Appearance of a white fluffy mycel ial growth . Firstly developing of a white sclerotia and later becoming black (Agrios 1 988) . I nfected fruit often fall but sometimes the infection will heal leaving an unsightly scar on the fruit (Sale 1 990) . Numerous leaf spots and blight dark brown to black colour. Progression of disease cause leaves to dry and fall off eventually (Agrios 1 988) . The fungus can be present in plants at all stages of growth . In leaves the fungus attacks the veins of the underside of the leaves causing lesions dark, brick-red to purpl ish (Agrios 1 988) . Development of necrotic spots on leaves and buds. When disease is advanced necrosis leads to blights and death of the plant partially or total (Agrios 1 988) . ..... ..... Chapter One 1 2 decaying plant debris and as sclerotia. Sclerotia are generally considered to be the most important structures involved in the survival of this fungus (Coley-Smith 1 980) . Sclerotia almost invariably germinate to produce mycelial threads that can infect plant tissue directly but in a few instances sclerotia have been found germinating to produce apothecia and ascospores (Agrios 1 980) . The conditions for producing the sexual stage form sclerotia has been studied and an appropriate laboratory procedure devised (Faretra & Antonnaci 1 987) . Pathogenicity of B. cinerea on different commodities B. cinerea is probably the most common and most widely distributed disease of vegetables, ornamentals, fruits and field crops throughout the world. Grey mould caused by B. cinerea is a serious disease of lettuce (Wheeler 1 969) , grapes (Mustonen 1 992) and several species of berries l ike raspberries (Knight 1 980) , and strawberries (Jarvis 1 962). Several species of Botrytis cause plant diseases, for example B. alii, B. byssoidea and B. squamosa cause neck rots of onions and B. tulipae, tulip fire. Leaf spot diseases of economic importance are chocolate spot of broad beans caused by B. fabae and B. cinerea (Wheeler 1 969). In apples, blossom-end rot caused by B. cinerea can lead to considerable economic losses (Lakshminarayana et al. 1 987). In the last decade, stem-end rot caused by B. cinerea has been reported as one of the major causes of rotting of stored kiwifruit (Hawthorne et al. 1 982) . B. cinerea is a common fungus of greenhouse-grown crops. Grey mould of ornamentals is reported as a serious disease of begonias, roses, cyclamen and several cut flower crops (Agrios 1 980; Fletcher 1 984; Hammer et al. 1 990) . The infection process by Botrytis Germination of Botrytis conidia on the host surface is the first step in the infection process from conidia. Certain conditions such as availability of nutrients on the p lant or fruit surface, production of toxic substances by the host, nutritional status of the plant etc. are required for the success or Chapter One 1 3 fai lure of Botrytis germination and penetration into the host (Kamoen 1 992) . Once the fungus has germinated, penetration is considered as the second important step of the infection process. Penetration of Botrytis is carried out by the formation of a narrow infection hypha which may form from an appressorium or from the tip of a germtube. Kamoen (1 992) reported this type of hyphal swelling during penetration in onions, tomatoes and gerbera ray flowers. Invasion of Botrytis can also occur through natural openings such as stomata and on carpels. B. fabae and B. cinerea on broad beans and strawberries respectively are examples of this type of penetration (Verhoeff 1 980) . Wounds produced on vegetable crops during cultural practices such as pruning and grafting and during the produce harvesting are also important points of entry for Botrytis. After penetration into the host, lesions expanded as a result of both intracellular and/or extracel lular hyphal growth, spreading throughout the whole plant organ at a rate that varies according to the compatibility of the host/pathogen combination (Kamoen 1 992) . Effect of environment during the infection process by Botrytis Relative humidity and temperature are considered the most important envi ronmental factors that affect the initial infection and spread of Botrytis. Infection by this fungus can take place in the field, greenhouse or storage rooms. In the latter although the development of disease occurs during storage of the produce, the infections most probably occur in the field and may involve a latent stage. Most reports of B. cinerea epidemiology mention that a high relative humidity (92%) is necessary for B. cinerea infection (Jarvis 1 980). Optimum temperatures for B. cinerea infection in the field or at greenhouses vary according to each specific crop and the time of exposure. Jarvis (1 980) gives as optimum temperatures for germination of B. cinerea on strawberries as 20°C to 30°C for 3-20h or 3°C to 20°C for 3h with high relative humidity in grapes (Nelson 1 951 ) . Thomas et al. ( 1 988) , found more conidial formation on the surface of grapes at 2 1 °C with 94% relative humidity and 0.6 m/sec wind speed than on grapes held at the -------- Chapter One 1 4 same temperature and relative humidity and zero wind speed. Conversely, in macadamia flowers B. cinerea was depressed at temperatures higher than 20°C and relative humidity of 95-1 00% (Hunter et al. 1 972) . In other crops such as tomatoes, tulips and onions, it was necessary for periods of h igh rainfall or long persistent water films and high humidity for aggressive lesions caused by this pathogen to develop (Price 1 970; Verhoeff 1 970; Jarvis 1 980) . Similar environmental conditions plus air movement were necessary for B. cinerea infection on greenhouse grown cucumbers (Yunis et al. 1 990) . B. cinerea can germinate at O°C and high relative humidities and cause infection. During storage of several vegetable crops growth of B. cinerea and infection were enhanced by the high relative humidity and condensed water (Jarvis 1 980) . In strawberries B. cinerea develops during transit and storage at O°C. Stem-end rot of kiwifruit during storage can also occur at O°C (Sharrock & Hallet 1 991 ) . Kiwifruit and B. cinerea Prior to 1 978 B. cinerea was not a commercial problem on kiwifruit but as the kiwifruit industry developed in New Zealand, it became a disease problem during storage (Pennycook 1 985; 1 990) . At present, Botrytis stem­ end rot is one of the major problems facing the kiwifruit industry (Manning & Pak 1 993) . For example, in 1 991 postharvest losses of kiwifruit from Botrytis rots were evaluated at NZ $6 mi l lion with labour and repacking costs at a further NZ $4.5 mil lion (Personal communication: Peter Bul l) . Botrytis is a major contributor to the wastage through fruit loss, lost revenue and loss of confidence in the marketplace (Anonymous 1 994a) . It has been widely reported that B. cinerea gains entrance into various fruits, including kiwifruit through the stem-end wound produced during the snapping of the fruit from the pedicel at harvest (Sommer et al. 1 983; Poole & McLeod 1 991 ) . Sharrock & Hallet ( 1 992); Hallet & Sharrock ( 1 993) Chapter One 1 5 reported that the primary route for postharvest infection from B. cinerea spores is through the stem-end vasculature which has been exposed by picking the fruit from the pedicel. Increased contamination of the fruit by this fungus, is likely during the subsequent handling operations such as grading and packing (Pennycook 1 990; Brook 1 991 ) . Numerous studies have shown that primary Botrytis rot in kiwifruit generally appears after 4-5 weeks but before 1 2- 13 weeks coolstorage at O°C. Secondary Botrytis rots (commonly termed "nesting") develop when the Botrytis hyphae spread across the trays from rotten fruit to adjacent healthy fruit (Lallu 1 989b; Beever 1 991 ; Sommer et al. 1 992) . CONTROL MEASURES Alternatives for control of B. cinerea Chemical control: Control of B. cinerea in the field and during storage has been achieved using various fungicides. The dicarboximides (iprodione, procymidone, vinclozolin) have been extensively used in spray programmes in the orchard but only for experimentation during coolstorage of the fruit. For example a sign ificant reduction of B. cinerea ( 1 . 1 %) after 1 2 weeks storage was achieved when 0.05% of vinclozolin as "Ronilan" was applied at harvest compared with control fruit (6.6%) (Pommer & Lorenz 1 982) . However there would be little future in postharvest use of such fungicides because of the ability of B. cinerea to develop resistance to them and the lack of a complete protection against B. cinerea during coolstorage (Brook 1 990a) . Postharvest fumigation methods under investigation for kiwifruit include treatments with ozone (03) and sulphur dioxide (S02) . In preliminary experiments application to the stem-scar of 250 ppm of 03 reduced stem­ end rot to 0.25% compared with 1 .5% in the control fruit (Brook 1 990a) . Cheah et al. ( 1 992a) reported complete inhibition of germination in vitro of B. cinerea spores at S02 concentrations of 400-3200 ppm applied for 1 0, Chapter One 1 6 20 or 30 min and reduction of stem-end rot with time of treatment increased within levels between 800 to 3200 ppm. Although the above mentioned methods have shown promise for control of B. cinerea during coolstorage of the fruit, the increasing worldwide trend to consume commodities free of chemical residues means such postharvest treatments do not now have general international approval (Hammer & Marois 1 989; Johnston 1 994). Moreover, in the near future there wil l be new legislations conceming the use of pesticides in the kiwifruit industry in New Zealand (Holland et al. 1 994) . Therefore at this stage, there is a trend to concentrate experimental work on the control of B. cinerea by means of non-chemical agents. Non-chemical control methods: Heat treatment by dipping the fruit in hot water or by dry air treatments at 44-50°C reduced stem-end rots during storage (Brook 1 990a), Similar Botrytis control (92%) but at temperatures between 50-54°C, for 2, 4 or 8 min, 42 and 46°C for 8 min (64%) , 46°C for 1 5 min and 48°C for 8 min ( 1 00%) have been reported by Cheah et al. ( 1 992b; 1 993a; 1 993b) but at the same time, they reported that certain disorders of the fruit such as physiological breakdown and rapid loss of firmness were encouraged at the highest temperatures. Reports associated with biological control experiments in which microorganism such as Cladosporium cladosporioides, Bacillus subtilis, Erwinia herbicola, Trichoderma spp. were applied to kiwifruit to stem scars before inoculation with B. cinerea spores, achieved a reduction of stem-end rots caused by this fungus (Harvey et al. 1 991 ) . A decrease of B. cinerea infection after 1 0-1 2 days incubation at 20-22°C was shown in fruit artificially inoculated at the stem scar by various isolates of B. subtilus and spores of B. cinerea previously grown on agar or in l iquid culture. B. subtilus isolates grown on agar gave . between 1 0 to 55% infection, while Chapter One 1 7 those grown on the l iquid media showed B. cinerea reduction between 0 to 1 5% (Brook 1 990a). Additional investigations carried out by Cheah et al. ( 1 992c) found an antagonist effect between various isolates of Trichoderma and B. cinerea. In that study five isolates of Trichoderma significantly reduced B. cinerea sporulation (0-1 3.9%) on kiwifruit stem scars after ten days incubation at 1 5°C. Manipulation of controlled atmosphere storage of kiwifruit to reduce B. cinerea infections is another approach still under experimentation (Manning & Lallu 1 993; Lallu & Manning 1 994) . However, in general results have shown an increase (2.0-2.9%) in both B. cinerea stem-end and other rots such as those caused by Phomosis, Fusarium and Crytosporiopsis in fruit stored under modified atmospheres compared with air stored fruit. Another natural method for control of stem-end rots of kiwifruit during coolstorage has been to hold the fruit at ambient temperature for several days before storage (Evans 1 992) . This preconditioning practice carried out soon after harvest is commonly named "curing" (Brook 1 990a; Hallet et al. 1 99 1 ; Beever 1 992; Pennycook & Manning 1 992) . Definition of curing Curing is a pre-storage practice widely used to reduce spoilage by microorganism during the normal storage period of a number of commodities such as fruits (Hopkins & Loucks 1 948; Ben-Y ehosua et al. 1 987a; Pennycook & Manning 1 992) and vegetables (Passam ef al. 1 976; Baudoin & Eckert 1 985). In root and tuber crops curing refers to the process of wound healing with the development and suberization of new epidermal tissue called wound periderm whereas in bulb crops, curing is the process of drying the external layers of those vegetables to reduce neck rot diseases (Kasmire & Cantwell 1 992) . Beever ( 1 99 1 ) has defined the term curing "as the encouragement Chapter One 1 8 of the natural processes of repair that take place in a fruit or vegetables, following physical damage". Commercial curing of fruits and vegetables is carried out by holding the produce in conditions of relative humidity and temperature which are different (usually higher temperatures) from those in which the commodity is held for long term storage (Kitinoja & Kader 1 994) . Impact of curing to control postharvest diseases. In tubers, bulbs and roots: Most tubers and roots have the abi l ity to heal cuts, wound and bruises if provided with a suitable environment (Ryall & Lipton 1 980) . Meijers ( 1 987) found that in root and tuber crops the curing process or wound healing is developed in a two stage process which result in the development of new epidermal tissue called wound periderm. A number of diseases can spread through potato tubers in storage, e.g . gangrene (Phoma spp.) , dry rot (Fusarium caerulum) , black scurf (Rhizoctonia solam) , late blight (Phytophthora infestans) , blackleg (Erwinia carotovora pv. atroseptica) and soft rot (Erwinia carotovora pv. carotovora). Curing is an important procedure in l imiting the losses caused by these postharvest disease problems. The development of gangrene in potato was studied by Malcomson & Gray ( 1 968) . They found suppression of gangrene disease when tubers were held for three, seven or ten days at 1 8-23°C and concluded that suppression of gangrene disease increase with length of the curing period. Similarly Adams & Griffith ( 1 978) found that a curing period of ten days, together with the time of harvest and previous wounding of the tubers were the main factors which affected the incidence of gangrene in potato. Stewart et al. ( 1 983) cured potatoes at 1 5°C and 1 00% relative humidity for one day to control tuber blight and also found that susceptibil ity to this Chapter One 1 9 disease decreased with late harvest. Morris et al. (1 989) found stimulation of suberin development and periderm formation when wounded potato tubers were held at 25°C and 98% relative humidity for seven days. Snowdon ( 1 991 ) reported differences in curing time according to the holding temperature. Three to six days were necessary to cure tubers at 20°C, one to two weeks at 1 0°C and three to six weeks at 5°C, while there was no curing effect at lower temperatures. Improvement in caladium tubers by means of curing has been reported by Marousky & Raulston ( 1 973) and Marousky & Harbaugh (1 976). They reported that the suberization process was increased in tubers incubated at 26.6°C and at relative humidities of 75% compared with tubers held at the same temperature and 90% relative humidity. Bulbs of various cultivars of the Dutch iris (Iris hollandica Hoog) plants were improved by means of ethephon treatments together with curing (Cascante & Doss 1 988) . The time to flowering was shortened and there was a reduction of the numbers and length of leaves when bulbs were dipped for 1 h in ethephon at 0.25 gil and then heat cured at 32°C for three days. Curing of zantedeschia tubers (Zantedeschia elliottiana) is also an important commercial practice to protect them against microorganisms and desiccation. Funnell et al. (1 987) reported development of suberized cells and periderm formation in tubers cured at 30°C and 80% relative humidity for periods longer than three days. Further research carried out by Funnell & McKay ( 1 988) reported that although suberization could take place at 30°C and at both 80 or 40% relative humidity, less weight loss was reported in those tubers incubated at the highest relative humidity. The beneficial effects of curing has also been reported in tropical roots, tubers and corms such as cassavas (Manihot esculenta C.) boniatos sweetpotatoes and yams (Dioscorea spp.) , for control of postharvest Chapter One 20 diseases such as black rot (Ceratocystis fimbriata Ell . & Haist.) and various storage rots (Rhizopus spp. and Dip/odia spp.) (Snowdon 1 99 1 ) . In sweetpotatoes decay was reduced by 1 9%, according to the harvest date and cultivar when held for 1 week at 30°C, 1 00% relative humidity (Delate et al. 1 985) , whilst Snowdon ( 1 99 1 ) reported the best curing temperature for this commodity as 35°C for two to ten days. Walter et a/. ( 1 989) , reported that wound healing in this commodity was closely related to the soil temperature in which the crop was grown. They found that the best curing results i .e . less rots and less weight loss, occurred on tubers of sweetpotato harvested at soil temperatures of 1 5-1 7°C and cured for one to seven days at 30°C and 90-95% relative humidity. Storage qual ity of commodities such as cormels (Xanthomona caracu Kouch and Bouche) can also be improved by curing. According to Mbonomo & Brecht ( 1 99 1 ) cormels cured at 30 or 35°C and 95-1 00% relative humidity for seven days had less weight loss and decay caused by soil-born pathogens such as Erwinia carotovora and Erwinia chrysanthemi during storage than uncured cormels. Snowdon ( 1 99 1 ) reported an increase in the quality of carrots (Daucus carota L.) following a brief curing period at ambient temperature and high relative humidity to promote wound healing. Postharvest diseases of shallots, onions and garlics such as black mould (Aspergillus nigerv. Tieghem) and bacterial rots (Erwinia spp. , Lactobacillus spp. and Pseudomonas spp.) can be successful ly controlled by curing (Snowdon 1 99 1 ) . According to Kasmire & Cantwell ( 1 992) and Kitinoja & Kader ( 1 994) , for these two commodities, five to ten days were necessary to allow the external layers of the skin and neck tissue to dry when held at ambient temperature, whilst one day or less was sufficient using forced air at 35 to 45°C with relative humidities at 60 to 75%. Snowdon ( 1 99 1 ) recommended 27-30°C as the most optimum temperature range for curing. Chapter One 21 In vegetables: Curing is recommended for vegetables such as squash and pumpkin. Snowdon ( 1 991 ) , reported curing temperatures of 20°C or higher for one week, to reduce rots caused by Alternaria alternata (Fr.) Keissler and Alternaria cucumerina (Ell . & Ev.) E lliot, and to develop wound healing. In leafy vegetables: Bae ( 1 989) found that leaf rotting caused by Botrytis spp. decreased in a number of cultivars of tobacco when leaves of this commodity were cured at temperatures of 25, 30 and 35°C and relatives humidities of 82, 92 and 1 00%. This researcher also found that development of Botrytis disease increased during the rainy season compared with the dry season. Other studies on this commodity have shown that content of leaf fatty acids such as palmitic, oleic, l inoleic and l inolenic increased with curing (Srivastova & Chaudhary 1 990) . In tropical and subtropical fruits: Control of green mould (Penicillium digitatum) in "Valencia" oranges (Brown & Barmore 1 983) , sealed lemons, and pummelo fruit (Citrus grandis L. Osbeck) (Ben Yehoshua et al. 1 987a 1 987b; 1 988b) was reduced about 1 2%, 2- 4% and 5% respectively when fruit were cured at 30°C for 24h in combination with high relative humidities. In lemons, the incidence of active lesions of sour rot (Geotrichum candidium Link ex Pers) were reduced about 8% fol lowing 5 days of curing at 25°C and 1 00% relative humid ity (Baudoin & Eckert 1 985; Predebon & Edwards 1 992) . Curing of sealed citrus (pummelo fruit) at 32-36°C enhanced lignification and antifungal metabolite production (Ben-Yehoshua et al. 1 988a). In grapefruits (Schiffmann-Nadel et al. 1 971 ) the incidence of storage rots during their shelf life and cold storage period was reduced about 5% when they had been cured at 1 2°C for several days or weeks. Chapter One 22 In temperate fruits: The process of wound heal ing in apples has also been studied (Skene 1 981 ; Lakshminarayana et al. 1 987) . Although in these studies the term curing was not used, In wounded apples, the development of periderm tissue, synthesis of phenolic substances, tannins, lignins and callose varied with temperature, time of incubation and harvest date. They reported that 38 days at 5°C were necessary to develop resistance compared with only 1 4 days when incubated at 20°C. They also reported less decay caused by B. cinerea and P. expansum inoculated onto healed wounds than onto fresh wounds. Curing as an alternative for control of B. cinerea during kiwifruit storage Several workers have found that a delay between harvesting the fruit and inoculation results in a decrease in infection . There is a considerable body of evidence that once the pedicel has been removed, the stem scar develops a high level of resistance (Sharrock & Hallet 1 99 1 ; 1 992) . Penycook & Manning (1 992) and Beever ( 1 992), reported that the incidence of stem-end rot in this commodity was reduced (from 46% to 6%) progressively with increasing duration of a holding period at ambient temperature. Microscope studies by Hallet et al. ( 1 991 ) showed that there was l ittle germination of Botrytis spores on stem scars of cured kiwifruit compared with those on uncured ones. Collapse of Botrytis spores on the stem scar were frequently observed in the cured fruits. Long-term storage conditions to reduce B. cinerea on kiwifruit. The postharvest storage life of most commodities for short or long-term storage is dependant on the environment. The storage system in use should be able to reduce or minimize postharvest losses due to the normal physiological responses of the fruit to environment and to infection by microorganisms. Chapter One 23 Among the principal objectives of much long-term storage research are the reduction of commodity metabolism to delay senescence, minimization of moisture loss to reduce shrinkage and loss of turgor and reduction of growth and spread of microorganisms. Davis ( 1 980), considered that produce should be kept at lower temperatures for long-term storage than those used for short-term storage. Once fruit or vegetable produce is separated from the plant or dug from the soil, the postharvest life is short at ambient temperature. Low temperatures serve to greatly extend the storage life of the commodity by suppressing disease and extending host resistance by means of delaying the commodity senescence (Sommer 1 985; 1 989) . In contrast to the uniformity of an optimum requirement for low temperatures for long-term storage, there are some contradictions about the optimum relative humidity for low temperature storage of vegetable crops. Mann ( 1 954) and Thompson ( 1 992) considered that the relative humidity should be maintained at 90-95% for most perishable commodities because higher levels of relative humidity in the storage room would encourage high levels of decay. Similar optimum storage relative humidities of 90-95% have been reported for potatoes, sugar beets, carrots and cabbages (Ryal l & Lipton 1 980) . However, studies on carrots showed that lower or equal levels of infection were found when they were stored at relative humidity ranges of 98-1 00% than at 92-96% (Van den Berg & Lentz 1 966; 1 973a; 1 973b; 1 974) . Further research (Van den Berg & Lentz 1 977a; 1 978) with parsnips, rutabagas, carrots, celery and other vegetable crops also showed that relative humidities between 98-1 00% reduced decay levels during storage. Studies on kiwifruit (McDonald 1 990; Mitchell 1 990; Lallu et al. 1 992) have shown that O°C and relative humidities above 95% are the most suitable environmental conditions to store this commodity in order to avoid a high incidence of disorders and high moisture loss. Differences in this Chapter One 24 recommended storage temperature are sl ight: Sale ( 1 990) recommended temperature ranges between -0.5 to O.SoC as the optimum for kiwifruit storage. It is unlikely that a more accurate control of temperature under commercial conditions could be achieved. GENERAL OBJECTIVES I n this study, three related areas of the kiwifruit - B. cinerea pathosystem were investigated: - First, some factors which could affect the pathogenicity of B. cinerea were investigated . These included an evaluation of isolate-to-isolate variability in pathogenicity to kiwifruit using isolates obtained from various infected crops from different areas of New Zealand, and several factors affecting inoculum potential (culture age, inoculum concentration and growing media) . - Second, in vitro survival of conidia of B. cinerea incubated over various periods of time at several temperatures and relative humidities that could be used during curing were studied. -Third, environmental prestorage curing conditions (temperature and relative humidity) were assessed for effects on general fruit characteristics during storage,for effects on stem scar structure and for suppression of storage rots of kiwifru it. CHAPTER TWO GENERAL MATERIALS AND METHODS The materials and methods described here are those used in most of the present study. Where procedures differed, or are specific to each chapter then they are explained in the materials and methods section of that chapter. Fruit harvesting Kiwifruit used in these experiments were harvested from vines trained on a winged T bar system at Massey University Fruit Crops Orchards, Palmerston North (Lat S 40 21 , Long E 1 75 37) , New Zealand. Fruit were harvested between 7:30 to 1 0:30 am and the relative humidity and temperature in the orchard were recorded at the start of harvesting. Fruit were snapped from the pedicel and placed into a picking bag which was emptied into a cardboard carton at the end of the row. The harvested fruit were taken to the laboratory and the sepals removed with a soft nail brush to provide a more uniform environment at the picking scar. One 1 7 JlI droplet of the appropriate spore suspension was placed on each picking scar wound. After the droplet had dried (about 30-45 minutes) fruit were placed in plastic plix trays in commercial cardboard kiwifruit trays with a polyethylene l iner. Unless otherwise stated they were immediately stored at O°C in the Massey University Plant Growth Unit coolstores. Preparation of inoculum A single spore isolate of B. cinerea (isolate K3) which was originally obtained from diseased kiwifruit at Massey University was maintained on malt agar and subcultured at 3-4 week intervals. Inoculum was prepared from 1 0- 14 day old cultures. Spore suspensions were prepared by flooding cultures with sterile 0.02% Tween 20 (Polyoxyethylene sorbitan monolaurate by Serva, Chapter Two 26 Feinbiochemica) , rubbing with a sterile bentglass rod and filtering through glass wool to remove hyphal fragments. Spore concentrations were measured using a haemocytometer and adjusted to the required spore concentrations. These varied between experiments but included one or more of 1 000, 5000, 25000 or 1 25000 per 1 7 III droplet of suspension corresponding to 5.9 x 1 04, 2.9 x 1 05, 1 .5 x 1 06 or 7.4 x 1 06 spores per m l respectively. Plastic dropper bottles fitted with a micropipette tip were much quicker to use than micropipettes and they del ivered a 1 7 III droplet. Since one droplet was placed on each stem scar the number of spores per droplet was a convenient way of expressing inoculum loadings (this technique was developed by Dr. Greg Tate of Crop Health Services, Hawkes Bay) . Quality measurements For each experiment initial firmness and total soluble sol ids of 1 0 to 1 5 fruits were measured at harvest on fruit taken one metre along the vine stem and one metre from the row. Firmness (kgf) was measured with a penetrometer (R. Bryce Mod. FT327) at the m id point of each side of each fruit after removing a 2-3 cm diameter disc of peel. Juice was squeezed from a slice cut from each end of the fruit and total soluble solids (TSS) concentration (%) was measured using a hand-held Atago N-20 refractometer (Mod. N. McCormick Fruit Tech. , brix range from 0-30%). Both fruit firmness and total soluble sol ids were calculated as the mean of the two measurements. Defined relative humidity The principle of incubating fruit in air of a defined relative humidity is simple. Air from an aquarium pump is bubbled through water or saturated salt solution , into an entry chamber where the input humidity can be measured, through the chamber containing the fruit and into an exit chamber where the relative humidity of the air leaving the fruit chamber can be checked. In practice, the aquarium pumps used in this work (Mod. El ite 802 or Etema I I ) produced flow rates of 2.5 or 3.5 I/min respectively as measured by Chapter Two 27 collecting air exiting the fruit chamber and collecting it over a specified time in a measuring cyl inder inverted in a water bath. The input and output chambers were 500 ml "Agee" preserving jars with input and output tubing fixed to the metal l ids and with a 2 cm hole that could be plugged with a rubber bung or with a humidity probe with a sealing strip to the same diameter. In itially, the water and salt solutions were placed in one litre "Agee" jars with 5-1 0 mm internal diameter plastic tubing as inlet and outlet tubes. The in let tubes ended at the bottom of the jars and the outlet tubes were flush with the l ids. Three problems were encountered with this design: 1 . Salt crystall ised in tubes which soon became blocked. 2. The vigorous bubbling of the salt solutions forced l iquid up into the tubes. 3. With a full flow rate from the pumps, the air bubbles were large with a smal l volume:surface ratio and were in contact with the salt solutions for such a short time that it was impossible to maintain a constant humidity in the desired range. At lower flow rates there was insufficient air passing through to cope with the water loss from the fruit. After considerable experimentation, these problems were overcome by: 1 . Using tubing with a larger ( 1 5-20 mm) internal diameter. 2. Placing the water or saturated salt solutions in vertical polythene tubes 1 m long and 35 mm diameter. With the tubes two thirds to three quarters ful l there was sufficient headspace to prevent l iquid bubbl ing up into the outlet tubing and this combined with the larger diameter outlet tubing eliminated the blocking by salt crystallisation . The great depth of l iquid through which the bubbles had to pass gave a longer contact time for the humidity in the bubble to stabil ise. 3. Connecting two tubes in series provided a stable, defined humidity output of 2.5 I/min for experiments in vitro and 3.5 Chapter Two 28 I/m in per series for the remaining experiments. Analytical grade Calcium Chloride (CaCI2) was used for the lowest relative humidity range, Sodium Chloride (NaCI) for the lower intermediate relative humidity range, Potassium Chloride (KCI) for the higher intermediate range and water for the saturated atmosphere. Relative humidity and temperature were monitored using a Squirrel meter/logger device mod. Grant 1 200 with a humidity probe (Gaffney 1 978; Talbot & Baird 1 991 ) or by a manual mercury thermometer respectively. Measurement of ethylene and carbon dioxide production Fruit were held overnight at 20°C to stabil ise their temperature . Ethylene and carbon d ioxide production were measured by placing individual fruit in separate air-tight glass jars, of approximately 700 ml capacity. Jars were sealed, incubated at 20°C for 1 h and 1 ml gas samples were then taken from each jar using an air tight syringe to withdraw the sample from the headspace above the fruit. Ethylene and carbon dioxide measurements were made from the same sample. A Varian 3400 or Pye Unicam series 1 04 gas chromatographer with flame ionization detector (FlO) and N2 as the carrier gas was used for ethylene and a GC-8A Shimadzu gas l iquid chromatographer for CO2• Respiration rate and ethylene production were calculated according to the fol lowing formulae (Shusi ri 1 992) : Carbon dioxide: �CQ [c02]final-[ CO2] initial . .,. 1000 60 II 2 x( Vjar- VfrUl'J x x- 100 wfruit T Chapter Two Ethylene: H [C2HJfinal-[C2HJinitial (VJ' Vl� ·,. 1 000 60 FC2 4- x 'jar- ,rUJ" X x- � OOO wfruft T Where: FC02 = rate of carbon dioxide production (cm3kg-1h-1 ) . FC2H4 = rate of ethylene production (/J.IKg-1h-1 ) . [C02]initial = initial carbon dioxide concentration (%) . [C2H4]initial = initial ethylene concentration (/J.rl [C02]final = final carbon dioxide concentration (%) . [C2H4]final = final ethylene concentration (/J.rl Vjar = jar volume (cm3) . wfruit = fruit weight (kg). T = time. 29 Titratable acidity was calculated as % (w/v) of citric acid in ju ice according to the following formulae (Shusiri 1 992) : where: %citric acid mINaOH*N(0.1 ) *64 1 ml juics* 1 0 64 = molecular weight of citric acid divided by 3 Fruit weight loss Total and daily weight loss was measured according to the following formulae: where: % weight loss wt initial-wt final initialx 1 00 wt wt initial = I nitial weight. wt final = Final weight. Chapter Two 30 Assessment of infection B. cinerea storage rot was assessed at six and 1 2 weeks of coolstorage. Infected fruit were removed at six weeks to prevent secondary spread of infections to neighbouring fruit. The Botrytis stem-end rot is quite distinctive but samples were tested for Botrytis to confirm diagnosis. Statistical Analysis The SAS System programmes (SAS/Stat User's Guide, 1 988) were used to analyze data from each experiment for Analysis of Variance (ANOVA), means, standard errors and standard deviations. If required, data were appropriately transformed to satisfy the basic assumptions for analysis of variance. Means separation was performed by Duncan's multiple range test (P < 0.05) . When data were unbalanced only means and standard errors were evaluated. CHAPTER THREE INOCULUM VARIABLES AFFECTING PATHOGENICITY OF BOTRYTIS CINEREA INFECTION OF KIWIFRUIT INTRODUCTION To induce Botrytis rot in kiwifruit during storage by artificial inoculation with spores or mycelium sti l l remains a problem. The incidence of rot can vary considerably from one experiment to another even when the same strain of Botrytis is used as inoculum at the same spore concentration. For experimental work on infection it is important to identify and quantify sources of variabi l ity. Nutrient status, age of spores, isolate to isolate variation and inoculum concentration can all affect infectivity of B. cinerea (Lorbeer 1 980) . Nutrient availabil ity is known to affect colony morphology and growth of B. cinerea. For example, studies in vitro with a range of B. cinerea isolates showed significant differences in sporulation and sclerotial formation according to the peptone concentration of the agar medium on which the fungus was grown (Stewart 1 986). Conidia of this fungus are dependent on exogenous nutrients for in vitro sporulation and germination (Leach & Moore 1 966; Lorbeer 1 980) . The pathogenicity of B. cinerea conidia is known to be stimulated by high levels of nutrients (Kosuge & Dutra 1 962; Kosuge & Hewitt 1 964; Koh l & Fokkema 1 994) . Nutrients required for infection can be supplied in the form of g lucose, fructose, potato dextrose agar (PDA) , malt extract agar (MA) or extracts of leaves or fruits (Clark & Lorbeer 1 977a) . Significant infection was recorded in cabbage (Brassica oleracea L) , onion leaves (Allium cepa L) , tomato (Lycopersicum esculentum M) and gerbera flowers (Gerbera Chapter Three 32 jamesonniJ) when glucose, pollen extracts or sucrose were added to the B. cinerea spore suspension (Chu-Chou & Preece 1 968; Chou 1 972a; Yoder & Whalen 1 975; Clark & Lorbeer 1 977b; Gorfu 1 986; Salinas et al. 1 989). A stimu lation of germination of B. cinerea conidia suspended in aqueous suspensions of pollen grain extracts of various small fruits, fruit trees and ornamentals was reported by Borecka & Millikan ( 1 973) . Van den Heuvel ( 1 981 ) reported that development of lesions on leaves of French beans caused by B. cinerea was dependant on the presence of various factors such as pH, type and molarity of the buffer, presence of g lucose and inoculum concentration. Further histological studies on bean, inoculated with B. cinerea spore suspension amended with glucose and other nutrients have shown variations in the penetration structures formed on the surface of the leaves according to the nutrients in which the fungus was suspended (Van den Heuvel & Waterreus 1 983). Recent studies on kiwifruit have also demonstrated a significant increase in disease incidence when the spore suspension was amended with a yeast extract (Long & Wurms 1 993) . The effect of spore age and inoculum level on germination and on infection has been studied by Last (1 960) . He compared B. fabae conidia of various ages for abil ity to germinate on leaves of a range of varieties of beans and found that percentage germination decreased with spore age. He also found that when the spore concentration reached a critical level , increased percentage germination was reduced due to self-inhibition of germination. This self-inhibition was not apparent on kiwifruit where high levels of inoculum were required to achieve good infectivity by B. cinerea (Long & Wurms 1 993) . Mansfield & Hutson ( 1 980) found that infections by high inoculum levels (200 conidia/droplet) of suspensions of B. fabae and B. tulipae on broad beans and tulip leaves grew at rapid linear rates compared with the low rate found when plants were inoculated with a lower spore concentration (20 conidia/droplet) . B. cinerea isolates may differ in their pathogenicity according to the origin Chapter Three 33 of isolates. In red raspberry canes (Rubus idaeus L.) significant differences were found in the lesion lengths inoculated with 31 different B. cinerea strains (Wil l iamson & Jennings 1 986). However, Bryk ( 1 985a) , inoculated apples with 80 B. cinerea strains from different hosts and found that all isolates caused apple fruit rot. OBJECTIVE To investigate the effect of Botrytis isolate, culture age, culture media and inoculum level on pathogenicity and infection of kiwifruit. MATERIALS AND METHODS Experiment No. 1 Title: Effect of B. cinerea isolates, culture age and inoculum concentration on in vitro conidial germination. The experiment was carried out in the Plant Health laboratory of Massey University, Palmerston North New Zealand. B. cinerea isolates were grown on malt agar (MA) for 7 or 28 days. Spore concentrations from each isolate/age combination were adjusted to 1 000 or 25000 spores/ 1 7 JlI droplet of suspension (equivalent to 5.9 x 1 04 and 1 .5 x 1 06 spores/ml). Inoculated malt agar disks (20 mm diameter) in Petri plates were incubated at 20°C for 1 2 hours to examine germination in vitro. One isolate of B. cinerea was obtained from each of infected strawberry (Palmerston North) , blueberry (Palmerston North) , grapes (Hawkes Bay) and camellias (Palmerston North) . The remainder of the isolates were obtained from infected kiwifruit at the New Zealand Institute for Crop and Food Research , Levin (CFRI) , at Massey University Fruit Crops Unit (Palmerston North) (OS) , Hort Research, Mt. Albert (Auckland) and Hort Research (Lincoln) . Chapter Three 34 Statistical Analysis The experiment was designed as a 8x2x2 factorial with three replications. Data required a square root transformation before Analysis of Variance. Experiment No.2 Title: Pathogenicity of B. cinerea isolates from a variety of sources on kiwifruit. The experiment was carried out at the Massey University Fruit Crops Orchard (MUFCO) , Palmerston North and duplicated at the New Zealand Institute for Crop and Food Research (CFRI) , Kimberley Road, Levin by Dr. L. Cheah. Fruit was harvested on June 2nd 1 992 at both sites. At harvest, temperature and relative humidity for MUFCO experiment were 1 3°C and 80% respectively while at the CFRI ambient environment was 1 3-1 5°C and 65-70% relative humidity. MUFCO fruit had a total soluble solids content (TSS) of 1 1 .6% and a firmness of 7.8 kgf, while those at CFRI had a TSS of 1 1 .0% and a firmness of 8.5 kgf. Cultures of B. cinerea isolated from infected kiwifrut were obtained from research centres in four different areas of New Zealand; at CFRI (Levin), Hort Research (Lincoln) , Hort Research, Mt. Albert (Auckland) and Massey University Fruit Crops Unit (Palmerston North) (OS) . A fifth isolate was obtained from diseased strawberry fruit and a sixth from diseased grapes (Hort Research , Hawkes Bay) . A laboratory-induced dicarboximide resistant strain (DR) of the Massey isolate was also used. All isolates were grown on MA for 1 0- 14 days. Spores suspensions were adjusted to the required concentration to give 1 000, 5000, 25000 and 1 25000 spores per 1 7JlI droplet of water (corresponding to 5.9 x 1 04, 2.9 X 1 05, 1 .5 X 1 06 and 7.4 x 1 06 spores/ml) . All cultures were prepared and grown at Massey University and those required at CFRI were couriered overnight. After inoculation , fruit were packed in commercial single layer 36 count trays and coolstored at O°C (90-98% R.H.) . B. cinerea storage rot from both Chapter Three 35 MUFCO and CFRI was assessed at 6 and 1 2 weeks of coolstorage. Statistical Analysis The MUFCO experiment was analyzed as a two factor experiment with four replications and the CFRI experiment was analyzed as a completely randomized (one factor) experiment since results for each tray within a treatment were not separated out. Square root transformation was carried for both MUFCO and CFRI data before analysis of variance. Experiment No.3 Title: Effect of growth media, culture age and inoculum level on pathogenicity of B. cinerea to kiwifruit. The experiment was carried out with fruit harvested at Massey University Fruit Crops Orchard (MUFCO) , Palmerston North and at the New Zealand Institute for Crop and Food Research (CFRI) , Kimberley Road, Levin by Dr. L. Cheah . Fruit was harvested on May 20th 1 992 at both sites. At harvest temperature and relative humidity from MUFCO were 1 1 °C and 80% relative humidity while at the CFRI ambient environment was 1 3-1 5°C and 65-70% relative humidity. MUFCO fruit had an average total soluble solids content (TSS) of 9.69% and a firmness of 8.5 kgf, while those at CFRI had a TSS of 7.6% and a firmness of 9.2 kgf. B. cinerea (Massey isolate K3) was grown on PDA, MA and autoclaved kiwifruit leaves (LVS) for 7, 1 8 or 28 days. Spore concentrations from each media/age combination were adjusted to 1 000, 5000 or 25000 spores per 1 7J.11 droplet of suspension (corresponding to 5.9 x 1 04, 2.9 x 1 05 and 1 .5 x 1 06 spores/ml) respectively. An additional concentration of 1 25000 per droplet (7.4 x 1 06 spores Iml) was also tested at CFRI . Aliquots of each spore suspension were incubated at 20°C on MA, PDA, L VS and water agar to evaluate in vitro percentage germination. Chapter Three 36 After inocu lation, fruit were packed in commercial single layer 36 count trays and coolstored at O°C (90-98% R.H.) . B. cinerea storage rot was assessed at 6 and 1 2 weeks of coolstorage at both research centres. Statistical Analysis The experiment was designed as a 3x3x3 factorial with three replications, at MUFCO and as a 4x3x3 with one replication at CFRI . Data from the MUFCO required a log transformation while data from CFRI required a square root transformation before Analysis of Variance. RESULTS Experiment No. 1 Significantly h igher (P < 0.001) conidial germination rates were found from cultures of B. cinerea isolated from diseased kiwifruit (Levin, Lincoln, Auckland and Palmerston North) than from cultures originating from other crops. The lowest germination rates were found in cultures isolated from diseased camellia and grapes (Table 3-1 ) . Spores prepared from young (seven day old) cultures of B. cinerea showed a higher germination rate (P < 0.001) compared with those from older (28 days) cultures (Table 3-1 ) . Spore concentrations significantly (P < 0.001) influenced germination . The lower spore concentration (1 k) gave the highest percentage germination (Table 3-1 ) . The lowest spore load gave highest percentage conidial germination for most isolates, a trend particularly marked with the strawberry and blueberry isolates (Fig.3-1 ) . The significant interaction between isolates and inoculum level in Table 3-1 is because two isolates (Lincoln and Massey) showed the reverse of this trend with most spores germinating at the higher Chapter Three 37 Table 3-1 . SUMMARY OF IN VITRO GERMINATION OF B. cinerea CONIDIA AFTER 1 2h INCUBATION AT 20°C ON MA. Source Main effects Isolates Cu lture Age (Ca) (days) Spore concentration (K) (nos. 1 7JlVdroplet) I nteractions Isolates x Ca Isolates x K Ca x K Level Auckland Blueberry Camellia Grape Levin Lincoln Massey OS Strawberry 7 28 1 000 25000 x = P values after square root transformation . Meanx germination (%) P < 0.001 76. 1 5 ( 1 .9)az 46.23 (3.7)C 34.61 (2.7)d 47.71 (4.0)d 92.27 ( 1 . 7)a 86.20 ( 1 . 7)b 90.74 ( 1 .3)a 74.81 (2.5? P < 0. 00 1 78.23 ( 1 .6)a 58.95 ( 1 .5)b P < 0.00 1 74.40 ( 1 .5)a 62.78 ( 1 .6)b P < 0.001 P < 0.001 P < 0.001 Z = Mean separation within columns by Duncan's multiple range test, P < 0. 05. Values enclosed in parenthesis indicate overall standard error of the mean. Chapter Three +oJ CO c E L.. � 8 Q) 0> CO +oJ C Q) � 6 CD 0.. +oJ o o L.. 2 � ..... . - . ----- ......... . -..,. 0-. . . . . ' .... . -.... .. . . �. --. --. 0 L\ \ 0 \ \ \ \ \ \ e \ \( : -. -6 e::r- _. _. -' -' -' • . -:: .. , \ \ \ '8 e 1 000 25000 I noculum load 0 !:::. e 6 0 EB ® 0 I -. -_. . . .. . . . . - -. - . _ . - - -----. . . . . 38 Auckland Blueberry Camellia Grape Levin Lincoln Massey OS Strawberry Figure 3-1 . Interaction between isolates and spore concentration during in vitro conidial germination on MA. Vertical bar indicates overall standard error of the mean (SEM). Chapter Three 39 concentration as showing in Fig . 3-1 . The lower overall germination of isolates from blueberry, grapes and camel lia can be clearly seen. With the exception of the isolate obtained from infected kiwifruit at Levin, there was h igher germination in vitro when conidia were obtained from seven day old cultures than when they were obtained from 28 day old cultures (Fig .3-2) . The spores obtained from seven day old cultures had a high percentage germination at both spore concentrations. Spores from 28 day cultures had a lower germination and this decreased at the h igh spore concentration (Fig.3-3) . Experiment No.2 At MUFCO most fruit (74.7%) ultimately diseased (Table 3-2) developed symptoms after six weeks coolstorage, but at CFRI most infected fruit (58.6%) developed disease symptoms within six weeks coolstorage (Table 3-3) . At MUFCO there was considerable variabil ity in the proportion of infected fruit showing symptoms after six weeks with a range from 36.2% (strawberry isolate) to 1 00% (Levin isolate) . The final disease incidence was considerable higher, especially at low inoculum levels, at CFRI than at MUFCO. After six weeks coolstorage there were significant differences between isolates at MUFCO (P < 0.05) and at CFRI (P < 0.0 1) (Tables 3-2 & 3-3) . At MUFCO B. cinerea isolates from infected kiwifruit at Lincoln and from infected grapes had significantly higher infection levels than that from strawberry. At CFRI , isolates from infected kiwifruit at Lincoln and Auckland caused significantly more disease than those from grapes, Levin and the Massey DR isolate. By 1 2 weeks of coolstorage, there were no statistical differences between isolates at either MUFCO or at CFRI . The relative ranking of isolates had also changed. Chapter Three ..... � c E � � 8 Q) 0> � ..... c Q) � 6 Q) 0- ..... o o � 2 � " " .-� .... .. .. ... .c-' .--.-- 9:<:. _ . _ . " " " . : . . '-. " ''® '-. '-. '- . o . E) 7 28 Cultu re age (days) 0 !:J. e 6 0 E9 ® 0 I _ . Auckland --, Blueberry . . . . . . . . Camellia -. -. - Grape .-.- Levin - Lincoln ----- Massey 0 s . . . . - Strawberry 40 , Figure 3-2. Interaction between isolates and culture age during in vitro conidial germination on MA. Vertical bar indicates overall standard error of the mean (SEM). Chapter Three ...... CO c E � � 8 Q) 0> CO ...... C Q) � 6 Q.) 0.. ...... o o � 2 e--� ___ _ -� s .. . 1 000 25000 I nocu lum load I EB - 7 days e · · · · · · · · 28 days 41 Figure 3-3. I nteraction between culture age and spore concentration during in vitro conidial germination on MA. Vertical bar indicates overall standard error of the mean (SEM). Chapter Three 42 Table 3-2. SUMMARY OF THE EFFECT OF B. cinerea ISOLATES AND SPORE CONCENTRATIONS ON PERCENTAGE INFECTION OF KIWIFRUIT AFTER STORAGE AT O°C AT MASSEY UNIVERSITY FRUIT CROPS ORCHARD. Source Main effects Isolates Spore concn. (K) (nos. 1 71l1/droplet) Interactions Isolates x K Level Auckland Grape Levin Lincoln Massey DR Massey OS Strawberry 1 000 5000 25000 1 25000 Mean x infection 6 weeks (%) P < 0.05 5. 1 8 (2.0)abZ 7.61 (2.9)a 4.51 (1 .6)ab 7.78 (2.6)a 3.96 ( 1 . 1 )ab 3.27 (1 .2)ab 1 . 1 8 (O.4)b P < 0.001 0.09 (0. 1 )a 0.87 (0.3)b 6.70 ( 1 .8)C 1 1 .47 (1 .5)C NS x = P values after square root transformation . NS = not significant. Mean x infection 1 2 weeks (%) NS 7.77 (2.6)a 1 1 .08 (4.0)a 4.51 ( 1 .8t 8.57 (3.0)a 5. 1 7 ( 1 .2)a 4.49 ( 1 .6)a 3.26 ( 1 .0)a P < 0.001 0.09 (0. 1 )a 1 . 1 7 (O.4)b 9.57 (2. 1 )C 1 3.55 (2.0)C P < 0.05 Z = Mean separation with in columns by Duncan's multiple range test, P < 0.05. Values enclosed in parenthesis indicate overall standard error of the mean. Chapter Three 43 Table 3-3. SUMMARY OF THE EFFECT OF DIFFERENT B. cinerea ISOLATES AND SPORE CONCENTRATIONS ON PERCENTAGE INFECTION OF KIWIFRUIT AT CROPS AND FOOD RESEARCH LEVIN AFTER STORAGE AT O°C. Source Main effects Isolates Spore concn. (K) (nos. 1 7J..lI/droplet) Level Auckland Grape Levin Lincoln Massey DR Massey DS Strawberry 1 000 5000 25000 1 25000 Mean x infection 6 weeks (%) P < 0.01 20. 1 2 (0.8)az 2.75 ( 1 .9)b 8.27 (6.5)b 21 .48 (7.2)a 4. 1 3 ( 1 .6)b 1 0.02 (6.2)ab 1 4.20 (7. 1 )ab P < 0.05 5.31 (3.0)b 5.91 (2.8)b 1 5.42 (4.8)a 1 9.62 (4.5)a Mean Y infection 1 2 weeks (%) NS 27.30 (3.0)a 1 3. 1 7 (4.5)ab 21 .87 ( 1 1 .9tb 26.72 (7.4)ab 1 3.55 (7.8)b 1 4.45 (5.3)ab 21 .20 (8.3)ab P < 0.001 1 0.31 (4.5)b 1 1 .30 (8.3)b 23.34 (1 1 .9)a 34.05 (5.3)a x Y = P values after log and square root transformation respectively. NS = not significant. Z = Mean separation within columns by Duncan's multiple range test, P < 0.05. Values enclosed in parenthesis indicate overall standard error of the mean. Chapter Three 44 Inoculum levels influenced disease incidence with significant differences (P < 0.001) at both, six and 12 weeks storage at MUFCO and at P < 0.05 (six weeks) and P < 0.001 ( 1 2 weeks) at CFRI (Tables 3-2 & 3-3). A significant interaction between isolates and concentration at P < 0.05 was found after 1 2 weeks of kiwifruit storage at MUFCO only (Table 3-2) . The highest spore load for each isolate gave the highest infection levels in kiwifruit, but the curve for inoculum vs d isease increased sharply at low inoculum levels and levelled off at high inoculum levels for some isolates (e.g. Levin and Massey DR) while the reverse happened with others (e.g. grape and Lincoln) hence the significant interaction (Fig.3-4) . Experiment No.3 The percentage germination of conidia tested in vitro on MA, PDA, L VS and water agar was 1 00% on all four media tested . There was rapid disease development at both sites with 60% of the fruit u ltimately diseased at MUFCO showing symptoms within six weeks coolstrage while at CFRI the corresponding figure was 77%. As in the previous experiments disease, incidence at CFRI was higher (four and a half more times) than at MUFCO (Tables 3-4 & 3-5) . At MUFCO, there were no significant differences from preparing inoculum cultures on different media when fruit had been coolstored for six weeks but after 1 2 weeks inoculum produced on malt agar had caused significantly more infection (P < 0.01) than that from the other two media. At CFRI inoculum prepared from colonies on autoclaved kiwifruit leaves caused significantly more infections (P < 0.001 , P < 0.01) than those prepared from the other media after both six and 1 2 weeks of coolstorage. There was no significant effect of the age of cultures from which inoculum had been prepared at either MUFCO or at CFRI . Chapter Three c o +-' 9 8 t> 7 CD - c � 6 CO +-' c � 5 1- CD Cl. +-' 0 4 e CD 1- � 3 0" (J) 2 1 ® ----- Auckland e ·· · · · · · · Grape <:) . . . . . Levin (B - Lincoln o ._.- Massey OS D,. --. Massey Resistant DR o . Strawberry � I I I I . /- . 0 0 · · · · · · ·/ • I I I I I I I I I / : • I . : • I !. : : �'--- . --- 0 .: �I • -;=-r:--6 . // / ./.� : // / :. : // 1/ ---• /1 ./ .: . I . : ;,/. / / ...-. � . : . . . . . e 1 000 5000 25000 1 25000 I nocu lum load (spore/stem scar) 45 Figure 3-4. Interaction between isolates and spore concentration on storage rot incidence at MUFCO. Vertical bar indicates overall standard error of the mean (SEM). Chapter Three 46 Table 3-4. SUMMARY OF THE EFFECTS OF MEDIA, CULTURE AGES AND INOCULUM LEVEL ON INCIDENCE OF STEM END ROT CAUSED BY B. cinerea AT MASSEY UN IVERSITY FRUIT CROPS ORCHARD AFTER STORAGE AT O°C. Source Main effects Media Culture Age (Ca) (days) Spore concn. (K) (nos. 1 7f.1l/droplet) Interactions Me x Ca Me x K Ca x K Level PDA MA Kwlv 7 1 8 28 1 000 5000 25000 x = P values after log transformation. NS = not significant. Mean x infection 6 weeks (%) NS 5.32 ( 1 .9)az 6.35 (2.0)a 3.58 ( 1 . 1 )a NS 4.60 ( 1 .3)a 5 .95 (2.0)a 4.71 ( 1 .7t P < 0.001 0. 1 0 (0. 1 )a 2.04 (0.9)b 1 3. 1 3 (2. 1 )C P < 0.001 NS NS Mean x infection 1 2 weeks (%) P < 0.01 7. 1 7 (2.3? 1 1 . 1 7 (2.3)a 6.95 (1 .4)b NS 8.1 (1 .5)a 9.4 (2.5)a 7.6 (2. 1 )a P < 0.00 1 2 .33 (O.4)a 6.95 (1 .3)b 1 6.0 (3.0)C P < 0.001 NS NS z = Mean separation within columns by Duncan's multiple range test, P < 0.05. Values enclosed in parenthesis indicate overall standard error of the mean. Chapter Three 47 Table 3-5. SUMMARY OF THE EFFECTS OF MEDIA, CULTURE AGES AND INOCULUM LEVEL ON INCIDENCE OF STEM END ROT CAUSED BY B. cinerea AT CROPS AND FOOD RESEARCH LEVIN AFTER STORAGE AT O°C. Source Main effects Media Culture Age (Ca) (days) Spore concn. (K) (nos. 1 7/-lI/droplet) Interactions Me x Ca Me x K Ca x K Level PDA MA Kwlv 7 1 8 28 1 000 5000 25000 1 25000 Mean x infection 6 weeks (%) P < 0.001 23.31 (6.4)bZ 23.75 (8.5)b 42.32 (6.4)a NS 29.04 (4.3)a 28.56 (7.7)a 3 1 .78 (7.0)a P < 0. 001 7.84 (2.8)C 1 8. 1 7 (4.8)b 28.67 (6.9)b 64.89 (4.2)a NS NS NS x = P values after square root transformation. NS = not significant. Mean x infection 1 2 weeks (%) P < 0. 01 31 .25 (6.3)b 34.28 (7.8)b 50.50 (5.7)a NS 37.63 (4.0)a 37.64 (7.3)a 40.76 (6.7)a P < 0.001 1 9.44 (3.6)C 27.33 (4.4)bC 37.66 (6.9)b 70.27 (3.7)a NS NS NS Z = Mean separation within columns by Duncan's multiple range test, P < 0.05. Values enclosed in parenthesis indicate overall standard error of the mean. Chapter Three c o .... () Q) -c Q) 0> CO .... c Q) � Q) 2 0. 0> 2 o ...J A - MA • .. . . .. . . PDA • . _ .- LVS •.......... • '_._ . .:::.-:::-.:::- .+-.-:::::::.-::::: ._. ...•. .... , " " " • ··· ............ . .... � . . � . .' r -.-.� .. 7'�· ... .. 7 1 8 28 Culture age (days) 48 a I b I Figure 3-5. Interaction between media and B. cinerea culture age on storage rot incidence at MUFCO. a) 6 weeks, b) 1 2 weeks of storage. Vertical bars indicates overall standard error of the mean (SEM) . Chapter Three 49 Inoculum level influenced disease incidence with significant differences at P < 0.001 after six and 1 2 weeks of storage at both MUFCO and at CFRI . The highest spore concentration gave the highest mean percent of diseased fruit at both locations (Tables 3-4 & 3-5) while the lowest spore load gave the lowest disease incidence. There was a significant interaction between media and culture age (P < 0.001) after 6 and 1 2 weeks of coolstorage at MUFCO (Table 3-4). Spores from 1 8 day-old cultures grown on malt agar caused significantly more infection than those from other age and media combinations (Fig .3-5) . At CFRI there were no significant interactions. DISCUSSION In this present study, the growing medium and inoculum levels influenced B. cinerea pathogenicity on harvested kiwifruit. Likewise, in vitro germination varied between isolates obtained from different infected crops. In Experiments 2 & 3, higher levels of infection were recorded in fruit harvested and inoculated at the CFRI than in fruit from MUFCO. The inoculum used at both sites was prepared at Massey University and inoculated at the packing shed from the same batch of Petri plates incubated under the same conditions than the CFRI plates and sent by overnight courier. Fruit were harvested on the same day and at approximately the same maturity as measured by TSS and firmness. The major d ifference in procedure was that the fruit were snapped off the pedicel at harvest at MUFCO but at CFRI the pedicels were cut off the vine and the pedicel snapped from the fruit immediately before inoculation . This fresh wound would be more susceptible than those several hours old (at MUFCO) and could be the reason for the different infection levels at the two locations. Differences in a microclimate at the two sites could also have an Chapter Three 50 influence on susceptibil ity of the fruit. Regional differences in levels of infection during kiwifruit storage have been reported by Brook ( 1 990a). Consistent differences in amounts of B. cinerea between regions and districts within regions were also reported by Hopkirk et al. ( 1 990a) who related this variabil ity in infection to climate variability. For example they found fruit from the Northland region had the highest losses (5.9%) while Nelson area had the lowest ( 1 .3%) . In that study infection levels in 1 989 in fruit from the Southern North Island (Manawatu, Wanganui etc . . ) were approximately 0.8%, while overall infections in this present study ( 1 992 experiments) after 1 2 weeks coolstorage were 6.4% and 20% at MUFCO and CFRI respectively. Knight (1 980) showed that the virulence of B. cinerea on red raspberries can be associated with the susceptibil ity of each red raspberry cultivar to B. cinerea. In this present study cultivar d ifferences in B. cinerea infection on kiwifruit can not be accounted for this type of host­ pathogen association because the Hayward cultivar is the most extensive cultivar grown in New Zealand orchards (Ferguson & Bollard 1 990) . On balance, the difference in incubation procedures would appear to be the most l ikely explanation for different infection levels at both sites of study. The isolates of B. cinerea evaluated in this study were all pathogenic to kiwifruit and caused almost 20% of inoculated fruit to become diseased. After 1 2 weeks coolstorage there were no significant differences between infection levels by different B. cinerea isolates at either site and the isolates did not rank in the same order (based on infection levels) at either site. The statistical d ifference between isolates after six weeks incubation were probably not real effects since the relative virulence of isolates was not the same at each site and significant differences had disappeared by 1 2 weeks coolstorage. Other studies, related with B. cinerea pathogenicity in different cultivars (Wil l iams & Jennings 1 986) , found that the length of disease lesions on red raspberry canes inoculated with 31 different isolates of B. cinerea obtained Chapter Three 51 from different localities, significantly varied between 665 to 42 mm. However in that study, the origin of the isolate was not associated with individual pathogenicity. Similar results were obtained evaluating several isolates of Didymella applanata on the same cultivar, all 32 isolates were pathogenic regardless the area where they were obtained (Pepin et al. 1 985). For experiments 2 & 3 disease incidence varied between the two locations. In those in vivo studies, it was evident that as the spore concentrations increased a greater number of infected fruit were recorded at both assessment periods. Similar results have been found by other researchers; Long & Wurms ( 1 993) have reported that in order to achieve a h igh percentage of infection in kiwifruit it is necessary to apply high doses of spore inoculum. In their experiment, 1 25000 spores per droplet in the picking scar wound of the fruit was needed to ach ieve a high percentage of d isease fruit. Hallet & Sharrock ( 1 993) reported that infection levels of kiwifruit inoculated after 4h after harvest with a dry spore mass (about 50,000 spores/fruit) were h igher (98%) compared with a l iquid spore suspension (4000 fruit) where fruit infection was approximately 20%. Segall & Newhall ( 1 960) found that blight disease in onions caused by B. alii gradually increased with spore load. They reported that inoculum load of 59, 1 250 spores/ml gave 222 spots per plants compared with 4 spots per plant observed when inoculum load was of 6678 spores/ml. Coinciding with those results, Last & Hamley ( 1 956) and Price ( 1 970) working with B. fabae on broad beans and B. tu/ipae on tulip leaves respectively, reported a direct correlation between spore load ( 1 02 to 1 06/ml) and droplet size (0.001 to 0.003 ml) with lesions numbers (0 to 500 and 1 to 5 respectively) , however in further work, contradictory results were reported by Last ( 1 960). This researcher found that at concentrations of 1 x1 05 conidialml or more of B. fabae infectivity decreased with culture age up to 40 days old. In this present study, the high spore concentrations required to infect kiwifruit suggests some type of antifungal activity present on the kiwifruit stem scar. The highest percentage of infected fruit came from fruit inoculated at CFRI Chapter Three 52 with spores from B. cinerea grown on autoclaved kiwifruit leaves. Exudates from portions of tomatoes, leaves of different tomato cultivars (Chou 1 972b), grape berries (Kosuge & Hewitt 1 964) and heads of safflower (Carthamus tinctorius L.) (Barash et al. 1 963; 1 964) have been shown to enhance B. cinerea germination and infection. Analysis of these exudates showed that carbohydrates such as glucose and fructose were the main compounds, although in tomato exudates inhibitory phenolic substances were also detected. Simi lar results were reported by Blakeman ( 1 975) when addition of exogenous nutrients equivalent to leaf exudates of beetroot (Beta vulgaris L) were added to conidial suspension of B. cinerea. In that study the pathogenicity of B. cinerea was related not only to the isolates but also to the addition to the growth media of certain amino acids and/or simple sugars such as fructose and sucrose. Possibly in this present study the autoclaving temperatures allowed denatural ization of inhibitors present in the leaves permitting the B. cinerea conidia to gain sufficient nutrients and reserves for the infection process. At MUFCO, the combination malt agar/1 8 old day culture gave the highest d isease incidence. Maas & Powelson ( 1 972) observed that growth of B. convoluta was best on a maltose and glucose medium. However Clark & Lorbeer ( 1 977) reported different nutrient dependency between B. squamosa and B. cinerea. They found that germination of B. squamosa conidia on onion leaves was equivalent in water or nutrients (glucose solution , standard nutrient solution, onion leaves diffusate or filtrate of cattai l pol len) , while germination of B. cinerea was lower in water than in nutrient solutions, however the number of lesions produced were higher with nutrients in both species. In that study conidia germination of both species declined with age (21 days) regardless water or nutrient solutions. Sal inas et al. ( 1 989) found that addition of gerbera pol len diffusates stimulated germination of stored (eight weeks) dry conidia of B. cinerea in both in vitro (20°C) and on gerbera flowers nevertheless incubation temperatures (4 or 25°C) . Choi et. al. ( 1 990) observed better spore production on cucumbers Chapter Three 53 when B. cinerea conidia were grown on PDA or Czapeck-Dox media compared with corn meal agar, nutrient agar or lime bean agar. Although 8ryk ( 1 985b) reported more B. cinerea mycel ial growth and sclerotia formation only when grown on PDA. Last & Hamley ( 1 956) reported that B. fabae 1 0 days old cultures produced more lesions (about 70) on broad beans than conidia from 25 days old cultures (about 5) . Choi et al. ( 1 990) reported disease severity on cucumbers was not affected by B. cinerea spore age (20 days old) . The difference in percentage germination of isolates on MA were not reflected by consistent d ifferences in virulence on kiwifruit at the two sites. In the absence of any one isolate of B. cinerea showing a marked characteristic of high viru lence compared with other isolates, the Massey isolate K3 was selected for future work since it has been used in other studies on kiwifruit at Massey University and would provide continuity between this and other work here. The choice of media on which to produce inoculum does not appear to be critical with MA giving best results at MUFCO and autoclaved kiwifruit leaves at CFRI . MA has been used in other studies at Massey is easier and more uniform over time (seasonal variation) than kiwifruit leaves and was therefore selected as the medium of choice for future work. The age of colonies from which inoculum was prepared did not have a significant effect on virulence of B. cinerea to kiwifruit but in view of the lower percentage germination on MA of spores from 28 compared with seven day old colonies and of the l iterature quoted above it was decided to use 7-1 4 day old colonies for future work. The high concentration of spores had a lower percentage germination on MA confirming reports of self-inhibition at high concentrations in vitro. However, this effect was not found in kiwifruit where increases in spore concentration resu lted in increased disease incidence. With no clear resolution to this aspect of inoculation it appeared prudent to continue to use a range of spore concentrations (inoculum loadings) . CHAPTER FOUR EFFECT OF RELATIVE HUMIDITY AND TIME OF EXPOSURE AT DIFFERENT TEMPERATURES ON SURVIVAL OF CONIDIA OF BOTRYTIS CINEREA. INTRODUCTION Several factors may influence spore production, survival, germination and infectivity of plant pathogenic fungi. They include inherent fungal factors such as isolate variability, age and nutrient status of the spores and environmental conditions such as temperature, moisture and external nutrient supply. McLaughlin & True ( 1 952) showed that conidia (on glass beads) and mycelium (in wood chips) of Chalara quercina survived longer at temperatures of 1 0 and 20°C than at h igher temperatures (25 and 30°C) . A relative humidity of 20% was more favourable for survival than h igher humidities. More extensive studies of the oak wilt pathogen by Merek & Fergus ( 1 954) using both endoconidia and ascospores confirmed that a cool , dry atmosphere was the most favourable condition for survival of this fungus. Conidia and sclerotia of B. squamosa survived longer in soil at low temperatures than in soi l held at high temperatures (El lerbrock & Lorbeer 1 977). Despite this consistent pattem in the above examples of an improved survival of fungi at low temperatures, the relationship between temperature, relative humidity and spore survival is not necessarily so straight forward. Thus Coley-Smith ( 1 980) found that urediniospores of Melampsora lini remained viable longer in mid-range humidities (40 and 60%) than in dry (20%) or humid (80%) conditions. A relative humidity of 75% dramatically Chapter Four 55 reduced survival of conidia of Aspergillus flavus and of A. terreus compared with humidities of 32% or 85% (Teitel l 1 958) . This lethal effect occurred over a very narrow humidity range since the effect of humidities of 73% and 77% were l ittle different from that of other humidities. In common with the general trend of temperature and survival discussed above, Teitel l ( 1 958) also found that spores of the two Aspergillus spp. survived for a shorter time at higher temperatures. Mycelium and conidia of B. cinerea and mycelium of Sclerotinia sclerotium survived longer at O°C than at 20°C in the absence of nutrients (Van den Berg & Lentz 1 968) . In contrast to the findings of Coley-Smith ( 1 980) and Teitel l ( 1 958) , survival was longer at relative humidities of 99-1 00% than at 85-90% but was dependant on type and strain of organism. A first step in studying the effect of temperature and relative humidity on infection of kiwifruit by B. cinerea is to ascertain the effect of these parameters on the pathogen itself. Given the variabi l ity between strains found by Van den Berg & Lentz (1 968) and the inconsistencies noted above, it was important to test the characteristics of the isolate of B. cinerea used in the rest of this work. OBJECTIVE The objective of this study was to evaluate the effect of relative humidity and temperature on survival of conidia of B. cinerea over time periods which could be used in a pre-coolstorage curing period for kiwifruit. MATERIALS AND METHODS Survival of conidia of B. cinerea isolate K3 were assessed in control led environment chambers at temperatures of O°C ± 1 , 1 0°C ± 2, 1 5°C ± 2, 20°C ± 1 , 25°C ± 2 and 30°C ± 2 . Chapter Four 56 The four relative humidity ranges of 40-50%, 65-75%, 80-90% and 95-1 00% were obtained by the methods explained in the General Materials and Methods. Glass microscope cover slips were sterilized by immersion in alcohol and passing through a bunsen flame. They were placed in plastic Petri plates, five per plate. A 1 4 day old culture of B. cinerea was inverted over each Petri plate and tapped to release spores so they fel l onto the cover slips. The Petri plates with cover slips were placed in humidity chambers (30 x 30 x 30 cm) (Fig.4-1 ) maintained at the appropriate temperature/humidity combination with an output ai r flow of 2.5 I/min. Relative humidities were monitored every day and the relative humidity values given in Fig.4-2 are the mean of at least three measurements per day. Temperature was monitored twice daily using a maximum and minimum thermometer placed on top of the relative humidity chamber. One plate per treatment combination was removed at each assessment time of 2, 4, and 6 days from the 20°C temperature and of 2, 4, 6, and 8 days for the other temperatures. The cover slips removed from the chambers were each inverted onto 20 mm diameter disks of malt agar and incubated at 20°C for 8 or 24 hours to test spore viability. They were then stained with lactophenol acid fuchsin and the percentage germination counted at 1 00x magnification. Statistical Analysis Experiments were carried out as a nested design with fixed effects. Arcsin­ transformation was required before analysis of variance. To compare differences in germination between 8 and 24h a two sample t-test was also carried out. RESULTS The relative humidities obtained during this experiment remained constant 6 2 3 8 4 1 .- Air pump 2 .-Salt containers 3.- Humidity chambers (4) 4 .- Jar 6.- Humidity probe 6 .- Tubing 7 .- Petri dishes 8.-Glass slides 7 Figure 4-1 . Relative humidity system. 90 � :i;:80 '2 E £ 70 .� ]j 80 £ 50 oOe ±2 .- . . . . • • . . . - ' . . . . . .. " .' • • • • 41 ...- -0- -0..... _ ..- _ 0- - 0- -0 .0L---�--�--���.��5�--=---�--�--J Days 200e ±2 l00r------------------------------------, 90 . . . . . . . .. . . . . . .. . . . . . . .. . . . . . .. . . . . . . .. 50 � _ -. _ _ .- - -4 _ _ ..... _ -4 .0L---�----7---�3----�.----�--�----� Days 1 00e ±2 1 5°e ±2 100 00 . . . . ... . . . . . . . . ... . . .•. . . . .• . . . . . ., 90 . .• . . .. . . . ..... . . . . .. . . . . ... . . . . .. . . . ... . . . .. . . . . .. 80 80 �--.--�--�--�--.---.--� 70 70 80 80 50 ..... _O""" .,. ..... - .- - ... - t- - � - O 50 .- -0- -0-- -0- _0- -0- -0_ -0 .0 .0 L---.---:2�--;---�.;---5S---.---:7---e�==�------------���-,;---;---3I---• .---55;-;-S---�---a---J 90 80 70 80 50 0 High Days Days t:>. Medium high 0 Medium low 0 Low 100 . . . . . .. . . . . .. . . . .. . . . .... . . . . .. . . . . .. . . . .. 90 . . . . . .. . . . . . . . . . .. . . . . .. . . . ... . . . .. . . . . .. 80 70 00 50 .... -0- -0- -0-- -0- --0_ -0- -0 .-. - 0- -0- -00- -0- _00-- _0 ..... .,..., 0 .0 L-----------------.----�5----------------� .0 • 5 Days Days Figure 4-2. Actual relative humidities attained at six temperatures. 01 ex> Chapter Four 59 and distinct in each chamber throughout the eight days. The humidity ranges were distinct at each temperature although there was some variation in absolute values from one temperature to another (Fig.4-2) . The highest RH range remained over 95% (except day 5 at 20°C) and the lowest was always below 50% RH. The second RH range was always between 80 and 92% RH but the third range showed considerable variation between temperatures. It was lowest at 1 0°C (60-69%) and h ighest at 30°C (74-79%) with RH at other temperatures falling within the range 70-79%. Highly significant overall d ifferences (P < 0.001) were observed among temperatures, relative humidities, days of treatment and incubation on MA (Table 4-1 ) . Percentage germination decreased with temperature after eight hours incubation. Spores survived better at lower temperatures than at h igh ones with an approximately 42.7% decrease in survival rates at 30°C compared with that at O°C after 8h incubation on MA and 1 4.0% after 24h. There was no similar trend of reduced spore survival with high or low relative humidities. More spores survived exposure to the lowest relative humidity range than any other but fewest spores survived in the medium high range (83-92%) at both germination incubation periods. There was a consistent reduction of spore survival with increasing length of treatment as assessed after both eight and 24h incubation on MA. There was approximately 35% (8h) and 20% (24h) decrease in survival rates after eight days compared with that after two days of treatment. Overall percentage spore germination was higher on MA after 24h (85.6%) than after eight hours (67.2%). The data in Table 4-2 shows spore survival at each relative humidity/duration combination grouped by temperature. The 20°C treatments were carried out first and on the basis of the results obtained it was decided to add an eight day duration and to assess spore survival for 24h as wel l as for eight hours for the remainder of the experiment. Chapter Four 60 Table 4-1 . SUMMARY OF B. cinerea CONID IAL SURVIVAL AT DIFFERENT TEMPERATURES AND HUMIDITIES AS ASSESSED BY SUBSEQUENT GERMINATION ON MA. Environment Temperature (OC) (P < 0. 001) 0 1 0 1 5 20 25 30 Relative humidity (0/0) (P < 0.001) H igh (90 - 1 00) Medium high (83 - 92) Medium low (70 - 79) Low (40 - 47) Time (days) (P < 0.001) 2 4 6 8 Incubation on MA (h) (P < 0.001) Lsmeans ± SE Percentage germination 8h on MA 86.9 ± 1 .6 82.2 ± 1 .6 61 .8 ± 1 .6 73.6 ± 1 .9 48.5 ± 1 .7 49.8 ± 1 .6 66.2 ± 1 .3 61 .0 ± 1 .4 66.9 ± 1 .3 74.6 ± 1 .3 79.4 ± 1 .3 73.2 ± 1 .3 64.5 ± 1 .3 51 .6 ± 1 .5 67.2 ± 0.7 24h on MA 89.6 ± 1 .4 92.6 ± 1 .4 90.5 ± 1 .4 78.4 ± 1 .5 77.0 ± 1 .4 88.8 ± 1 .2 77.3 ± 1 .3 86. 1 ± 1 .3 90.3 ± 1 .3 93.5 ± 1 .2 88.0 ± 1 .3 86.2 ± 1 .2 74.8 ± 1 .3 85.6 ± 0.6 P values after Arcsin transformation. - = Missing data. Chapter Four 61 Table 4-2. SURVIVAL OF B. cinerea CONIDIA AS INDICATED BY GERMINATION ON MA AFTER 8h OR 24h FOLLOWING INCUBATION AT VARIOUS COMBINATION OF RELATIVE HUMID ITI ES AND TEMPERATURES FOR DIFFERENT PERIODS OF TIME (P < 0.001Y. Lsmeans Duration Percentage germination Temp. Relative of Incubation (h) t-test 8h as (OC) Humidity (%) treatment % (days) 8 vs of 24h 8 24 24h 0 High 2 49.0 1 00.0 ...... 49.0 ( 1 00) 4 96.4 99.0 NS 97.3 6 98.6 1 00.0 NS 98.6 8 97.2 98.2 NS 98.9 Medium high 2 88.0 94.2 NS 93.4 (89-92) 4 82.8 84.5 NS 98.0 6 55.6 57.0 NS 97.5 8 39.3 77. 1 NS 50.9 Medium low 2 97.3 98.2 NS 99.0 (73-75) 4 95.3 1 00.0 NS 95.3 6 97.9 98.7 NS 99. 1 8 95.8 98.5 NS 97.2 Low 2 69.5 89.6 NS 77.5 (40-45) 4 88. 1 96.6 NS 9 1 .2 6 91 .6 99.5 NS 92.0 8 93.7 94.9 NS 98.7 1 0 High 2 90. 1 97.9 NS 92.0 (97-98) 4 93.5 93.7 NS 99.7 6 76.3 96.0 NS 79.4 8 88.5 1 00.0 .. 88.5 Medium high 2 92.0 96.2 NS 95.6 (86-90) 4 77.9 91 . 1 NS 85.5 6 79.5 87. 1 NS 91 .2 8 48. 1 86.2 .. 55.8 Medium low 2 82.0 1 00.0 ...... 82.0 (60-69) 4 87.4 92.9 NS 94.0 6 92.9 94.6 NS 98.2 8 48.7 87.6 NS 55.2 Low 2 98. 1 1 00.0 NS 98. 1 (45-48) 4 90.5 95.8 NS 94.4 6 65.2 97.2 .. 67.0 8 80. 1 87. 1 NS 9 1 .9 z = P values, after Arcsin transformation . NS, ', " , ••• = Not significant or significant P < 0.05, 0.0 1 or 0.001 respectively. Chapter Four 62 Table 4-2 (Cont.) Lsmeans Duration Percentage germination Temp. Relative of Incubation (h) 8h as (OC) Humidity (%) treatment t-test % (days) 8 vs of 24h 8 24 24h 1 5 High 2 56.3 90.0 NS 62.6 ( 1 00) 4 68.9 1 00.0 .. ,. .. 68.9 6 67.8 67.8 NS 75.2 8 58. 1 90.3 NS 64.3 Medium high 2 54.6 95. 1 .. 57.4 (83-84) 4 75.4 83.3 NS 90.5 6 59.0 84. 1 NS 70. 1 8 38.6 70.5 NS 54.8 Medium low 2 56.2 95.8 ,. 58.7 (75-77) 4 76.3 90. 1 NS 84.6 6 66.6 1 00.0 ,.,.,. 66.6 8 55.2 1 00.0 ...... 55.2 Low 2 46.7 92. 1 ,. 50.7 (45-48) 4 91 .5 97.9 NS 93.5 6 46.6 95.9 ,. .. 48.6 8 71 .4 73. 1 NS 97.6 20 High 2 89.8 (90-95) 4 57.8 6 73.8 Medium high 2 97.2 (84-86) 4 73. 1 6 56.0 Medium low 2 91 .6 (75-77) 4 96.2 6 92.0 Low 2 74.2 (44-45) 4 81 .6 6 62.7 NS, ·, •• , ••• = Not significant or significant P < 0.05, 0.0 1 or 0.001 respectively. - = Missing data. Chapter Four 63 Table 4-2 (Cont.) Lsmeans Duration Percentage germination Temp. Relative of Incubation (h) 8h as (OC) Humidity (%) treatment t-test % (days) 8 vs of 24h 8 24 24h 25 High 2 92.3 1 00.0 NS 92.3 (98-1 00) 4 66.5 83.5 NS 79.6 6 1 5. 1 81 .4 * 1 8.5 8 7.4 81 . 1 ** 9 . 1 Medium high 2 58.0 94. 1 * 61 .6 (88-90) 4 54.0 61 .8 NS 87.3 6 4 . 1 67.5 ** 6.0 8 1 .6 74.5 *** 2 . 1 Medium low 2 79.5 85.2 NS 93.3 (70-72) 4 67.3 85.2 NS 78.9 6 58.0 58. 1 NS 99.8 8 5 .4 26.7 * 20.3 Low 2 84.9 89.9 NS 94.4 (45-49) 4 54.8 1 00.0 *** 54.8 6 80.6 93.0 NS 86.7 8 1 2. 1 43.0 NS 28. 1 30 High 2 86.5 91 .7 NS 94.3 (98- 1 00) 4 21 .0 79.4 NS 26.4 6 36.6 87.8 ** 41 .6 8 23.6 23.6 NS 1 00.0 Medium h igh 2 81 .3 93.4 NS 87.0 (87-89) 4 42.4 58.3 NS 72.7 6 50.3 93. 1 * 54.0 8 1 5.6 73.0 NS 2 1 .3 Medium low 2 81 . 1 84.7 NS 85.7 (74-79) 4 20.2 95.3 *** 2 1 .2 6 2 . 1 73.5 *** 2.8 8 0.6 40.3 NS 1 .5 Low 2 93.8 97.9 NS 95.8 (45-47) 4 66.2 95. 1 NS 69.6 6 87.2 89. 1 NS 97.9 8 63.5 79.9 NS 79.5 NS, ', " , ••• = Not significant or significant P < 0.05, 0.0 1 or 0.001 respectively. Chapter Four 64 After treatment at O°C or at 1 0°C, spore survival was high and most spores germinated quickly within eight hours on MA i rrespective of the test relative humidity. Treatment at 1 5°C did not have a marked effect on survival but spores were slower to germinate as shown by the difference in percent germination after eight and 24h . The available data for 20°C is similar to that for 1 5°C but after treatment at 20 or 30°C there was a decrease in overall germination, especially after 8 days of treatment, and spores were slower to germinate. Of particular note is the very slow germination of spores exposed to the high and medium high humidities at 25°C and to the medium high and medium low humidities at 30°C. DISCUSSION Conidia of B. cinerea are usually considered to be short-l ived although there is evidence that they can survive for some time (Coley-Smith 1 980) . In substrates such as soi l , they are subject not only to the influence of temperature and humidity but also to fungi stasis (Ellerbrock & Lorbeer 1 977) and to attack by soil microorganisms such as amoeba (Coley-Smith 1 980) . The effect of temperature and humidity in this type of environment could be either a direct effect on the B. cinerea itself or an indirect effect on the general soi l microbial population and its interaction with Botrytis. The infection court on a kiwifruit is the stem scar where the fruit has been snapped from the pedicel. This site is 'virgin ground' when first exposed and spores deposited on it afterwards, for example in the picking bag, would not encounter a wel l established microbial community. The effect of the physical environment at this time would therefore probably be primarily to the host, to the pathogen and to thei r interactions. In this present study B. cinerea conidia survived exposure to a very wide range of temperatures (0 to 30°C) and relative humidities (40-1 00%) for the eight days of the experiment. Spores of B. cinerea do not germinate on glass sl ides but could well do so on kiwifruit stem scars during the curing Chapter Four 65 period when the surface of the scar could provide a suitable, moist substrate. High temperatures can be used to kill fungal spores but there may be a small margin between the heat treatment required to kil l the pathogen and that which damages the host. Choi et al. ( 1 990) found that cucumber grey mould disease did not develop at temperatures greater than 25°C but Coley-Smith (1 980) has shown that conidia of B. cinerea can survive for a short time at temperatures of 33-50°C. Temperatures of this magnitude are unlikely to be used for curing kiwifruit because of possible adverse effects on the fruit (Chea et al. 1 993b) It is generally recognised that there is a marked effect of temperature on survival of fungal spores. For example, Teitell ( 1 958) found that survival of Aspergillus flavus decreased with increasing temperature in the range 29- 48°C. Lidell & Burgess ( 1 985) showed that microconidia of Fusarium moniliforme Sheldon can survive for extremely long times in soi l at 5°C and 75% relative humidity (at least 900 days) . At higher temperatures (25°C and 35°C), survival was reduced to less than 70 days - a period sti l l well in excess of those that would be used to cure kiwifruit for resistance to B. cinerea. Ellerbrock & Lorbeer ( 1 977) studied the survival of conidia of B. squamosa in soil. They found that conidia could be detected in field plots up to two months after infestation . If infested soil was held in the laboratory at 30°C then nearly half had lost viabil ity within one day and almost all were non-viable by 1 0 days. At 21 °C, one third were stil l viable after 1 0 days and at 3°C two thirds remained viable. Alternating moist and dry conditions dramatically reduced survival - after six days no conidia had survived alternating wet and dry conditions whereas over half had survived continuously dry or continuously wet conditions. McLaughlin & True ( 1 952) evaluated (using agar plates) the longevity of Chalara quercina previously incubated at various temperatures and relative humidities on glass beads or in oak twigs. They found that fewer conidia survived at high temperatures Chapter Four 66 (25°C and 30°C) than at 1 0°C or at 20°C where conidia survived for 1 1 1 and 1 73 days respectively. They also reported that conidia on glass sl ides survived longer than those in plant tissue. Merek & Fergus ( 1 954) evaluated the survival of endoconidia (on glass beads) and ascospores (on g lass cover slips) of Endoconidiophora fagacearum Brets exposed to temperature and humidity ranges of 3-37°C and 95- 1 00% respectively. The lower the relative humidity and temperature, the greater the survival of these fungal structures - 1 73 days for endoconidia at 3-9°C and 232 days for ascospores. Wetting the ascospores halved their longevity. In view of these results the possibi l ity that conidia of B. cinerea would not survive on kiwifruit stem scars as long as on cover glasses must be borne in mind when interpreting the results obtained in the current study. Nevertheless there is a clear indication from these present results that with an increase in temperature from 0 to 30°C and duration of treatment from two to eight days of exposure, B. cinerea spore survival decreased. The relationship between relative humidity and fungal spore survival is not as simple as that between temperature and survival (Teitell 1 958) . While endoconidia of Endoconidiophora fagacearum survived best under dry conditions, at 1 2-24°C the ascospores remained viable longer at 95% relative humidity than at 75% (Merek & Fergus 1 954) . Coley-Smith ( 1 980) showed that urediniospores of Melampsora lini remained viable for longer at 40-60% relative humidity than at 20 or 80%. Teitell ( 1 958) found a small band of relative humidity around 75% that reduced viabil ity of Aspergillus flavus and of A. terreus to zero after one month . In the case of A. flavus, at 29°C, viabil ity dropped from 90% to 8% within six days, a time period comparable with that used in this work on B. cinerea, and to 0 after 1 3 days. Viabi l ity of spores of B. cinerea held for six days at 30°C and 74-79% relative humidity dropped from 1 00% to 73.5% when viabil ity was assessed after 24h incubation on MA. However, the speed of germination was also affected and only 2. 1 % of the spores had germinated when examined after 8h incubation on MA. T eitell assessed germination after one incubation time Chapter Four 67 only (9-1 4h) on potato dextrose agar and would not have been able to assess speed of germination. His assessment time of 9-1 4h was intermediate between the two (8h and 24h) used in the current work and the survival rate of A. flavus (8%) was intermediate between those found for B. cinerea (2. 1 and 73.5%) . After eight days (the maximum tested) the figures for B. cinerea were 0.6 and 40.3. It would appear l ikely that survival of conidia of B. cinerea and of A. flavus is affected to a similar extent by relative humidity. The effect of temperature and humidity on fungal spore germination is quite well documented and a few key issues will be summarised here. Dennis & Cohen ( 1 976) working with various strains of B. cinerea observed different rates of germination and mycelial growth on potato dextrose agar over a temperature ranges of 0-25°C. Percentage germination increased up to 95% as temperature increased. Studies carried out by Yoder & Whalen ( 1 975) found a slow rate of growth at temperatures below 1 5°C or above 25°C in two strains of B. cinerea. Temperatures greater than those tested in the present study have shown a similar tendency for reduced spore germination (in vitro) with increased temperature. Kramer & Eversmeyer (1 992) recorded different rates of germination of u rediniospores of P. recondita and P. graminis on water agar which varied to the previous incubation temperature. They found no germination of either P. recondita or P. graminis at 2°C for the first 4h of incubation but after 1 7h germination was 98% and 80%. In both species germination was about 98% after 2h incubation at 6°C to 28°C but at 3 1 °C 47% had germinated after 4h and there was no further germination after 1 7h. There was no germination at 35°C or above. Greater spore germination with increased temperature has been reported by Estrada et al. ( 1 993) for two isolates C. gloesporoides. They found a Chapter Four 68 higher germination from spores incubated on cel lulose membranes for about 1 2h at 25 or 30°C than from those incubated at 20°C. Similarly Van Roermund et al. ( 1 984) concluded that germination of Zoophthora radicans on different media (Sabourad's maltose yeast extract agar and disti l led water agar) was greatly influenced by temperature together with other external factors such as media, l ight and pH. They found that spores of this fungus when incubated at 0 to 36°C could germinate but no germination took place at a very high (40°C) temperature. They also observed that germ-tubes did not develop at temperatures below 24°C. Inhibition of B. cinerea germination on MA following a low temperature period did not occur in this present study as has been reported in many other fungi . Matsumoto & Sommer ( 1 967) reported a direct effect between spore germination of R. stolonifer on potato-dextrose broth (PDB) with low temperatures (-1 , 0, 2.5 and 5°C) and time of incubation on spore survival. They reported that spore germination during incubation at 25°C was less than 5% when they were previously held at O°C for six days compared with a 1 0% germination at 2.5°C. In that study they also reported that spores of R. stolonifer gradually decreased from 1 00 to 5% with increased incubation period at DoC from 0 to 48h. Diversity in results on in vitro studies of exposure periods to different temperatures among other fungal species have also been reported by Wells & Forbes ( 1 967) ; Zentmyer et al. ( 1 976); Beckman & Payne ( 1 983) and Phi l l ips & Weste ( 1 985). These authors working on spore germination of G. cingulata, germ tube elongation and spore germination of C. zea-maydis and colony diameters of various strains of P. cinnamomi respectively, found in these fungi a broad optimum temperatures of growth according to time of exposure. Couey & Uota ( 1961 ) have studied the effect of relative humidity on spore germination of B. cinerea on cold gelatin. They found that conidial Chapter Four 69 germination was greatly influenced by the combinations of relative humidity, 802 concentration and time of exposure. In their study, relative humidities of 93 to 96% and 802 concentrations of 1 00ppm for 0 to 1 2 min reduced germination by about 24%. Percentage germination gradually increased with a decrease in relative humidity and/or increase in 802 concentration i .e. 87% spores germinated when incubated in a relative humidity of 1 9-20% and 802 concentration of 6800 ppm for eight hours compared with 39% in a relative humidity of 93-96% and 802 concentration of 1 00 ppm. Estrada et al. ( 1 993) working with two isolates of C. gloesporoides (12 and 14) showed that germination of both isolates took place at relative humidities of 97.5 and 1 00% after 20h incubation while only 1 1 % (C. gloesporoides 12) and 21 % (C. gloesporoides 14) of conidia were able to produce germ tubes after incubation at 95% relative humidity for 30h . They also reported that at relative humidities of 39% for one week no germination of C. gloesporoides occurred with either isolates. Germination was significantly delayed when spores were incubated at relative humidities at 62 and 86%. for one to two weeks. In the present study germination of B. cinerea conidia increased when spores were held to 24h on MA following the treatment period . Good & Zathureczky ( 1 967) observed that spores of B. cinerea germinated 1 00% on moist d ialysing membranes while Cercospora musae and Monilinia fructicola germinated about 83-93% after periods of drying for seven, 1 2 or 1 9 hours. A second period of drying showed that spore survived was 87- 90% for B. cinerea, 67-88% for C. musae and 6 1 -76% for M. fructicola. They reported that spore rehydration occurred in some minutes when placed on di luted ju ice agar or in a saturated atmosphere for one hour. The effects of combinations of temperature/relative humidity/time of treatment on germination of B. cinerea, Sclerotium sclerotia and Aspergillus spp. has been reported by others. Van den Berg & Lentz ( 1 968) studying Chapter Four 70 envi ronmental effects on various strains of B. cinerea and S. sclerotium germination found that survival of Botrytis strains at ooe and 200e decreased as relative humidity decreased. They also reported survival of B. cinerea conidia for more than one year when they were stored at ooe, 90- 1 00% relative humidity. Rewal & Grewal ( 1 989), reported that three different strains of B. cinerea conidia (81 , 84 or 85) showed differences in germination on filter papers according to incubation temperature, relative humidity and type of strain. For example, they found the h ighest conidial germination at 20°C compared with no germination at ooe or 35°e regardless of type of strain. The minimum for germination of 81 was 93% of 82 was 81 % and of 85 was 75%. An environment of 95% relative humidity at 25°e was required for the conidial growth and appressorium formation of C. zea-maydis. In that study, continuous relative humidities of 60%, 70%, 80% and 90% for 6, 8, 1 0 and 1 5 days respectively, d id not reduce the conidial viabi l ity (Thorson & Martinson 1 993). The isolate K3 of B. cinerea used in this experiment and in subsequent work can survive under a wide range of environmental conditions and percentage spore germination is unlikely to be severely reduced by the temperature and humidity conditions l ikely to be used for curing kiwifruit. However, the infectivity could be reduced and a slower spore germination wou ld give more time for effective defense mechanisms to develop and thus affect the probabil ity of successful infections taking place. CHAPTER FIVE CURING OF KIWIFRUIT TO CONTROL BOTRYTIS CINEREA DURING STORAGE. INTRODUCTION The curing process in fruits and vegetables involves several complex events including physiological, anatomical and histochemical mechanisms outlined in the General Introduction. Physiological considerations of curing It has long been recognized that temperature and relative humidity can affect commodity quality. Weight loss, ethylene production , rate of respiration, firmness and soluble solids are among some of the factors that can be greatly influenced by these two factors not only during the time of exposure but also during the subsequent storage l ife of the commodity. (Bourne 1 982; Mitchell 1 986; Amand & Randle 1 989; Mi ller et al. 1 990; Vano & Hasegawa 1 992; 1 993a; 1 993b; Sentana et al. 1 993) . Picha ( 1 986b) reported a higher rate of weight loss during a ten day curing period than in the subsequent 20 weeks of coolstorage of 6 cultivars of sweet potato (Ipomea batatas) . Transpiration was the major cause of overall weight loss during curing but respiration was more important in the latter part of coolstorage. In lemons previously held at 2, 5, 8 or 1 4°C for several weeks, the rate of respiration increased as temperature increased and there was a low ethylene production at all temperatures (Cohen and Schiffmann­ Nadel 1 978) . Studies on caladium tubers (Marousky & Harbaugh 1 976) showed that weight loss during storage at 24°C for eight weeks of tubers cured at low Chapter Five 72 relative humidities (30-45% and 60-75%) was higher than with those previously cured at 90-1 00% relative humidity. Mbonomo & Brecht ( 1 99 1 ) reported that in cormels, curing temperatures (25, 30, 35 o r 40°C) and 95- 1 00% relative humidity for seven days, affected weight loss both during curing and subsequent storage. They reported a direct relation between curing temperature and weight loss during this conditioning period whi le after four weeks storage at 25°C and 75% relative humidity those cormels cured at 25 or 40°C showed higher weight loss than those cured at 30 or 35°C. They concluded that cured cormels showed 1 0.8% less weight loss than those uncured. Morris et al. ( 1 989) measured weight loss of potatoes during curing at a range of temperatures from 1 0 to 30°C and of relative humidities from 50 to 98%. Weight loss was inversely related to relative humidity. They also concluded that the best curing temperature/relative humidity combination was 25°C and 98% although weight loss increased more than 50% at temperatures above 1 0°C. Studies on sweet potatoes concluded that weight loss during storage at 25°C and 70% relative humidity was affected by curing i .e less weight loss was observed in those cured (30°C and 95-1 00% relative humidity) and wrapped than in the not cured (Delate et al. 1 985) . Other external factors such as soil temperature at harvest, degree of dissection, bruising and peeling affected weight loss as wel l (Walter et al. 1 988; 1 989). Lurie & Klein ( 1 991 ) observed that ethylene production in tomatoes incubated at 36, 38 or 40°C was lower after three days incubation, compared with the fi rst day incubation. Atta-Aly ( 1 992) reported that maximum ethylene production in greenhouse tomatoes was registered at 20°C, declining with increasing temperatures up to 30°C. Maxie et al. ( 1 974) reported similar tendency in pears incubated at 20, 30 or 40°C. In that study, ethylene production was inhibited when fruit were incubated at 30 or 40°C. The combination of some physiological factors such as ethylene Chapter Five 73 concentration and carbon dioxide production on fruit qual ity has also been studied. Metzidakis & Sfakiotakis ( 1 988) reported that firmness of avocado was influenced not only by storage temperature but also by carbon dioxide concentration and internal ethylene. Similarly An & Paull ( 1 990) reported differences in papaya fruit firmness according to incubation temperature and exogenous application of ethylene. They reported that fruit incubated at temperatures of up to 30°C showed abnormal softening, while appl ications of ethylene (:::: 1 00 Illf1) improved fruit quality. Curing may improve the physiological quality of stored fruit. Fruit of four apple cultivars pretreated for two or four days at 40°C were fi rmer after 2-4 weeks storage at 2 1 °C than untreated fruit (Liu 1 978) . Klein & Lurie (1 990; 1 992) also found apple fruit were firmer after heat treatment whether stored at O°C or at 20°C. There is l imited information about the physiological implications of a curing period on subsequent storage of kiwifruit. In some preliminary work Beever ( 1 991 ) , reported a high percentage weight loss and a rapid in itial loss of firmness when coolstorage of kiwifruit was delayed after harvest. Because of the importance of quality for the sweet potato and potato processing industry, the carbohydrate content of these commodities has been widely investigated not only at harvest but also during curing and subsequent storage. In studies on raw sweetpotato carried out by Lambou ( 1 958) sucrose was the main component of the solid solubles not only at harvest but also during the curing (30°C, 80-85% RH for 1 0 days) and storage periods ( 1 5°C for six months) . Morris & Mann ( 1 955) obtained similar results for this commodity when they evaluated three different methods of curing (warm-house, field pile and storage house) at a range of temperatures (4.4, 1 0 and 26.6°C) and relative humidities during curing (30- 80%) and subsequent storage (70-90%). However, Walter (1 992) reported that curing of sweetpotatoes at 29.5°C, and 90-95% relative humidity for Chapter Five 74 seven days did not increase the sugar content compared with those non­ cured and stored at 1 4.5°C, 85% relative humidity for 8, 1 5, 29 and 57 days. This researcher found that differences in sugar content were associated with cultivar rather than with curing. This researcher also reported that sucrose was the major sugar in all cultivars, with glucose and fructose present in lower amounts. However, Picha (1 986a) found that the content of sucrose was only high at harvest while fructose and glucose were the main sugars detected in sweetpotatoes when previously cured for 1 0 days at 30°C, 90% relative humidity and then stored at 1 5.5°C, 90% relative humidity for four to 46 weeks. Picha ( 1 986a) also found that differences in the amount of sugars associated with different variety but that, in general, sugars increased during both curing and storage periods. Previous studies carried out by McCombs & Pope ( 1 958) did not find changes in the amount of sweetpotato glucose with curing treatment (four or 1 0 days) , but they reported higher glucose content during storage at 1 2°C in those cultivars cured for 1 0 days at 30°C than those stored at temperatures of 1 5.5 and 1 8°C. Differences in sugar content of different cu ltivars have also been reported by Sistrunk et al. ( 1 953); Scott & Mathews ( 1 957) and Hammet ( 1 96 1 ) . They concluded that there were general distinct varietal differences in sugar content and that levels changed at harvest, during curing and during storage. In further research , the effect of external factors such as ethylene exposure on the quality of sweetpotatoes has also been evaluated. In those studies, it has been reported that ethylene exposures in cured or non-cured tubers of two cultivars of sweetpotatoes influenced sugar content (Chegeh & Picha 1 993) . They found that both sucrose and total sugars increased with increasing ethylene (0, 1 , 1 0, 1 00 and 1 000 ppm) for 1 5 days at 2 1 °C, but there was no effect on fructose, g lucose or maltose concentration. Samotus et al. ( 1 974) studied the effect of storage temperatures, varieties strains and seasonal variations on reducing sugar accumulation in potatoes. Chapter Five 75 They found that storage at 6°C (after curing at 1 5-20°C) did not enhance the levels of reducing sugars as occurred if potatoes were stored at the lower temperatures of 1 and 2°C. They also found that glucose and sucrose were the main reducing sugars compared with the non-reducing sugar sucrose. In that study accumulation of sugars depended on the variety, period of low temperature storage and year of production. Dogras et al. ( 1 989) also reported h igher reducing sugars in potatoes (cured at 1 5-1 6°C for one week) stored at a low (6°C) than at higher ( 1 0°C) temperature. Simi larly they found differences in sugar concentrations both between, cultivars and between growing regions. Further work to investigate storage temperatures and sugar accumulation was carried out by Cottrel et al. ( 1 993) who found that concentrations of glucose and fructose in potatoes stored at 4°C for 3 to 1 39 days were significantly higher than in those stored at 1 0°C. They also found differences in sugar levels between cultivars. Nagao et al. ( 1 991 ) studied the effects of curing squash (Cucurbita maxima Duch.) at 25°C for 1 2 days and subsequent storage temperatures of 7.5, 1 0, 1 2.5 and 1 5°C for 1 2 to 1 00 days. They reported that reducing sugar content increased more during storage temperatures, although apparently there was no differences in sugar content among storage temperatures. In that study they also reported that the best curing temperature to increase sugars was 30°C compared to those temperatures at 20 or 25°C. In other commodities such as red beet, parsnip and carrots although no curing practice was carried out, the effects of long-term storage on the chemical composition has also been studied. Phan et al. ( 1 973) reported that in raw carrots reducing sugars fluctuated throughout the storage period, ( 1 °C and 98% relative humidity) for a period of time. They found that after eight weeks storage reducing sugars decreased and increased again between 1 4 and 1 8 weeks storage. Rutherford ( 1 977) reported differences in glucose and fructose contents of both red beet and parsnips stored at Chapter Five 76 4°e for six to seven months. In that study no differences were found in sugar content when the whole plant root of either commodity was analyzed, but in the parsnip root dissected plant roots were analyzed and a higher content of sugars, (about 84%) was found in the stele than in the cortex. Shattuck et al. ( 1 991 ) working with field grown parsnip roots stored at O°C and 93% relative humidity, reported that in parsnips stored at O°C for two and four weeks the concentration of both fructose and glucose decreased during the coolstorage period . Control of postharvest diseases by curing One of the main objectives of the curing practice is to control postharvest diseases during commodity storage, hence the importance of studies associated with effects of temperature and/or relative humidity on infection levels. For example, the influence of various temperature ranges on infections caused by pathogens such as those caused by B. cinerea, A. brassicae, R. stolonipher, Mucor piriformis has been widely reported. Pierson ( 1 965) observed reduction of Rhizopus rots of peaches of about 1 0% or 60% when held at O°C for 7 or 1 4 days respectively while Spotts & Chen ( 1 987) observed significant reduction of storage rots such as P. malorum, P. expansum, M. piriformes and B. cinerea on pears held at 2 1 - 38°C for 1 -7 days. I n tomatoes, the pathogenicity of various isolates of Fusarium equiseti, F. chlamydosporum, A. solani, G. candidum, Acremonium recifei and others was affected by temperature and relative humidity (Oladiran & Iwu 1 993) . They reported that the optimum temperature for maximum fruit rot by al l isolates tested was between 30 to 35°e compared with the lower rot development at 5 or 1 0°C. Likewise, the optimum relative humidity to for rots to develop was in the range of 70% to 90% rather than 50% to 60%. In other commodities such as cape gooseberry (Physalis peruviana l.) , it was reported that increasing the storage duration from 1 5 to 33 days and increasing temperatures from aoc to re, increased storage rots caused by Alternaria, Botrytis, Cladosporium and Penicillium spp. following storage at 1 8°e for seven days (Lizana & Chapter Five 77 Espina 1 993) . There is general agreement that curing and fi lm wrapping gives a significant reduction of citrus storage rots such as those caused by P. digitatum Sacc. , P. italicum, A. citri and G. candidum during fruit storage (Mil ler et al. 1 987; Chun et al. 1 988; Waks et al. 1 988; Ben-Yehoshua et al. 1 987a 1 987b; 1 988a; 1 988b; 1 989;) . Curing condition varied from one citrus species to another but in general, the best curing conditions were achieved with temperatures in the range of 30-34°C and relative humidities of 95% for about three days with subsequent storage at 1 2°C. Nagao et al. ( 1 991 ) , reported that during storage at 1 0°C squash fruit developed fewer rots when previously cured at 30°C for 1 6 days compared with lower temperatures of 20 or 25°C. OBJECTIVE To define the environmental conditions that are required to minimize Botrytis rot in kiwifruit during a curing and to determine the principal physiological responses of fruit to this preconditioning practice and to the subsequent effects on fruit qual ity during storage. MATERIALS AND METHODS Experiment No. 1 Title: Curing temperatures, physiological changes and incidence of B. cinerea stem-end rot during subsequent coolstorage in 1 992. Fruit harvesting Fruit were harvested from the Massey University Fruit Crops Unit on 1 1 th Chapter Five 78 May 1 992. Temperature and relative humidity in the orchard at harvest were 1 5°C and 85% RH respectively. The in itial total soluble solids and firmness of fruit were 1 0.8% and 6.6 kgf. Inoculum Inoculum of B. cinerea (K3) was applied to the kiwifruit stem scars as 1 7 j..L1 droplets each containing 1 25000 spores (equivalent to 7.4 x 1 06 spores/ml) . Preparation of treatments The experiment was carried out in incubator rooms at temperatures of 1 0°C ± 1 , 20°C ± 2 or 30°C ± 1 . Inoculated fruit were placed in commercial kiwifruit trays stem scars uppermost. Five trays were exposed to each temperature for each of the three incubation periods. A further five inoculated and uninoculated trays of fruit were closed and placed in the cool room at O°C as per normal commercial practice to evaluate natural and artificially induced levels of infection . Physiological changes in inoculated fruit was also evaluated and only percentage disease from uninoculated trays. Assessments Healthy fruit were selected from each temperature and curing period to evaluate the following physiological parameters: Weight loss (%), ethylene production (C2H4) (j..LI ·kg-1 ·h-1 ) and carbon dioxide production (C02) (cm3·kg- 1 ·h-1 ) , total soluble solids (% TSS) and firmness (kgf) were the parameters assessed. Biochemical assays Five fruit previously cured at each temperature were taken for analysis of pH, titratable acidity (TTA-calculated as citric acid equivalent) , sucrose, fructose and glucose content. Chapter Five 79 pH and Titratable acidity (TT A) pH and TTA were measured using a Mettler DL21 auto-titrator. One ml of fruit ju ice mixed with 40 ml of disti l led water was titrated against 0 . 1 M sodium hydroxide (NaOH) to an endpoint of pH 8.2 (Shusiri 1 992) . Analysis of sugars Aliquots of the same batches of ju ice used for pH and titratable acidity measurements were used to prepare ethanol extracts for separation and estimation of sugars by high pressure liquid chromatography (HPLC) (Pesis et al. 1 99 1 ) . The aliquots were mixed with 95% ethanol (1 :5 v/v), heated to boi l ing in a microwave oven, fi ltered and stored at -20°C for 6 months. G lucose, fructose and sucrose were analyzed by HPLC equipped with a modified C 18 column (ODS-224 220 x 4.6mm) and a Waters 490E UVNIS Detector. Samples of 20 III were injected and eluted isocratically with 0.01 3N sulphuric acid at a continuous increasing flow of 0.4-0.7 ml/min for 20 min, at 22°C and were detected at 2 10 nm. Results were expressed in mg/1 00ml juice. Assessment period At harvest 1 5 fruits were weighed and marked to follow weight loss of individual fruit during the curing period. Ethylene production , rate of respiration, TSS and firmness were also assessed in a further ten fruits. Similar assessments were carried out on ten fruits, on control inoculated fruit and on fruit cured for 2, 4 or 6 days at 1 0°C,' for 2 or 4 days at 20°C and in fruit held for 2 days at 30°C. Similar evaluations in ten fruits for each combination temperature/curing period were carried out after 1 , 3 and 6 months of coolstorage. Analyses of sucrose, glucose, fructose, pH and acidity in five fruits were also carried out for each combination of temperature/curing time during the first and third month of storage. Percentage of infection was evaluated after 1 2 weeks of storage at O°C. Chapter Five 80 Statistical Analysis Curing period data were analyzed separately for each temperature as a complete randomized design to test differences between curing periods for the physiological parameters (except ethylene) and infection levels. There were too many zeros in the ethylene production data to use analysis of variance so one and two sample t-tests were used for this parameter. Data from the storage period were analyzed as a nested design . Before analysis of variance a square root transformation was carried out for firmness data from the curing period and a log transformation for ethylene production , rate of respiration, sugars, pH and acidity from storage data. Experiment No.2 Title: Curing temperature and incidence of B. cinerea stem-end rot of kiwifruit during subsequent coolstorage in 1 994. Fruit harvesting Fruit were harvested from a commercial orchard near Levin (Lat S 40 37 Long E 1 75 1 7) , on 1 6th, 23rd and 27th May 1 994. Orchard temperature and relative humidity were 1 2°C and 78.5% for the first harvest, 1 1 °C and 80% for the second and 1 4°C and 90.5% for the third. The initial total soluble solids and firmness for each harvest were 6.8% and 9.5kgf, 7.0% and 7.4kgf and 8.3% and 8.7kgf respectively. Inoculum The inoculum applied to the kiwifruit stem scar was 25000 spores in a 1 7 JlI droplet (equivalent to 1 .5 x 1 06 spores/ml) . Preparation of treatments The experiment was carried out in control temperature rooms at O°C, 5°C, 1 0°C, 1 5°C or 20°C. Inoculated fruit were placed in the commercial kiwifruit trays stem scars uppermost. To maintain a h igh relative humidity during the three day curing period trays were wrapped (but not sealed) in black Chapter Five 81 polyethylene bags. After three days of curing, commercial polyethylene l iners were placed in the trays which were closed and stored at O°C as per normal commercial practice. There were four repl icate trays per treatment. Assessments Three trays per treatment were used to evaluate levels of infection after 1 2 weeks of storage and fruit from the fourth replicate were used to evaluate percentage weight loss every day during curing, to assess firmness at the end of curing and after 6 or 1 2 weeks coolstorage. Statistical Analysis Analysis of variance was performed on data using a completely randomized design. Before analysis of variance square root transformation was carried out for percentage infection at both storage periods and for all harvest dates. The experiment was repeated three times. Experiment No. 3 Title: Relative humidity during curing and B. cinerea stem-end rot incidence in kiwifruit during subsequent coolstorage in 1 993. Fruit harvesting Fruit were harvested from the Massey University Fruit Crop Unit on 23rd May and 4th June 1 993. Orchard temperature was 1 5°C or 1 7°C respectively and relative humidity as 82-85% at both harvest periods. In itial total soluble solids and firmness at the first harvest were 8.8% and 9 . 1 kgf and from the second were 1 0.6% and 9.2 kgf respectively. Inoculum The inoculum was as described for Experiment No.2. Treatment preparation The experiments were carried out in a controlled temperature room at 1 0°C Chapter Five 82 and three relative humidities ranges were assessed: 40-59%, 60-80% and 92-97% corresponding to vapour pressure deficits (VPD) of 0.70-0.49, 0.48- 0.24 and 0.09-0.03 Kpa respectively. Polythene pipes 2 .20 m long x 1 5 cm wide were used as relative humidity chambers (Fig.5-1 ) . A series of six holes (2.5 mm diameter) were drilled equidistant down the length of each pipe to allow access for relative humidity measurements. Each hole was blocked with a rubber bung that could be removed and replaced with a relative humidity probe inserted through another bung of the same external diameter. Commercial plastic plix trays were cut into single cup strips and stapled end to end to form chains 36 cups long . Fruits were placed stem scar uppermost in alternate cups, inoculated, and when dry, the entire string of cups was carefully pulled into one of the pipes using two, long wires as cords. The ends of the pipes were capped and connected to the tube from the inlet chamber at one end and to the outlet chamber at the other. Relative humidity of inlet, outlet and tube access points were checked every day over the three day curing period. Assessments There were eight repl icate pipes per treatment. Fruit from seven were packed and coolstored as per normal commercial practice and fruit from the eight was used for measurement of weight loss, TSS and firmness immediately after curing . Statistical Analysis The experiment was analyzed as a completely randomized design. Square root transformation was carried out before analysis of variance of percentage infection at both storage periods and the two harvest dates. Experiment No. 4 Title: Relative humidity, type of inoculum and harvest maturity on incidence of B. cinerea stem-end rot in kiwifruit during subsequent coolstorage in 1 994. l .- Air pump 2 .- Salt containers 3.- Jar (input) 4 .- Kiwifruit 5.- Relative humidity chamber 6.- Plix 7 .- Ruber bung 8 .- Jar (output) 9 .- Relative humidity probe 2 Figure 5-1 . Relative humidity system. Chapter Five 84 Fruit harvesting Fruit were harvested on four occasions from a commercial orchard at Levin. Harvest dates were the same as those of Experiment No.2 but with an additional harvest on 1 st June. The orchard temperature and relative humidity for this harvest were 1 00G and 56% and the initial TSS and firmness were 9.2% and 8 . 1 kgf respectively. Inoculum Half the fruit were inoculated with a spore suspension prepared from 1 0-1 4 day old cultures as in Experiments 2 and 3. The remaining half were inoculated with dry conidia by touching a fine artist paint brush on a sporulating 1 2 day old culture of B. cinerea growing on MA and then touching the stem scar of a kiwifruit. An estimate of the inoculum deposited by this procedure was obtained by touching a glass slide in a similar manner to the stem scar and then counting the number of conidia. The mean of three attempts was equivalent to 6700 spores per stem scar. Treatments and experimental procedure The experiments were carried out at 1 00e using three humidity ranges. Sodium chloride was replaced by potassium ch loride to produce a middle humidity range that was h igher than that in Experiment 3. The ranges achieved were 50-80%, 80-93% and 95-98% resulting in a range of vapour pressure deficits (VPD) of 0.59-0.24, 0.24-0.08 and 0.05-0.02 KPa respectively. Subsequent packing, coolstorage and measurement of fruit parameters were as described for Experiment 3. Statistical Analysis The experiment was designed as a 4x3x2 factorial with seven repl ications per each relative humidity. Data from percentage weight loss and infection levels at both storage periods required square root transformation before Chapter Five 85 analysis of variance. RESULTS Experiment No. 1 Physiological c�anges of fruit during curing and coolstorage in 1992 Temperature and time of curing greatly influenced most of the physiological parameters evaluated in this study. Weight loss was greatest at the highest temperature (0.79%) and least at O°C (Table 5-1 ) . The differences in percentage weight loss of fruit held at 0, 1 0 or 20°C were h ighly significant (P < 0.001) . Weight loss increased with time of curing at each temperature. Subsequently measurement of weight loss during storage was not possible because most of the fruit marked for this purpose showed symptoms of Botrytis. Table 5-1 . 1 992: PERCENTAGE WEIGHT LOSS (MEAN ± SE) OF KIWIFRUIT CURED AT FOUR TEMPERATURES FOR UP TO SIX DAYS. Weight loss (%) (days) Treatment 2 4 6 Control I noculated O°C 0. 1 2 ± 0.01 0.29 ± 0.07 0.58 ± 0.06 P < 0.001 1 0°C 0 .38 ± 0.01 0.66 ± 0.03 1 . 1 0 ± 0.04 P < 0.001 20°C 0.53 ± 0.01 1 .05 ± 0.02 P < 0.001 30°C* 0.79 ± 0.02 * Only mean and standard error were evaluated for this treatment. Chapter Five 86 After two days curing there was little difference in firmness between fruit held at 1 0, 20 or 30°C but those at O°C were softer (Table 5-2). Firmness decreased with time of curing and the decrease over six days was greater (P < 0.001) in the inoculated control fruit than it was in fruit cured at 1 0°C ( 1 9%: 0.05). Table 5-2. 1 992: FIRMNESS (MEAN ± SE) OF KIWIFRUIT CURED AT FOUR TEMPERATURES FOR UP TO SIX DAYS. Treatment Control Inoculated O°C P < 0.001 1 0°C P < 0.05 20°C NS NS = No significant. 2 7. 1 5 ± 0.54 8.05 ± 0.35 8.9 ± 0.61 8.0 ± 0.30 Firmness (kgf) (days)Z 4 6.74 ± 0.32 8.38 ± 0.39 7.71 ± 0.75 6 4.50 ± 0.24 6.53 ± 0.32 * Only mean and standard error were evaluated for this treatment. Z P values after square root transformation. Within one month of coolstorage, firmness of fruit in all treatments including the inoculated controls, had diminished by about two thi rds (Table 5-3). Firmness of fruit cured at 30°C for two days was less than the other temperatures after one, three and six months but there were no consistent differences between the remainder of the treatments. There was no further overal l reduction in firmness between one month (mean = 1 .92) and six months (mean = 2.02) of coolstorage. Chapter Five 87 Table 5-3. 1 992: F IRMNESS (LSMEAN ± SE) OF KIWIFRUIT CURED FOR UP TO SIX DAYS THEN STORED AT O°C FOR UP TO SIX MONTHS. Storage Time Temperature & (months) Curing period 1 3 6 (P < 0.001) Firmness (kgf) 1 0°C + 2 days 1 .62 ± 0.05 2. 1 2 ± 0.05 2.23 ± 0.05 4 days 2 .42 ± 0.05 2.35 ± 0.05 2. 1 3 ± 0.05 6 days 2.23 ± 0.05 2.47 ± 0.05 2 . 1 1 ± 0.05 20°C + 2 days 1 .90 ± 0.05 2. 1 2 ± 0.05 1 .89 ± 0.05 4 days 2.38 ± 0.05 2.60 ± 0.05 2.03 ± 0.05 30°C + 2 days 0 .90 ± 0.05 1 .83 ± 0.05 1 .77 ± 0.05 Control 2 .02 ± 0.59 2.00 ± 0.03 1 .98 ± 0.06 Inoculated O°C* * Only means and standard error were evaluated for this treatment. Table 5-4. 1 992: ETHYLENE PRODUCTION (MEAN ± SE) OF KIWIFRUIT CURED AT FOUR TEMPERATURES FOR UP TO SIX DAYS. Treatment Control Inoculated O°C P < 0.001 1 0°C P < 0.01 20°C NS Ethylene production (JlI ·kg-1 ·h-1 )Z (days) 2 4 6 0.00 0.00 0.468 ± 0.045 0.00 0.29 ± 0.08 0.462 ± 0.020 0.00 1 .095 ± 0.71 30°C 0.295 ± 0.28 NS NS = not significant. Z t-test analyses. C2H4 at harvest = zero. Chapter Five 88 There was no ethylene production at harvest and after two days of curing only fruit held at 30°C produced ethylene (Table 5-4) . After four days curing fruit held at 1 0°C and at 20°C produced ethylene but at O°C it was six days before ethylene was detected. Ethylene production was significantly greater after six than after four days at 1 0°C (P < 0.00 1) . After two and four days at 20°C and at 30°C respectively, the ethylene production was not significant. Although there was no significant difference between fruit held for two or for four days at 20°C, fruit from these treatments showed the h ighest levels of ethylene production. H ighly significant differences (P < 0.001) in ethylene production were found during the subsequent coolstorage (Table 5-5) . Table 5-5. 1 992: ETHYLENE PRODUCTION (LSMEAN ± SE) OF KIWIFRUIT CURED FOR UP TO SIX DAYS THEN STORED AT O°C FOR UP TO SIX MONTHS. Temperature & Curing period 1 (P < 0.001)Z 1 0°C + 2 days 1 . 1 22 ± 0.25 4 days 0.00 6 days 0.573 ± 0 .25 20°C + 2 days 0.899 ± 0 .25 4 days 0.384 ± 0.25 30°C + 2 days 0.359 ± 0.25 Control 0.00 Inoculated O°C* Storage Time (months) 3 C2H4 (1l1 ·kg·1 h1 ) 0.492 ± 0.25 0.456 ± 0.25 0.260 ± 0.25 0.032 ± 0.25 0.376 ± 0.25 0. 1 29 ± 0.25 0.00 6 0 . 1 57 ± 0.25 0.257 ± 0.25 0.061 ± 0.25 0.295 ± 0.25 0.261 ± 0.25 0.959 ± 0.25 3.383 ± 2. 1 5 * Only means and standard error were evaluated for this treatment. Z P values after log transformation. Ethylene production was not detected in fruit from the inoculated control unti l the six month assessment when it was greater than in any other treatment. Fruit from all treatments except those cured at 1 0°C for four days Chapter Five 89 produced ethylene within one month of coolstorage and production was detected in that treatment after three months. There was no consistent relationship between ethylene production, curing temperature and incubation time. Respiration as measured by CO2 production was greatest in fruit held at O°C and decreased with an increase in curing temperature (Table 5-6). Table 5-6. 1 992: RATE OF RESPIRATION (MEAN ± SE) OF KIWIFRUIT CURED AT FOUR TEMPERATURES FOR UP TO SIX DAYS. CO2 production (cm3·kg-1 ·h-1 ) (days) Treatment Control Inoculated O°C P < 0.001 1 0°C P < 0.001 2 1 8.97 ± 0.78 1 5.03 ± 0.38 1 3.53 ± 0.42 30°C* 1 3.61 ± 0.23 NS = not significant. 4 6 27.84 ± 1 . 1 3 23. 1 7 ± 1 .25 23.93 ± 1 .46 26.86 ± 0.50 1 4.54 ± 1 . 1 1 * Only mean and standard error were evaluated in this treatment. CO2 at harvest = 1 3.65. Over a six day curing period the respiration rate continued to increase and fruit held at 1 0°C were respiring more after six days than fruit held at O°C. Overall differences in respiration rate were highly significant (P < 0.001) (Table 5-7) . After one month of coolstorage respiration rate of fruit from the Chapter Five 90 inoculated control were less than those from all treatments and 59% less than the treatment average. At three months coolstorage, fruit from the control and curing treatments were comparable and by six months fruit respiration from the control was 35.6% greater than those of the treatment averages. Table 5-7. 1 992: RATE OF RESPIRATION (LSMEAN ± SE) OF KIWIFRUIT CURED FOR UP TO SIX DAYS THEN STORED AT O°C FOR UP TO SIX MONTHS. Temperature & Curing period (P < 0.001)Z 1 0°C + 2 days 4 days 6 days 20°C + 2 days 4 days 30°C + 2 days Control Inoculated O°C* 1 22. 1 4 ± 0.79 1 8.88 ± 0.79 1 6.92 ± 0.79 1 8.94 ± 0.79 1 7.36 ± 0.79 1 2.64 ± 0.79 1 0.59 ± 0.31 Storage Time (months) 3 CO2 production 1 6.05 ± 0.79 1 7. 1 7 ± 0.79 1 4.68 ± 0.79 1 8. 1 6 ± 0.79 1 6. 1 7 ± 0.79 1 7.02 ± 0.79 1 6.26 ± 0.46 6 1 4.78 ± 0.79 1 0. 1 1 ± 0.79 1 6.46 ± 0.79 1 4.08 ± 0.79 1 5.49 ± 0.79 1 5.57 ± 0.79 1 9.55 ± 1 .35 * Only means and standard error were evaluated for this treatment. Z P values after log transformation. Chemical composition of fruit There was little effect of curing on total soluble solids at any temperature (Table 5-8) . Overall differences in total soluble solids were significant at P < 0.05 (Table 5-9) . TSS of fruit after one month coolstorage was higher than that immediately after curing but there was no consistent effect of curing temperature or of time of incubation on TSS during storage. As the period of storage increased, glucose and fructose content of fruit increased in 1 0 of the 1 2 treatments. Chapter Five 91 Table 5-8. 1 992: TOTAL SOLUBLE SOLIDS (MEAN ± SE) OF KIWIFRUIT CURED AT FOUR TEMPERATURES FOR UP TO SIX DAYS. Total soluble solids (%) (days) Treatment 2 4 6 Control Inoculated O°C 1 1 . 1 ± 1 .38 1 1 .8 ± 0.99 1 1 .5 ± 1 . 1 3 NS 1 0°C 1 0.6 ± 1 .45 1 0.6 ± 1 .64 1 1 .4 ± 1 .40 NS 20°C 1 0.6 ± 1 .67 1 1 .4 ± 1 .25 NS 30°C* 1 0.3 ± 1 .66 NS = not significant. * Only mean and standard error were evaluated in this treatment. Table 5-9. 1 992: TOTAL SOLUBLE SOLIDS (LSMEAN ± SE) CONTENT OF KIWIFRUIT CURED FOR UP TO SIX DAYS THEN STORED AT O°C FOR UP TO SIX MONTHS. Temperature & Curing period 1 (P < 0.05) 1 0°C + 2 days 1 2.66 ± 0.21 4 days 1 2.79 ± 0.20 6 days 1 2.57 ± 0 .20 20°C + 2 days 1 3.29 ± 0.20 4 days 1 3. 1 1 ± 0.21 30°C + 2 days 1 2.77 ± 0.20 Control 1 4.02 ± 0 .59 Inoculated O°C* Storage Time (months) 3 TSS (%) 1 2.79 ± 0.21 1 2.37 ± 0.20 1 2.63 ± 0.20 1 2.62 ± 0.20 1 3.01 ± 0.20 1 2.48 ± 0.20 1 4.05 ± 0.01 6 1 2.78 ± 0.20 1 2. 1 6 ± 0.20 1 1 .93 ± 0.20 1 2.43 ± 0.20 1 2.55 ± 0.21 1 2.39 ± 0.20 1 4.92 ± 0.32 * Only means and standard error were evaluated for this treatment. Chapter Five 92 Although the overall d ifference was not significant, sucrose content of fruit cured at 1 0°C increased significantly with storage time but this increase was not found in fruit cured at other temperatures. Fruit content of sucrose, glucose and fructose was generally lower in cured than in uncured fruit (Table 5-1 0) . Fruit cured for two days at 30°C had lower sugar levels than those cured for two days at 20°C which in tum had lower levels than those cured for two days at 1 0°C. A similar comparison can be made between fruit cured at 20°C and at 1 0°C for four days. There was an inverse relationship between duration of curing and fruit sugar content. Only the data for the sucrose treatment was significant overall at P < 0.001. pH values of fruit were lower after three months storage than after one month (Table 5-1 1 ) . Although significant differences (P < 0.001) were observed, in general pH remained relatively constant in a range of 3.3 to 4.0 in fruit from all the treatments at both storage assessments. Fruit acidity evaluated as a citric acid equivalence increased as the coolstorage period increased. Overall significant d ifferences were P < 0.001. There was no consistent relationship between content of citric acid and curing temperature/incubation time. Infection levels during coolstorage Fruit held at 20°C for six days and at 30°C for up to two days showed a rapid onset of Botrytis rot and a high loss of firmness during the incubation period. These fruit had no potential storage l ife. Percentage of infection after three months coolstorage was lowest in fruit cured at 1 0°C (Table 5- 1 2) . This was the only treatment with a lower incidence of disease than the inoculated controls. The disease incidence at 20°C was more then double after a four day compared with a two day curing period. Table 5-1 0. 1 992: SUGAR CONTENT OF CURED KIWIFRUIT DURING SUBSEQUENT STORAGE AT O°C. Temperature Sucrose (P < O.OO1)z and Curing time 1 month 1 0°C + 2 days * 4 days 34.89 ± 7.4 6 days 2 1 .92 ± 7.4 20°C + 2 days 46. 1 8 ± 8.3 4 days 34 .27 ± 7.7 30°C + 2 days 42 .02 ± 7.7 Control InoculatedY 66.69 O°C ± 1 4.8 * Missing data. Z P values after log transformation. NS = not significant. 3 months 73.33 ± 7.4 46.68 ± 8.3 53.73 ± 7.7 42 .31 ± 8.7 30.63 ± 7.4 32 .39 ± 8.3 63.20 ± 1 8.20 Lsmeans ± SE Glucose (NS)z mg/1 00ml of ju ice 1 month 3 months * 226.54 ± 20.5 1 44.48 1 54.59 ± 20.5 ± 22.9 86.66 1 73.21 ± 20.5 ± 2 1 .2 1 53.50 1 73.46 ± 22.9 ± 23.9 1 38.64 1 1 7 .41 ± 21 .2 ± 20.5 1 38.76 145.40 ± 2 1 .2 ± 22.9 1 66.88 21 5.94 ± 32.45 ± 58.38 Y Only means and standard error were evaluated for this treatment. Fructose (NS)Z 1 month 3 months * 224.30 ± 20.9 1 39 .23 1 56.32 ± 20.9 ± 23.4 88.36 1 67.32 ± 20.9 ± 21 .7 1 5 1 .81 1 76.59 ± 23.4 ± 24.4 1 35.85 1 1 9 .25 ± 2 1 .7 ± 20.9 1 36 .82 1 44.47 ± 2 1 .7 ± 23.4 1 72 .52 227.94 ± 34. 1 ± 58.80 Table 5-1 1 . 1 992: ACIDITY OF CURED KIWIFRU IT DURING SUBSEQUENT STORAGE AT O°C. Temperature and Curing period 1 0°C + 2 days 4 days 6 days 20°C + 2 days 4 days 30°C + 2 days Control InoculatedY O°C * Missing data. Lsmeans ± SE pH (P < O.00 1)Z Citric Acid equ ivalent (%w/v) (P < O.001t 1 month 3 months 4.0 ± 0.07 3.7 ± 0.07 3.7 ± 0.07 3.7 ± 0.07 4.0 ± 0.07 3.7 ± 0.07 3.6 ± 0.07 3.8 ± 0.07 * 3.7 ± 0.07 3.4 ± 0.07 3.7 ± 0.07 3.3 ± 0.21 3.8 ± 0.07 1 month 3 months 0.0949 ± 0.01 0. 1 598 ± 0.01 0.09 1 3 ± 0.01 0 . 1 948 ± 0.01 0.0584 ± 0.01 0 . 1 646 ± 0.01 0 . 1 586 ± 0.01 0. 1 252 ± 0.01 * 0 . 1 779 ± 0.01 0. 1 699 ± 0.01 0. 1 530 ± 0.01 0. 1 563 ± 0.01 0.2072 ± 0.02 Y Only means and standard error were evaluated for this treatment. Z P values after log transformation . Chapter Five 95 Table 5-1 2. 1 992: PERCENTAGE INFECTION (MEAN ± SE) OF CURED KIWIFRUIT AFTER 1 2 WEEKS COOLSTORAGE. Treatment 2 6.48 ± 1 .00 1 2.03 ± 3.21 30°C* 1 5.50 ± 2.95 Control inoculated O°C = 7.96 ± 0.56 Control uninoculated O°C = 0 NS = not significant. Curing period (days) 4 5 .78 ± 1 .62 28.56 ± 1 .00 6 5.78 ± 0.83 * Only means and standard error were evaluated in this treatment. Experiment No. 2 Fruit quality after the curing and coo/storage in 1994 Measurement of physiological changes in fruit during curing and coolstorage periods showed that cumulative and daily weight loss during curing and subsequent coolstorage periods were significantly d ifferent (P < 0.05) for all temperatures at each harvest date (Tables 5-1 3 & 5-1 4) . In general , weight loss increased with an increased curing temperature. The lowest cumulative and daily weight loss was observed in fruit from the inoculated control , while the highest weight loss was in fruit cured at 20°C. Cumulative weight loss during the three days of curing was highest after the first day of curing than the subsequent two days of incubation for all fruit from three harvests (Table 5-1 3) . Chapter Five 96 Table 5-1 3. 1 994: DAILY PERCENTAGE WEIGHT LOSS OF INOCULATED KIWIFRUIT FROM THREE HARVESTS CURED AT FIVE TEMPERATURES. Harvest 1 1 6 May Harvest 2 23 May Harvest 3 27 May o 5 1 0 1 5 20 C. inoc. o 5 1 0 1 5 20 C. inoc. o 5 1 0 1 5 20 C. inoc. Mean weight loss (%) Fi rsf O.OSC (0.01 ) 0. 1 2bC (0.02) 0. 1 7b (0.03) 0.43a (0.04) 0.42a (0.06) Curing days SecondX 0.07C (0.01 ) 0. 1 2c (0.02) 0. 1 0c (0.02) 0.23b (0.04) 0.41 a (0.05) 0. 1 1 C (O.OOS) 0.07d (0.005) 0. 1 9b (0.02) 0.41 C (0.01 ) 0.42c (O.OOS) 0.04C (0.0 1 ) 0.04c (0.01 ) 0.0 1 e (0.003) 0 .39ab (0.04) 0. 1 2c (0.02) 0.04d (0.006) 0.3Sab (0.02) 0.3Sab (0.05) 0.30b (0.03) 0.49c (0.0 1 ) 0.39ab (0.07) 0.03d (0.02) 0.20b (0.04) 0.2Sab (0.02) 0.32a (0.02) 0.04d (0.01 ) 0.02de (0.006) 0. 1 2c (0.01 ) 0.20b (O.OOS) 0.2Sa (0.01 ) 0.01 e (0.007) 0 .2Sd (0.01 ) 0.04c (0.005) 0.01 c (0.004) 0.29cd (0.0 1 ) 0.03c (0.007) 0.39ab (0.2) 0. 1 0bc (0.0 1 ) 0.34bC (0.2) O.44a (0.2) 0.23d (0.02) 0. 1 5b (0.02) 0.3Sa (0.06) 0.01 c (0.003) 0.04C (0.01 ) 0.03c (0.01 ) 0. 1 2b (0.01 ) 0 . 1 1 b (0.04) 0.2Sa (0.005) x Mean separation within columns by Duncan's multiple range test at P < 0. 05. Values enclosed in parenthesis indicate mean standard error (SEM) . Chapter Five 97 Table 5-1 4. 1 994: CUMULATIVE PERCENTAGE WEIGHT LOSS OF INOCULATED KIWIFRUIT FROM THREE HARVESTS AFTER CURING AND COOLSTORAGE AT ODC. Mean weight loss (%) Temperature Curing Storage ODC (DC) 3 daysX 6 weeksx 1 2 weeksY Harvest 1 1 6 May 0 0.24e (0.01 ) 0.92b (0.09) * 5 0.32d (0.01 ) 0.72b (0.07) 1 .40 (0. 1 ) 1 0 0.46c (0.02) 0.77b (0.05) 1 .86 (0. 1 ) 1 5 1 .07b (0.02) 1 .50a (0.07) 2 . 1 1 (0. 1 ) 20 1 .25a (0.03) 1 .55a (0.06) 1 .93 (0. 1 ) C. inoc. 0.09' (0.01 ) 0.77b (0.08) * Harvest 2 23 May 0 0 .56c (0.03) 0.79c (0.08) 0.94 (0. 1 ) 5 0.45c (0.02) 0.96bC (0.06) 1 .28 (0. 1 ) 1 0 0.71 b (0.05) 1 .24a (0.09) 1 .35 (0. 1 ) 1 5 0.76b (0.04) 1 . 1 0ab (0.06) 1 .23 (0. 1 ) 20 1 .0r (0.04) 1 .29a (0.07) 2 .49 (0. 1 ) C. inoc. 0.42c (0.07) 0.52d (0.07) 0.58 (0. 1 ) Harvest 3 27 May 0 0.34c (0.02) 0.93c (0.09) 1 .54 (0.2) 5 0.36c (0.01 ) 0.73c (0.05) 1 .05 (0. 1 ) 1 0 0.62b (0.03) 1 .57ab (0. 1 ) 1 .78 (0. 1 ) 1 5 0.61 b (0.03) 1 .84b (0.09) 2.50 (0. 1 ) 20 1 . 1 1 a (0. 1 ) 1 .33a (0. 1 ) 2.34 (0. 1 ) C. inoc. 0.26c (0.02) 0.88c (0.07) 1 .37 (0.2) x Mean separation within columns by Duncan's multiple range test at P < 0.05. Y As number of fruit were unbalanced due to losses from disease, only Ismeans and standard errors are shown at this storage period . * Fruit from these treatments completely rotten . Values enclosed in parenthesis indicate mean standard error (SEM). Chapter Five 98 Daily weight loss decreased the curing period for harvests two and three but there was no consistent pattem at harvest one. Total weight loss increased during both curing and subsequent coolstorage. Fruit coolstored for 1 2 weeks showed the highest weight loss (Table 5-1 4) . Firmness was significantly different (P < 0.05) after both curing and coolstorage, at the second and third harvests with the exception of harvest two after six weeks coolstorage, but at the first harvest significant differences were found only in fruit coolstored for six weeks (Table 5-1 5) . Fruit firmness decreased with curing and storage at all temperatures and at all three harvests. There was little change in firmness over the curing period itself but a drop of 39% (8.6-5.3 kgf) occurred in the first six weeks of coolstorage followed by a further 1 5.4% in the second six weeks. After 1 2 weeks coolstorage the average fruit firmness was 3.9 kgf irrespective of harvest date. Infection levels during fruit coolstorage Overall infection levels were high in fruit from the first harvest and considerably lower in fruit from the second and third harvests (Figs. 5-2 & 5-3) . Most diseased fruit showed symptoms by the six week assessment. There was a significant difference (P < 0.05) between temperature treatments after both six and 1 2 weeks coolstorage for each harvest date. Fruit cured at O°C and 5°C always had a higher incidence of disease than the inoculated control fruit and in 1 0 of 1 2 instances this difference was significant. Fruit cured at 1 0°C had a lower d isease than the inoculated and this was significant in four of the six comparisons. The disease incidence in fruit cured at 1 5°C was not significantly different from that in the inoculated controls except for the 1 2 weeks assessment of the third harvest. Fruit cured at 20°C had significantly less disease than the controls at harvest one after six weeks and harvest two after 1 2 weeks. Chapter Five 99 Table 5-1 5. 1 994: F IRMNESS OF INOCULATED KIWIFRUIT FROM THREE HARVESTS AFTER CURING AND COOLSTORAGE AT O°C. Mean firmness (Kg/f) Temperature Curing Storage O°C (OC) 3 daysX 6 weeksx 1 2 weeks Harvest 1 1 6 May 0 8.8a (0.2) 4 .6cd (0.2) * 5 9.2a (0.09) 5.2bC (0.2) 3.5 (0. 1 )Y 1 0 9.2a (0. 1 ) 6 .0b (0.03) 4.0 (0. 1 ) 1 S 8.6a (0.2) 4.2d (0. 1 ) 4.0 (0. 1 ) 20 9 . 1 a (0.2) 7. 1 a (0.4) 3.7 (0. 1 ) C. inoc. 9 .0a (0.2) 6.0b (0.2) * Harvest 2 23 May 0 9 .2a (0. 1 ) 4.Sa (0.3) 3.Sc (0. 1 ) S 8.SbC (0.2) S .Oa (0.4) 4.0ab (0. 1 ) 1 0 8.Sbc (0.2) 4 .Sa (O.S) 4 . 1 ab (0. 1 ) 1 5 8. 1 C (0. 1 ) 4.r (0.3) 3.9a bc (0. 1 ) 20 7.2d (0.2) 5.0a (0.4) 4.3a (0.2) C. inoc. 9.0ab (0. 1 ) 4.8a (0.2) 3.7bC (0.09) Harvest 3 27 May 0 9 .0a (0. 1 ) 5.9b (0.3) 3.8 bc (0. 1 ) 5 8 .3ab (0.2) 5.4Cb (0.2) 3.9abc (0. 1 ) 1 0 8.6a (0.2) 6.0b (0.2) 4.3ab (0. 1 ) 1 5 7.6b (0.3) 4.r (0.2) 3.4c (0. 1 ) 20 7.6b (0.2) 7 . 1 a (0.2) 4.4a (0.2) C. inoc. 8.Sb (0.2) S .4Cb (0.3) 3.5c (0.2) x Mean separation within columns by Duncan's multiple range test at P < 0.05. Y As number of fruit were unbalanced at this harvest and storage period, only Ismeans and standard errors were calcu lated . * Fruit from these treatments completely rotten . Values enclosed in parenthesis indicate mean standard error (SEM). 1 00 c o � (,) Q) -c Q) 0> $ C Q) � Q) 0.. 80 60 40 20 o May 1 6 � C. inoc. I11III OoC � SoC � 1 0°C � 1 SOC � 20°C May 27 Figure 5-2. 1 994: Percentage infection of kiwifruit from three harvests after curing at a range of temperatures and coolstorage for six weeks. Letters a,b c d & e refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. ...... o o 1 00 1m! C . inoc. liliiii OoC � SoC C 0 80 � 1 0°C :;::::::; � 1 SoC <.> Q) � 20°C --c 60 Q) 0> ctS ..-c Q) 40 � Q) a.. 20 o May 27 Figure 5-3. 1 994: Percentage infection of kiwifruit from three harvests after curing at a range of temperatures and coolstorage for 1 2 weeks. Letters a, b, c, d & e refer to Duncan's test (P < 0. 05). Vertical bars indicate SEM. 9 Q) � (b .., ::n � ..... o ..... Chapter Five 1 02 Experiment No.3 Relative humidity behaviour during the curing period in 1993 At both harvest dates the input/output relative humidity ranges remained constant throughout the three days of curing (Figs. 5-4 & 5-5) . With small differences between the relative humidity ranges from the first and second harvest, the lowest relative humidity varied from 40% to 60% (input) and from 4 1% to 59% (output) . The medium range varied from 6 1% to 80% and 62 to 81 % (input/output respectively) , while the highest varied from 94-98% and 92-98%. Fruit quality after the curing period: After three days curing at 1 0°C weight loss of fruit was inversely proportional to the relative humidity for all treatments (Fig.5-6) . Weight loss of fruit in the high relative humidity treatment was sign ificantly less (P < 0.05) than that in the other two treatments at both harvest dates. The highest percentage weight loss was in fruit cured at 40-59% RH although this difference was significantly greater than that in the mid-range (60-80% RH) at the second harvest only. Firmness of fruit from the first harvest was not affected by the relative humidity during curing (Fig .5-7) . At the second harvest, fi rmness of cured fruit at the highest relative humidity range was significantly higher (P < 0.05) than that in the other two treatments. Total solid solubles were not affected by relative humidity conditions during curing of fruit at either harvest (Fig.5-8) . Infection levels during coolstorage: Most infections of fruit by B. cinerea could be detected after 'six weeks coolstorage in the h igh relative humidity treatment but only two thirds of that in the low relative humidity treatment was detected at this time (Figs.5-9 & Chapter Five 100 80 60 40 20 - � 0 -- � 1 00 ...... "'0 E 80 ::J ..c ...... � 40 . 100 ...... "0 E 80 :::J ..c (l) 60 > :;:::. CO 40 (l) a: 20 1 00 80 60 40 20 • - • --- ------------... • • • Low r.h . (41 -59%) ------------... • • • Med. r.h . (62-81%) --------------.... • • • H igh r.h . (92-98%) 2 3 days 1 04 Input Output Figure 5-5. 1 993: Second harvest relative humidity during a three day incubation at 1 0°C (Mean ± SE). 3. 0 2.5 en � :: 2.0 ..c C> 'm 3: 1 .5 Q) C> ctI ..-C Q) 1 .0 � Q) a... h (40-59%) . � Low r. . 60-80%) o Med. r.h . (�2-97%) !Ba High r.h . c ------ �=� th t 1 0°C and one 0 'ng for t ree days a ------�. =�II�atted kiwifruit af�er l c ��rs indicate SEM. --- . I s of Inocu 05) Vertlca 1 993: Weight os ' test (P < O. . Figure 5-6. c refer to Duncan s . humidities. f three relative Letters a, b & 1 4 I&l Low r. h . (40-59%) 1 2 � Med . r.h . (60-80%) � High r.h . (92-97%) 1 0 ...-- C) � -- 8 en en Q) c 6 E � i..L. 4 2 0 a a a �?� a b b �/.� % �% % It % % % 0 � I� � � � � � % /. � � � � � /. �I % � � �- May 23 June 4 Figure 5-7. 1 993: Firmness of inocu lated kiwifruit after curing for three days at 1 00e and one of three relative humidities. Letters a & b refer to Duncan's test (P < 0.05) . Vertical bars indicate SEM. ...... o CJ) 1 4 1 2 a a a a , 1 0 " ./ '� ....- � 8 [2J Low r.h . (40-59%) 0 x --- E2l Med. r.h . (60-80%) (f) (f) � High r.h . (92-97%) J- 6 4 2 0 May 23 June 4 Figure 5-8. 1 993: TSS of inoculated kiwifruit after curing for three days at 1 aoe and one of three relative humidities. Letter a refers to Duncan's test (P < 0.05). Vertical bars indicate SEM. 1 00 90 80 c ,0 70 ...... <.> 2 60 c � 50 CO ...... � 40 <.> "- Q) 30 a.. 20 1 0 o a a � /'/- W3. • a May 23 � Low r. h . (40-59%) � Med. r.h . (60-80%) � High r.h . (92-97%) - Control a .�� � � � b � ;..; .� % T C . . ;..; June 4 Figure 5-9. 1 993: Effect of curing at 1 00e on incidence of B. cinerea infection of kiwifruit after six weeks of coolstorage. Letters a, b & c refer to Duncan's test (P < 0.05) . Vertical bars indicate SEM. -'" o 00 1 00 ,------------------------------------------------------------, 90 80 c o 70 :.;:::. u � 60 c � 50 CO ....... c 40 Q) � Q) 30 a... a a ab � Low r.h . (40-59%) 8a Med. r.h . (60-80%) � High r.h . (92-97%) --- Control Figure 5-1 0. 1 993: Effect of curing at 1 0°C on incidence of B. cinerea infection of kiwifru it after 1 2 weeks of coolstorage. Letters a, b & c refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. ...... o <0 Chapter Five 1 1 0 5-1 0) . Differences in disease incidence were not significant after six weeks of incubation of fruit from the first harvest but the final incidence as assessed at 1 2 weeks showed that there were significantly more infections (P < 0.05) in the fruit cured at the low than at the h ighest relative humidity (Fig.5-1 0) . At the second harvest there was the same pattem of results but this time the differences between humidity treatments were already significant at the first assessment. Experiment No. 4 Relative humidity behaviour during curing in 1994: With the exception of the lowest relative humidity range, the overall relative humidity during curing of fruit from the four harvests remained constant (Fig. 5-1 1 ) . At the lowest relative humidity range the input was 50% while the output was in itially 70% but increased during the curing period to 80%. The medium relative humidity range input remained constant at 80% and the output increased from 93% to 95%. The high range input was 95% relative humidity and the output 98-1 00% relative humidity. Fruit quality after the curing period Overall weight loss, firmness and total solid solubles were affected by fruit maturity at harvest with significant differences at P < 0.001 (Table 5-1 6) . The highest significant (P < 0.05) weight loss was observed at the first and second harvest, while the lowest was in fruit from fourth harvest. Firmness fluctuated from harvest to harvest but TSS increased progressively with each successive harvest (Table 5-1 6) . Relative humidity conditions during curing significantly affected weight loss (low weight loss at high relative humidity) but did not affect firmness or TSS (Table 5-1 6) . Chapter Five 1 00 80 60 40 20 - � 0 .......... >. 1 00 ...... "C E 80 ::J ..c Q) 60 > ...... ct:S 40 Q) a: 20 1 00 80 60 40 20 • - I nput • --. Output .--... ------. --------- • • • Low r.h . (50-80%) --- ----.... -----.... • • • Med. r.h . (80-93%) ------.... -----:::1 • • High r.h . (95-98%) 2 3 days 1 1 1 Figure 5-1 1 . 1 994: Range of relative humidities attained during three days incubation at 1 aoe for each of four harvests. Chapter Five 1 1 2 Table 5-1 6. 1 994: WEIGHT LOSS, FI RMNESS AND TOTAL SOLUBLE SOLIDS OF INOCULATED KIWIFRU IT HARVESTED AT DIFFERENT MATURITIES AND CURED FOR THREE DAYS AT 1 0°C AND ONE OF THREE RELATIVE HUMIDITIES. Source Level Mean Mean Mean TSS weight loss firmness (%) (%y (Kg/f) Main effects Maturity P < 0.001 P < 0.00 1 P < 0.001 1 st. harvest 0.58a (0.05) 8 .6a (0. 1 ) 7.5c (0. 1 ) 2nd. harvest 0.69a (0.05) 7.5c (0. 1 ) 8.5b 0.2) 3rd. harvest 0.54b (0.05) 8 .0b (0. 1 ) 9 .7a (0.2) 4th. harvest OA7c (0.05) 7.3c (0. 1 ) 1 0. 1 a (0. 1 ) Relative P < 0.001 NS NS humidity (RH) Low 0.93a (0.02) 7.7a (0. 1 ) 8.9a (0.2) Medium 0.66b (0.0 1 ) 7.9a (0. 1 ) 9.0a (0.2) H igh 0. 1 2c (0.03) 8.0a (0. 1 ) 9 .0a (0. 1 ) Type Inoculum NS NS NS ( lnoe) Spore O.SSa (0.03) 7.8a (0. 1 ) 9 .0a (0. 1 ) suspension Dry conidia 0.S8a (0.03) 8.0a (0. 1 ) 8 .9a (0. 1 ) Interactions Maturity * RH NS P < 0.01 P < 0.001 Maturity * Inoc NS NS NS RH * Inoe NS NS NS x P values after square root transformation. Values enclosed in parenthesis indicate mean standard error (SEM). NS = not significant. Chapter Five 1 1 3 The type of inoculum (spore suspension or dry con idia) did not affect weight loss, firmness or TSS (Table 5-1 6) . There was a significant interaction between maturity and relative humidity which affected firmness and TSS but not weight loss. The highest and medium relative humidity ranges gave a higher firmness than the low relative humidity at the first harvest date but the position were reversed at the fourth harvest (Fig.5- 1 2) . The relative TSS of the h igh and medium relative humidity treated fruit alternated at each successive harvest hence the significant interaction. Infection levels during coolstorage: Eighty three percent of the infections which ultimately developed were detected after six weeks of storage (Table 5-1 7) . At both assessments, overall significant differences in infection levels were found between harvest maturities and type of inoculum (Table 5-1 7) . There were progressively lower levels of disease over the 1 5 days from the first to the fourth harvests. Fruit treated at the highest relative humidity had the lowest percentage infection after six and 1 2 weeks coolstorage while those treated at the lowest relative humidity range had most disease. Fruit treated in the medium relative humidity range had a level of disease intermediate between that of the other two relative humidity ranges. Application of dry conidia caused nearly double the disease incidence of the spore suspension. There were significant interactions between maturity and relative humidity, maturity and type of inoculum and between relative humidity and type of inoculum (Table 5-1 7) . Fruit cured at the low and medium relative humidity showed a consistent drop in infection levels at each successive harvest but this pattern was not found in fruit cured at the high relative humidity where fruit from the first harvest had a low disease incidence (Fig.5-1 3) . Chapter Five 1 0 9 8 - 0,7 � --- (/) 6 (/) � 5 E .: 4 U- 3 2 1 0 9 8 7 -o �6 Cf) Cf) S I- 4 3 2 1 :x: I . --'--. • - 95-98% r.h . ... . . . . . . 80-93% r.h. • ._. - 50-80% r.h . .... ..•. . . . . . . . . . . ;�;:..: .<:� .... . ."., . ...--- . ... ,/ .. ',/' .Y' /.' . . --::-:-: :::-.::: � ... :. ;: ... 1 2 3 Harvest dates 4 1 1 4 Figure 5-1 2. 1 994: Interaction between maturity and relative humidity on firmness and total soluble solids of inoculated kiwifruit cured at one of three rh's and four harvest maturities. Vertical bars indicate overall standard error of the mean (SEM) . Chapter Five 1 1 5 Table 5-1 7. 1 994: INFECTION LEVELS OF B. CINEREA DEVELOPED DURING COOLSTORAGE OF KIWIFRU IT INOCULATED WITH SPORE SUSPENSION OR DRY CONIDIA BEFORE CURING AT 1 0°C AND A RANGE OF RELATIVE HUMIDITIES. Source Main effects Maturity � Relative humidity (RH) Inoculum (Inoc) Interactions Maturity * RH Maturity * Inoc RH * Inoc Level 1 st . harvest 2nd. harvest 3rd. harvest 4th. harvest Low Medium High Spore suspension Dry conidia Mean infection 6 weeks (%) P < 0.001 46.5a (2.7) 35.7b (3.5) 25.3c (3.9) 1 7.9d (3. 1 ) P < 0.00 1 42.4a (3.8) 32.7b (3.4) 1 9.0c (2.4) P < 0.001 21 .4a (2.5) 41 .3b (2.2) P < 0.01 P < 0.001 P < 0.00 1 Mean infection 1 2 weeks (%) P < 0.001 57.8a (3.3) 39.9b (3.6) 29.6c (4.0) 22.4d (3.5) P < 0.001 50.5a (4.2) 39.8a (3.5) 22.0b (2.8) P < 0.001 27. 1 a (2.8) 47.8b (2.5) P < 0.05 P < 0.00 1 P < 0.0 1 Overall means of inoculated control: 6 weeks storage, 32.8 ± 4.4; 1 2 weeks storage, 38. 1 ± 4.4. Values enclosed in parenthesis indicate mean standard error (SEM) . P values after square root transformation. NS = not significant. Chapter Five 9 8 7 6 5 c 4 o :;:::; � 3 - c Q) 2 C> 19 1 c Q) () � Q) 0.. 9 ..... o e 8 Q) � � 7 ::J 0- 00 6 5 4 3 2 1 I I ....... 1 2 3 Harvest dates 1 1 6 6 weeks • - 95-98% r.h. ... . . . . . . 80-93% r.h. • . _ . - 50-80% r.h. 1 2 weeks 4 Figure 5-13. 1 994: Interaction between maturity and relative humidity on B. cinerea storage rot incidence of kiwifruit cured at 1 0°C. Vertical bars indicate overall standad error of the mean (SEM) . Chapter Five 7 .... 6 ..•. 5 4 I c o 3 +-' () a> C 2 a> g> 1 +-' C a> () l0.- a> c.. • - Spore suspension .... . . . . . . . . Dry conidia . ... 6 weeks +-' 7 o o lo.- . . . · · · · ·· .. · · · · · · · .. ..J2 weeks 4 I 3 2 1 1 .... 2 3 Harvest dates . ... 4 1 1 7 Figure 5-1 4. 1 994: I nteraction between maturity and type of inoculum on B. cinerea storage rot incidence of kiwifruit cured at 1 0oe. Vertical bars indicate overall standard error of the mean (SEM). Chapter Five 9 8 7 6 5 c 4 0 +-' () 3 Q) - c Q) 2 0) ro 1 +-' C Q) () � Q) 0. 9 +-' 0 0 8 � Q) � ro 7 ::l 0'" (f) 6 5 4 3 2 1 I I 6 weeks •• • • • • • • • • • • • • _ _ • • • • • ••• • • • • • • • A,. ' .. • - Spore suspension & . . . . . . . . Dry conidia 1 2 weeks .... . . . . . ... Low Medium High Relative humidity 1 1 8 Figure 5-1 5. 1 994: Interaction between relative humidity and type of inoculum on B. cinerea storage rot incidence of kiwifruit cured at 1 aoc. Vertical bars indicate overall standard error of the mean (SEM). Chapter Five 1 1 9 Spore suspensions resulted in fewer infected fruit with each successive harvest but dry conidial inoculation did not continue this trend (Fig.5- 14) after harvest two. The h igher significant level of interaction between inoculum type and relative humidity at six than at 1 2 weeks coolstorage was caused by the greater divergence of disease levels in the medium relative humidity at six weeks coolstorage (Fig.5-1 5). DISCUSSION Physiological response during curing and coolstorage periods: In the present study, temperature, relative humidity and length of curing influenced fruit weight loss during both the curing process and subsequent coolstorage. In general , the percentage weight loss observed in kiwifruit from all experiments increased at higher curing temperatures and with longer curing periods (Experiments No. 1 & 2), and decreased with increased relative humidity during curing (Experiments NO.3 & 4) . The effect of curing on weight loss has been widely studied in tuber crops such as sweet potatoes (Kushman 1 975; Delate et al. 1 985 ; Walter et al. 1 989) , potatoes (Picha 1 986b; Kitinoja 1 987; Morris et al. 1 989) , yams (Passam et al. 1 976) caladium and Zantedeschia tubers (Marousky & Harbaugh 1 976; Funnell 1 988) and also in crops such as citrus (Ben-Yehoshua et al. 1 987a) . There is general agreement from these studies with the current work that the curing environment, especially temperature and relative humidity, influence weight loss both during the curing process and in subsequent coolstorage. In the present study, high weight loss was found after two days of incubation at temperatures of 1 aoc and above. Similar commercially adverse effects on fruit quality by high temperatures have been reported in citrus (Ben-Yehoshua et al. 1 987a), although in that study an additional Chapter Five 1 20 practice such as sealing played an important role in fruit weight loss since they reported 1 8% of weight loss during storage at 1 rc over a period of 32 days of cured and not sealed citrus, compared with 3 . 1 % in cured and sealed fruit. Kushman ( 1 975) reported that weight loss of artificially injured and cured sweetpotatoes at 28°C for nine days decreased with increasing relative humidities from 80, 90, 97 and 1 00% but the weight loss recorded during the subsequent storage of five months at 1 5°C , 85% relative humidity was similar to fruit from all treatments. In this present study, weight loss after the three days of curing for all temperatures and harvest dates, was less compared with weight loss values recorded after six or 1 2 weeks storage at O°C. However daily percentage weight loss for the first curing day, for all temperatures (second and third harvests) was higher compared with the second and third curing days. Similar findings to this every day weight loss behaviour have been reported by Walter et al. ( 1 989) . They reported that in general the highest percentage daily weight loss of sweet potatoes was recorded during the first days of curing. Funnell & MacKay ( 1 988) reported similar weight loss behaviour during the first days of curing (30°C) regardless of relative humid ity (80-84% or 40-44%). In this present study with exception of the first harvest (Experiment No.3) differences in weight loss between the medium and low relative humidities were clear. Although in other experiments carried out in vegetable crops such as peppers (Capsicum annuum L) (Lownds et al. 1 994) and fruit crops such as guava (Vazquez-Ochoa & Colinas-Leon 1 990) , apples (Lidster & Porrit 1 978; Lidster 1 990) durians (Durio zibethinus M.) (Ketsa & Pangkool 1 994) , carambolas (Averrhoa carambola L.) (Mil ler et al. 1 990); yellow passion fruit (Passiflora edulis f. f/avicarpa D.) (Arjona et al. 1 992) and papaya (Carica papaya L.) (An & Paull 1 990) , the effects of temperature and/or relative humidity as a curing practice were not evaluated on weight loss, similar tendencies to those found in the current study were reported ie. weight loss Chapter Five 1 21 increased at higher temperature and at lower relative humidities. Changes in firmness of cured fruit is another attribute of fruit quality to consider during curing and subsequent coolstorage of fruit. In 1 992 (Experiment NO. 1), inoculated, cured kiwifruit, cured at any temperature were firmer than the corresponding inoculated, uncured fruit during the curing period but the differences had disappeared by the end of coolstorage. Similar results were obtained in 1 994 (Experiment No.:!) although in this case the fruit cured at 20°C from the second and third harvests and at 1 0°C from the second harvest were still significantly firmer than uncured fruit after three months coolstorage. Curing appears to have had a beneficial rather than adverse effects on fruit firmness except when fruit was cured at 30°C. At this temperature there was a loss of firmness and other adverse effects (e.g. increased rots) . Clearly 30°C is a temperature which is marginal for fruit treatment. In this respect kiwifruit would appear unable to tolerate the temperatures to which various cultivars of apples can be exposed since there are reports of increased firmness with increased temperatu re from 21 to 48°C ((Liu 1 978; Porrit & Lidster 1 978; Klein & Lurie 1 990; Lurie & Klein 1 990; Klein & Lurie 1 992). The effect of temperature during long-term storage may be quite different from that during curing. Vazquez-Ochoa & Colinas Leon ( 1 990) found that guavas kept at ambient temperature (20°C) had a lower firmness than those stored at 3, 4, 7 or 1 1 °C. Commercially, kiwifruit are always stored at O°C. Other studies carried out in apples showed retention in fruit firmness regardless of relative humidities (Lidster & Porrit 1 978) . Further studies, found that firmness in apples gradually decreases with decrease in relative humidity after storage at 3°C for various months (Lidster 1 990) . I n the present study, with exception of fruit from the second harvest (Experiment No.3) fruit firmness after three days curing was not influenced by relative humidity (Experiments No.3 & 4) . As with the current study, no significant difference was reported in fruit firmness of durians when stored at three Chapter Five 1 22 different relative humidity levels 75, 83 or 93% at 30°C for six days (Ketsa & Pangkool 1 994) . However other studies carried out in guavas, reported significantly higher firmness on guavas stored at 80 or 88% relative humidity compared with 55% ambient relative humidity (Vazquez-Ochoa & Colinas-Leon 1 990) In the current study changes in fruit firmness during storage from one season to another were observed (Experiments No. 1 & 2). Results of this study showed that harvest date probably had more influence over fruit firmness during storage than curing environment. For example, in Experiment No.2 although there were significant differences among treatments, the low firmness recorded during storage in fruit from all curing temperatures could be explained not only by the curing period but also by the initial low firmness (6.6 kgf) recorded at harvest as wel l . Kiwifruit (Experiment No.2) during coolstorage showed the typical curve of firmness loss reported by Reid & Harris ( 1 977) and Crisosto et al. ( 1 984) . In the present study the combination of those ambient factors maturity and relative humidity influenced fruit fi rmness. Although fruit firmness was affected by harvest date (Experiment No.4) a consistent decrease of firmness with harvest maturity was not apparent in this study either at harvest or after the curing period . Differences in yearly firmness in kiwifruit at harvest have been already explained by Reid & Harris ( 1 977) . In this study the effects of curing temperature/incubation time were evident on ethylene production of kiwifruit (Experiment No. 1) since h igher curing temperatures increased ethylene production in kiwifruit. Porrit & Lidster ( 1 978) reported that ethylene production in apples gradually increased with incubation time ( 1 0 days) at temperatures at 21 °C while apples incubated at 38°C had lower ethylene production. They reported that at 38°C ethylene tended to decrease during the 1 0 days incubation. Similar inhibition of ethylene production with h igh temperatures was reported by Klein ( 1 988) & Klein & Lurie ( 1 990) . However they found that with high temperatures Chapter Five 1 23 (38°C) apples showed low ethylene production, when they were stored at O°C but when retumed to ambient temperature (20°C) ethylene production was reactivated. In the present study, with the exception of fruit from the inoculated control during subsequent coolstorage, ethylene production did not follow any specific pattern according to the previous temperature/incubation period . Apparently in the current study, ethylene produced during curing did not affect ethylene production of fruit during storage. For example in fruit cured for four days at 1 0°C there was ethylene production (0.29 J.l1) , however fruit from this treatment coolstoraged for one month did not produce ethylene. In the current study, except for fruit cured for six days at 1 0°C, the rate of respiration in kiwifruit increased proportionately to curing time but incubated kiwifruit showed a decline in respiration rate with increased temperatures. Carbon dioxide production during coolstorage did not show any specific trend respect to the curing temperature/time of incubation. Probably the lack of a climacteric respiratory peak observed in this study can be explained by differences in the climacteric rise behaviour typical of this fruit as reported by Reid et al. ( 1 982) and M itchell ( 1 990) . Apparently there was no relationship between ethylene concentration and carbon dioxide production as has been reported in other fruit crops such as pears (Maxie et al. 1 974) . In their study, respiration rates of pears exposed to 1 00 ppm ethylene were increased. Similarly in other studies, sweetpotatoes cured at 30°C with ethylene for three days increased carbon dioxide production by 30% more than the control during curing while no effect between ethylene and rate of respiration was reported during storage at 1 5°C for eight days (Kitinoja 1 987) . I n the present study, respiration rates of fruit incubated at 0, 1 0 or 20°C for two days did not correspond to the zero ethylene concentration observed in these treatments. I n the current study the pattern of ethylene production did not correspond Chapter Five 1 24 with the pattern of loss of firmness (Experiment No. 1) . For example kiwifruit firmness from the inoculated control decreased from the second to the fourth incubation day, while there was no ethylene production at either of these two incubation periods. Chemical composition of fruit In general neither curing temperature nor relative humidity affected the total solid solubles concentration of kiwifruit. There were significant differences in TSS in fruit from different curing treatments in subsequent storage periods (Experiment No. 1) but in general those values were high among all treatments. S imilar results to this present study have been reported in other fruit crops such as apples, passion fruits and durians (Porrit and Lidster 1 978; Klein & Lurie 1 990; Lidster 1 990; Lurie & Klein 1 990; Arjona et al. 1 992; Ketsa & Pangkool 1 994). In those studies, any significant changes in TSS levels were reported during heat or relative humidity treatments and subsequent storage. Clearly in this current study, those changes observed in TSS were more related to the harvest date and fruit maturity than temperature and/or relative humidity combinations. In this present study TSS increased with fruit maturity (Experiments No.3 & 4) . The high TSS values in 1 99 1 (Experiment No. 1) during curing and storage periods can be explained by the initial high values at harvest ( 1 0.8%) in spite of the harvesting date (1 1 th May) . These values differed from those recommended for export purposes. According to Lallu ( 1 989a) ; M itchell ( 1 990) and Hopkirk ( 1 992) the more suitable TSS at harvest in New Zealand to avoid postharvest problems during storage should be 6.2%, although a range between 7 to 1 0% is acceptable. In the present study, as reported by others (Hopkirk et al. 1 986) , seasonal differences were also observed in fruit at harvest from 1 992 to 1 994. Fruit TSS at harvest in the three years of experimentation varied from 1 0.8% the first year, 8.8 to 1 0.6% the second and 6.8 to 9 .2% in the third year. I n the current study the content of sugars during fruit coolstorage varied Chapter Five 1 25 regardless of the previous curing temperature. I n general glucose and fructose increased with storage period. Studies on kiwifruit carried out by N icolas et al. ( 1 988) reported that the amount of reducing sugars stored at O°C for a period of 26 weeks increased during the first five to 1 0 weeks of storage but remained constant for the remain ing storage period. Similar findings were reported by Mac Rae et al. ( 1 989a; 1 989b) in fruit from different regions and maturities or in fruit stored at 4 or O°C for up to 1 2 weeks. In both studies they found glucose was the main individual sugar, fol lowed by fructose and sucrose. With the exception of the glucose content of fruit from the inoculated control after three months storage, the amount of sugars (sucrose, glucose and fructose) in fruit from the inoculated control was higher than the treated fruit. In general agreement with these results, Heatherbell ( 1 975); Okuse & Ryugo ( 1 981 ) ; Reid et al. ( 1 982) ; Ben-Arie et al. ( 1 982) ; Matsumoto et al. ( 1 983) and MacRae et al. ( 1 989b) concluded that glucose was the main individual sugar of kiwifruit, fol lowed by fructose and in lesser amount, sucrose during fruit ripening, fully mature fruit or in fruit previously incubated at low temperatures at 4 or O°C for a period of time. Similarly Pesis et al. ( 1 99 1 ) , reported that the amount of glucose was higher in the non-infected tissue zone (by B. cinerea) of kiwifruit stored at O°C for a period of 1 5 to 20 weeks, compared with lower amounts of the invaded or infected zone of the fruit. In the present study kiwifruit were artificially inoculated but only fruit showing no symptoms of diseases were taken for chemical analysis. In the present study pH decreased and acidity increased with time during fruit coolstorage. There were significant differences in pH levels of fruit during the coolstorage but in general the range of pH was between 3.3 to 4.0. Ben-Arie et al. ( 1 982) reported that stored kiwifruit cv Bruno at -1 °C cv pH values significantly increased for about 7% ( 3.7 to 4.0) and percentage titratable acidity decreased about 40% (1 .01 to 0.5%) . Matsumoto et al. ( 1 983) found similar pH ranges in kiwifruit harvested at mature green stage and kept at 20°C for 20 days for ripening. In that study non-treated fruit had Chapter Five 1 26 a pH of about 3.3 and 1 .4% of total acidity. However Pesis et al. (1 991 ) reported a similar pH (3.5) in fruit from infected tissue compared with 4 . 1 from the non-infected tissue. In that study, acidity levels were lower than those found in the current study. Infection levels during coolstorage In this study curing conditions influenced percentage disease of kiwifruit during coolstorage. In 1 99 1 (Experiment No. 1) a curing temperature of 1 0°C for two to six days reduced B. cinerea infection of fruit during storage, compared with those held at 0, 20 or 30°C. I n 1 994 (Experiment No.2) the same temperature of 1 0°C reduce B. cinerea in fruit from the first harvest, but higher curing temperatures at 1 5°C or 20°C also reduced B. cinerea in fruit harvested at the second and third harvests. In both 1 993 and 1 994 (Experiments No.3 & 4) fruit cured at the lowest relative humidity ranges gave the highest infection levels during fruit coolstorage. Although in other studies the effect of temperature was not studied as a curing practice, similar findings were reported by Hyre ( 1 972) on incubated geranium leaves (Pelargonium hortorum) infected by B. cinerea. This researcher observed smallers lesions ( 1 .5 cm) when incubated at 1 0°C for three to seven days, compared with 25°C (8.5 cm) . Mridha & Wheeler ( 1 993) found significantly lower infection by A. brassicae on old leaves of oilseed rape incubated at 1 0°C compared with temperatures of 1 5, 20, 25 and 29°C for 8h. They observed the greatest number of spore penetrations at 25°C and the highest number of lesions at 20°C with a 24h period of wetness. In studies on kiwifruit in which fruit were cured for two or seven days at ambient temperature, Sharrock & Hallet ( 1 991 ; 1 992) observed lower spore germination and germ tube development on the stem scar surface of those fruit cured for seven days than when cured for only two days. In the present study less percentage infection was not observed when incubation time increased since h igher infection levels were recorded in fruit incubated for four or six days at 20°C or 30°C compared with levels of infection after two days curing. Other studies carried out by Pennycook & Manning ( 1 992) Chapter Five 1 27 reported that in kiwifruit held, at 1 4°C ambient temperature for one, two, four and seven days, B. cinerea infection were gradually reduced with an increase in the duration of curing. In the present study the higher infection levels that developed after curing at temperatures higher than 1 0°C and the rapid onset of symptoms in fruit cured at 20°C for six days or at 30°C for up to two days, may be associated with heat damage to the fruit which would facil itate B. cinerea infections. Bartz et al. ( 1 991 ) found that bacterial soft rot on inoculated tomatoes began earlier and progressed more rapidly in fruit stored at 30 or 35°C for 24h than in fruit stored at 1 5 or 25°C. After 72h most of the fruit stored at 25, 30 or 35°C showed bacterial d isease though those at 1 5°C were relatively disease free. Ben-Yehoshua ( 1 987b) suggested that the faster growth of P. digitatum on pummelos and lemons cured at 30°C for one day compared with the non-cured fruit can be associated with the curing conditions that affect the fruit membranes in a manner that made nutrients more available to the pathogen which could then grow faster. Alderman & Nutter ( 1 994) reported that in order to increase leaf spot disease (Cercosporidium persona tum Berlc & M.A. Curtis) of peanuts (Arachis hypogaea) it was necessary to incubate peanut plants in an additional treatment at 50-60% relative humidity and 1 6h/day fluorescence l ight photoperiod . However in other reports, it was observed complete inhibition of sporulation of Helminthosporium maydis sporulation on com plants (Zea mays) when incubated at relative humidities lower than 93% (Hyre 1 974) . Probably in the current study, temperatures of O°C and 5°C and the lowest humidity ranges, suppressed the development of resistance mechanisms which suppress B. cinerea in kiwifruit as reported by Poole et al. ( 1 993) . Mechanisms such as enzyme activity, production of l ignin and changes in tissue chemical composition were slowed down compared with those in fruit held at higher temperatures and h igher relative humidities. For example, in those studies, high enzymatic activity such as l ipoxygenase, phenylanaline ammonia lyase and peroxidase were observed immediately Chapter Five 1 28 after harvest and during curing. Another explanation for the infection levels recorded in the current study could be related to the d istribution of B. cinerea toxins and kiwifruit phenolic compounds produced during the infection process as has been reported by Harrison ( 1 980). This researcher studied the influence of relative humidity on the diffusion of toxins produced by B. fabae on leaves of broad beans. He suggested that toxins produced by B. fabae at 1 00% relative humidity diffused throughout the leaf while at lower relative humidities (75%) , they concentrated in certain areas of the leaf, hence the highest disease severity was found at the highest relative humidity. In the present study, the highest infection levels were at the lowest relative humidity and could be associated with the highest weight loss of fruit held at these relative humidities, hence enhancing infection levels. In other studies, a positive correlation between weight loss and infection levels during sweetpotatoe storage has been reported (Kushman 1 975) . In that study, curing and storage conditions at 26°C for seven days and later at 1 5°C for five months respectively, showed that percentage decay increased with weight loss increased (above 6%) . Conversely to the present results, Marois et al. (1 988) reported an overall l inear and inverse correlation with vapour pressure deficits between the susceptibil ity of greenhouse grown rosa flowers (Rosa hybrida) to B. cinerea. They reported higher susceptibil ity with a VPD of 0.7 Kpa compared with 1 .05 Kpa. In the present study in both, Experiments No.3 & 4, infection levels significantly increased with VPD increase: the highest infection levels were recorded in these VPD ranges of 0.70 - 0.24 Kpa compared with the lowest infection in fruit held at a VPD of 0.09 - 0.02 Kpa. The difference in infection levels between Marois et al. ( 1 988) study and the present results, can also be accounted for by the environmental temperature. For example, Marois ef al. (1 988) reported, that greenhouse temperatures during the two day treatment fluctuated between 1 5 to 22°C, Chapter Five 1 29 while the temperature used in this study was 1 COC for three days. Another important environmental factor such as dew period or period of wetness on onion leaves, inoculated with B. squamosa, has been extensively investigated by Shoemaker & Lorbeer ( 1 971 ; 1 977) ; Alderman & Lacy ( 1 983) and Alderman et al. ( 1 985). They reported that the longer the wet periods the greater the infection levels regardless of the type of inoculum (spore suspension or dry conidia) . Although in those studies the relative humidity of the dew chamber was not controlled it was in general h igh. Similarly, Mansfield & Deverall ( 1 974) reported that infections of B. cinerea on leaves of broad beans only took place if droplets of water were present to permit establishment of a spreading d isease. They reported h igher infection levels (75%) after six days at 1 7 ± 2°C when droplets of B. cinerea suspension persisted throughout the first days compared with a lower infection ( 1 7%) in sites where droplets were absent after the first day of inoculation. In the present study, there was no constant wet period during the curing treatments, but during the subsequent kiwifruit coolstorage, there was some condensation of water, since fruit was wrapped in polyethylene l iners. This condensation was on the liners and may not have affected the infection process since curing was completed at this stage. In the current study, contrary to the general belief (Sharrock & Hallet 1 991 ) infection levels were h igher at both storage periods (93% and 76% respectively) when dry conidia were applied to the stem scar compared with those recorded from inoculations with spore suspensions. Snow ( 1 949) and Jarvis ( 1 962) reported that although spore germination of B. cinerea on strawberries was faster in the presence of a droplet of water compared with germination in a saturated atmosphere, eventually, dry conidia could germinate. As with other studies on kiwifruit (Beever 1 979; Pennycook 1 981 ) , in this study, in general, fruit maturity influenced infection levels during fruit Chapter Five 1 30 coolstorage. For example in Experiment No.4 °brix levels increased from 6.8% to 9.2% (first and fourth harvests respectively) while infection levels after 1 2 weeks coolstorage decreased from 57.8% to 22.4%. Throughout this study, it was also observed that infection levels varied from year to year, for example in 1 991 fruit from the inoculated control showed 7.9 percentage infection while percentage infection of fruit from the 1 994 season was 38. 1 %. Similar seasonal differences in infection levels of kiwifruit during coolstorage have been reported in kiwifruit (Brook 1 990a; Hopkirk et al. 1 990b) and in other commodities such as potatoes (Malcomson & Gray 1 968; Adams & Griffith 1 978; Stewart et al. 1 983; Hide & Boorer 1 991 ; Merida & Loria 1 994). In general agreement, these authors mentioned weather conditions in the orchard and pre and postharvest handling operations as the main reasons for these fluctuations. CHAPTER SIX ANATOMICAL AND HISTOCHEMICAL STUDY OF INOCULATED KIWIFRUIT STEM SCARS DURING CURING INTRODUCTION Curing and wound healing It is well established that in many diseases of fruits and vegetables the pathogens gain entrance through wounds. According to Davies ( 1 987) wounding includes physical damage to the plant tissue and cells, especially mechanical d isruption of cell membranes, that eventually lead to loss of compartmentation of ions. It is well known that many of these wounds have the abil ity to heal by a number of different mechanisms (Bostock & Stermer 1 989) . One of the main objectives of curing is to encourage the repair of wounded areas as soon as possible thereby rebuilding the mechanisms of defense such as structural barriers and/or production of antifungal substances near to the damaged site. The more rapid the curing process the more chance of avoiding pathogen attack or arresting further invasion from incipient infections. Structural changes have been reported as an important aspects involved in wound repair as a mechanism of defence against plant pathogens (Ride 1 975; 1 978; Passam et al. 1 976; Cline 1 983; Biggs 1 984; 1 986; Rittinger et al. 1 987; Larson 1 994) . The process of wound heal ing in plants has been classified by Bostock & Stermer (1 989) according to the cell activity involved . They considered that three types of tissue repair can exist: I n one type there is an autolysis of the infected dead cells as occurs in carrots; In another type there is both regeneration of cells and cellular depositions as in kohlrabi tubers and in the third there is a more complex mechanism of Chapter Six 1 32 wound repair that includes these two cel lular mechanisms plus redifferentiation of parenchyma cell walls to form a suberized periderm layer in the damaged site, as occurs in potatoes. Anatomical and/or histochemical responses during wound healing The induction of structural barriers is one of the most common processes that occur in response to pathogen invasion and during wound heal ing. For example, the reported response to infection by different pathogens such as Phytophthora spp, Verticillium spp or Ceratocystis ulmi, include: alterations in the vascular system e.g. (as gum duct formation in Citrus trees) , cell wall multi layering and tyloses development e.g. (in american elm, Ulmus americana L.) and in avocado rootstocks, (Persea americana M.) and vessel coatings and plug formation in leaves of various vegetables e.g. (snapdragons, Anthirrhinun majus L. , eggplants, Solanum melogena L. and potatoes) and in shoots and branches of trees e.g. (hop plants, Humulus lupulus L. , sycamore maple, Acer pseudoplatanis L. and hedge maple, Acer campestris L.) . (Ouel lette 1 978; 1 980; 1 981 , Robb et al. 1 979; 1 982, Phi ll ips et al. 1 987) . Periderm development and cellular suberization and lignification have been reported as a response to damage by mechanical activities and to infection by pathogens such as Fusarium roseum f.sp. sambucinum, Cladosporium cucumerinum, Col/etotrichum lagenarium in various vegetables such as potatoes, yams, sweet potatoes and leaves and seedlings of cucumbers, (Passam et al. 1 976; Hammershmidt & Kuc 1 982; Hammershmidt 1 984; Dean & Kuc 1 987) . In kiwifruit, preliminary anatomical studies on stem scars of fruit infected by B. cinerea and incubated at various temperatures showed no apparent evidence of structural changes of the vasculature and surrounding tissues (Poole & McLeod 1 991 ; Sharrock & Hallet 1 99 1 ) . Similar studies in kiwifruit suggested the presence of antifungal compounds such as tannins, phenolics and alkyl aldehydes as a mechanism of defense against B. cinerea in the stem-end of the fruit (Poole & McLeod 1 99 1 ) . Chapter Six 1 33 Additional histochemical studies in onions (Moon et al. 1 984) , carrots (Garrod et al. 1 982) , in a range of avocado rootstocks (Phil l ips 1 993) , cucumber (Walter et al. 1 990) and in some wheat varieties (Ride 1 975) have demonstrated the activation of suberin, l ignin or phenolic compounds, when plants were artificially wounded ancllor inoculated by various pathogens. On inoculated apples, peaches and almond bark similar histochemical components were observed to develop in response to infection by various pathogens such as Leucostoma cincta, L. persooni, Phytophthora spp, Botryosphaeria obtusa, B. dothidea (Biggs 1 986; Doster & Bostock 1 988; Biggs & Britton 1 988) . Factors affecting response to wound healing The process of wound repair is closely linked to external factors such as temperature, relative humidity, light, oxygen, carbon dioxide production and harvesting period . Biggs ( 1 993) reported that in general wound healing is more rapid at 20 to 25°C with relative humidities between 70% and 1 00% and at 1 0°C between 80% and 1 00% relative humidity. He also suggested that the type of anatomical changes developed were determined by the relative humidity. Biggs ( 1 993) also considered that temperature is one of the most important environmental factors to influence the rate of wound healing in woody plant species and fruit trees. He found a reduction in wound repair time (Le. in development of Iigno-suberized layers) in potted plants with an increase in temperature. Studies on uninoculated, wounded apples, have shown that meristematic cell d ivision can vary according to harvest date and temperature. Apples harvested at the beginning of the picking season showed complete healing, but healing was delayed on those harvested late and no healing was observed in those kept at 3°C after harvest (Skene 1 981 ) . Further studies in mature apple fruit showed that development of resistance to B. cinerea Chapter Six 1 34 and to P. expansum occurred in those fruit incubated for 38 days at SoC or for 1 4 days at 20°C (Lakshminarayana et al. 1 987) . In potato tubers the most suitable environmental conditions for the wound repair process may be different. For example, Wigginton ( 1 974) observed a stimulation of cell d ivision and inhibition of suberization in cut potato tubers held at relative humidities closest to 1 00% and also a similar inhibition of suberization at low relative humidity. This researcher considered the most appropriate range of relative humidity and temperature for the process of suberization was 70%-1 00% at 20°C. Meijers ( 1 987) , found that rates of wound heal ing on potatoes can vary according to the temperature and time of curing. Results of this study showed that the highest temperature (30°C) and 80% relative humidity accelerated the healing process. Morris et al. ( 1 989) reported a temperature/relative humidity combination of 2SoC and 98% relative humidity as optimum for wound healing to induce resistance against Fusarium oxysporium and Erwinia carotovora pv carotovora. They also found that temperature was a more important factor for wound repair than relative humidity. OBJECTIVE To ascertain whether physical barriers and/or antifungal substances develop on inoculated kiwifruit stem scars during the process of wound healing. MATERIALS AND METHODS Fruit samples Five samples of healthy fruit from Experiment No. 1, Chapter 5 were incubated for two, four and six days at 0 and 1 0°C, for two and four days at 20°C and for two days at 30°C to carry out the anatomical and histochemical study. Since fruit from the 20 and 30°C incubation treatments for six and two days respectively showed early symptoms of softening and Chapter Six 1 35 of disease, and since sectioning of fresh or paraplast embedded stem scars showing mycelial growth was difficult because of d isintegration of the tissue, these treatments were not included in the present study. Tissue preparation for anatomical and histochemical study Stem scar tissue samples of each fruit were longitudinally hand sectioned to blocks about 1 to 2 mm (Plate 6-1 ) and fixed by vacuum infi ltration overnight in a solution of formalin: acetic acid: and alcohol (FAA). Samples were washed with water 4-5 times to remove excess fixative before dehydration. By the modified procedure of Feder & O'Brien ( 1 968), samples were then dehydrated and infiltrated with Paraplast. They were sectioned at 5-8�m thickness with a Jung Rotary microtome. Anatomical and Histochemical staining After removal of the paraffin by xylene and passage of slides through an ethanol series (Bautista-Banos 1 989) the following two stain ing procedures were used as a general staining procedure to highlight different tissues: a double stain of 1 % methyl violet and 1 % eosin and a safran in-anil ine blue combination (Johansen 1 940) . Specific tissue stains were a) Phloroglucinol­ HCI and Toluidine Blue (Peacock & Bradbury 1 973) for l ignin; b) Safranin and Fast Green (Bautista-Banos 1 989) for l ignin and cellu lose respectively; c) Sudan IV (Johansen 1 940) and Sudan Black B (Peacock & Bradbury 1 973) for suberin and cutin respectively and d) Glycerine-ferricyanide (Sherwood & Vance 1 976) for reducing compounds. Slides for histochemical studies were mounted in a 2% CaCI2 and glycerine solution (Herr 1 992) and specimens for anatomical studies were mounted in D .P .X. Light micrographs were taken with a Nikon fx-35wa camera mounted on a Reichert Diapan phase contrast microscope and on an Olympus dissecting microscope. Details of fixative and stain preparation and procedures are given in the Appendix. Plate 6-1 Plate 6-2 Longitudinal section of a fresh kiwifruit. ( Plate 6-2 stained with Phl orog l ucinol-Hel. mg. x 1 . 8 ) . a) stem s c a r su rface ( P lates 6-9 - 6-1 4) e) i n n e r pericarp b) suberized shoulder f) oute r pericarp c) sclerified plug g ) core d) vasc u l a r strands h) seeds Chapter Six 1 37 RESULTS Anatomical components of stem scar tissues The kiwifruit stem scar is a circular area of broken tissue about 3-4 mm diameter surrounded by a raised shoulder of suberised skin (Plate 6- 1 ) . The surface consists of parenchyma cells with a small number of vascular bundles arranged in a circular pattern . These vascular bundles contain l ignified xylem vessels that spread out over a woody, sclerified plug (P late 6-2) . The main body of the kiwifru it is composed of a central core, an inner pericarp where the seeds are formed, an outer pericarp and hairy skin (Plates 6-1 & 6-2) . The pedicel attachment i s very narrow at the point where i t is snapped off the fruit (Plate 6-3) . The central vascular core of the pedicel d iverges into three vascular bundles just above this point (Plate 6-4) and these i n turn are further subdivided to g ive five or six at the level of the t ip of the sclerified plug (Plate 6-5) . The upper surface of the sclerified plug is not smooth but is formed by a number of i rregular ridges (Plate 6-6) . The vascu lar tissues now consist of a very large number of small units. Part way down the p lug , the vasculature is intimately associated with the stem plug but b ranches spread out and ramify between the inner and outer pericarp (Plate 6-6 & 6-7) . After this paint, the vascular strands separate from the plug and cont inue through the fruit between the core and the inner pericarp. The developing seeds are supplied by side branches of these vascular strands (Plate 6-8) . The kiwifruit stem scar above the sclerified p lug consists of two main tissue systems: Fundamental or ground tissue and vascular tissue. The g round tissue consists of parenchyma, collenchyma and sclerified cells (Plate 6-9) . I dioblasts containing depositions of elongated calcium oxalate crystals ( raphides) were particularly d istributed among the parenchyma cel ls and along and paral lel to the xylem vessels (Plate 6- 1 0) . Chapter Six 1 38 P late 6-3. Longitud i n al section of a fresh kiwifruit with pedicel attached stained with P h l o roglucinol - H C I . (mg. xO.7) . Positions of the cross-sections shown i n Pl ates 6-4 -6-7 are i n d i cated with horizontal l ines. Section A 1 ( P l ate 6-4) , section A2 ( P l ate 6-5), section A3 ( P l ate 6-6) , section A4 ( Plate 6-7) and section AS ( P late 6-7) . a) stem scar su rface b) su berized shou lder c) sclerified p l u g d ) vascular strands e) inner pericarp f) outer pericarp g) pedicel h) point at which pedicel vascular tissue divides i nto three strands. Chapter Six 1 39 Plate 6-4. Cross section (A 1 ) of a fresh kiwifruit stem scar stained with Phloroglucinol-HCI (mg. x4.0) . At the union between pedicel and fruit, there i s a circular arrangement of three vascular b u ndles (v) su rrounded by, parenchyma and the suberized tissue from the fruit shoulder ( b ) . Plate 6-5. Cross section (A2) o f a fresh kiwifruit stem scar stained with Ph lorogluci nol-HCI ( m g . x 1 .8) . The orig inal three vascular bun dles ( v ) have divided t o form five. T h e upper most point of the sclerified p l ug (p) can be seen i n the centre. --- ---- Chapter Six 1 40 Plate 6-6. Cross section (A3) of a fresh kiwifruit stained with Phlorogl ucinol-HCI ( m g . xO.7). showing radial development of sclerified tissue (s) on the upper su rface of the plug and vascular b u ndles (v) . Plate 6-7. Cross section (A4) of a fresh kiwifruit stained with Phl orog l ucinol-HCI (mg. xO.7). The sclerified plug (p) is wel l defined and vascular bundles (v) are diverg i n g between the inner a n d outer pericarp. Chapter Six 1 4 1 Plate 6-8. Cross section (AS) of a fresh kiwifruit stained with Phlorogl ucinol-HCI (mg. xO.?). The lower, compacted region of the sclerified plug (p) i s su rrounded by well defined vascular b un d l es (v) and seeds (s). Plate 6-9. Longitudinal section of a kiwifruit stem scar stained with safran i n-fast green showi ng parenchyma (p), sclereids (s) and collenchyma (q) cells. Bar indicates 0 . 2 mm. Chapter Six 1 42 I n general , the vascular tissue system was composed of xylem (with helical thickening) (Plate 6-1 1 ) , phloem and cambium. At 20 and at SO°C spore germination and penetration of hyphae of B. cinerea through the xylem vessels of kiwifruit were observed (95%) after 2 , 4 and 6 days of incubation (Plate 6- 1 2) . By contrast, after 2 days (P late 6- 1 S) at 0 or 1 0°C most of the spores scattered on the surface of the xylem vessels and parenchyma tissue had not germinated (75%) and of those spores that had germinated l ittle hyphal growth had occurred. After 4 and 6 days at 1 0°C germ ination was sti l l rare but was common at O°C . There was no development of tyloses from neighbouring parenchyma cells at any temperature. At 1 0°C there was evidence of cel lu lar thickenings of the parenchyma cel l walls where they were in contact with the conidia of B. cinerea which had commenced germination (P late 6- 1 4) . Histochemical tests H istochemical tests showed some differences between fruit incubated at 0, 20 or SO°C and those incubated at 1 0°C but the sole d ifference between t imes of incubation was with the test for reducing compounds (Table 6- 1 ) . a ) Lignin . The Phloroglucinol and Tol uidine Blue tests gave a deeper colour reaction in the xylem vessels and in the parenchyma cell wall th ickenings of the 1 0°C samples and a faint colour in the axial parenchyma of the vascular system . The double staining procedure with Safranin and Fast-Green stained l ign ified tissue in xylem vessels and scleroids red and cel lu lose in parenchyma and phloem cells, green but with no d istinction between treatments. b) Suberin . A positive reaction was found in the cel l wal ls of the vascular vessels in the stem scar of fruit from al l temperatures using Sudan IV , but Sudan Black B i ndicated the presence of suberin on ly in samples held at 1 0°C, where it was concentrated in the vessel wal ls and in th ickenings where the parenchyma cel l walls were in contact with Botrytis hyphae or spores. Chapter Six 1 43 r - Plate 6-1 0. Longitudinal section of a kiwifruit stem scar stained with methyl violet eosin showi ng idioblast containing cal cium oxalate crystals (raphides) (r) and parenchyma (p) cel ls. Bar i n d icates 0.2 m m . x - � p , , Plate 6-1 1 . Longitu dinal section of a kiwifruit stem scar stained with methyl violet eosin showi ng xylem vessels (x) with helicoidal secondary wall thickening and parenchyma (p) cells. Bar i n d icates 0 . 02 mm. Chapter Six 1 44 Plate 6-1 2. Longitudinal section of a kiwifruit stem scar stained with methyl violet eosin after two days curing at 20 or 30°C. Most of the spores (s) scattered on the su rface of the xylem vessels (x) have germinated. Bar i n dicates 0.2 m m . Plate 6-1 3. Longitudinal section o f a kiwifruit stem scar stained with methyl violet e o s i n after two days curing at 1 0°C . Most of the spores (s) scattered on the su rface of the xyl e m vessels (x) have not germinated. Bar indicates 0.2 m m . Plate 6-1 4. Longitudinal section of a kiwifruit stem scar cured at either 0 or 1 Qoe for two days stained with methyl violet eosin. Parenchyma cell wal l s (p) i n contact with hyphae (h) are thicker than norma l . Bar indicates 0.2 mm. Table 6-1 . H ISTOCHEMICAL TEST OF K IWI FRUIT STEM SCARS INOCULATED WITH B. cinerea AND I NCUBATED AT () :::r VARIOUS TEMPERATURES FOR UP TO S IX DAYS. III '0 . 1 00 ..... "'C E 80 ::J ..c Q) 60 > ..... CO 40 Q) a: 20 100 80 60 40 20 • - I nput • --. Output ---�-------------- Low r.h. (34-80%) �--�----------�--- • • • • I Med. r.h . (75-90%) • ..... = .. High r.h. (1 00%) • • 2 3 4 5 6 Weeks of incubation 1 60 Figure 7-2. 1 992: Input and output relative humidity over a six week period at O°C. en en o 2 .5 ...... 2 .0 .c C> Q) � Q) 1 .5 C> en ...... c Q) 1 .0 � Q) a.. 0.5 001 Control uninocu lated � Control inoculated � Low r.h . (34-80%) � Med. r.h . (75-90%) � High r.h . ( 1 00%) 1 4 Weeks of incubation a b 6 Figure 7-3. 1 992 : Percentage weight loss of inoculated kiwifruit over a six week period at O°C and one of three relative humidities. Letters a, b, c & d refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. ...... 0> ...... Chapter Seven 1 62 Fruit firmness was maintained after one week of incubation for all treatments and for both controls but as the incubation period increased to 4 and 6 weeks, firmness in all fruit decreased markedly to 2-4.5 kgf (Fig.7- 4) . The effect of relative humidity/incubation time/storage time, showed high significant differences (P < 0.00 1) . By three months of coolstorage firmness had decreased to 2-2.5 kg/f where it remained for the second three month period of incubation (Fig .7-5) . The total soluble solids of fruit in all treatments and controls increased during the one to six week incubation period with most of the increase taking place in the one to four week period (Fig.7-6) . Ethylene production was not detected at harvest on uninoculated control fruit at any time during the experiment (Fig.7-7) . Inoculated control fruit produced more ethylene after four weeks incubation than those in any of the treatments. Ethylene production by fruit in the low relative humidity treatment was not detected after one week incubation, it was low after four weeks incubation but rose dramatically after six weeks incubation when production by some fruit only was extremely h igh contributing to both a high average production and a large standard error. In the medium relative humidity treatment, there was some ethylene detected after one and four weeks incubation and this had doubled after six weeks. In the high humidity treatment there was some ethylene production by fruit after one week incubation , a considerable amount after four weeks but one after six weeks. There was no definite pattem to changes in respiration rate as measured by CO2 production and there appeared to be l ittle overall differences between one and six weeks incubation (Fig.7-8) . At harvest The control inoculated fruit had the h ighest respiration rate after four weeks incubation . Infection levels during coolstorage Fruit from the uninoculated control developed less than 5% incidence of B. 1 2 a C- 1 0 0) � --- en 8 en C E 6 � i.L 4 2 1 a b 4 8) Control uninoculated � Control inoculated I&l Low r.h . (34-80%) � Med. r.h . (75-90%) � High r.h . ( 1 00%) 6 Weeks of i ncubation ", a Figure 7-4. 1 992 : Firmness of inoculated kiwifruit over a six week period at ooe and one of three relative humidities. Letters a & b refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. Firmness at harvest = 9.9 . Chapter Seven 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1 .0 0.5 4.5 4.0 3.5 3.0 2.5 2.0 ..-- 0> 1.5 � - 1.0 CJ) 0.5 CJ) Q) c E 4.5 � iI 4.0 3.5 3.0 2.5 2.0 1 .5 1 .0 0.5 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1 .0 0.5 (Controls) (34-80%) (75-90%) (1 00%) I!:il Inoculated rzl Uninoc. 0 1 week IiJ 4 weeks E:l 6 weeks 0 1 week 8J 4 weeks o 6 weeks IEl 1 week o 4 weeks III 6 weeks Months of coolstora e 1 64 Figure 7-5. 1 992: Mean (± SE) firmness of inocu lated kiwifruit stored at O°C after incubation at different relative humidities for up to six weeks. (Overall P < 0.001). 1 2 1 0 --. cf2. - 8 Cf) Cf) I- 6 4 2 1 4 Weeks of incubation 6 b Control un inoculated Control inocu lated Low r.h . (34-80%) Med. r.h . (75-90%) High r.h . ( 1 00%) Figure 7-6. 1 992: Total soluble solids of inoculated kiwifruit over a six week period at aoc and one of three relative humidities. Letters a & b refer to Duncan's test (P < 0. 05) . Vertical bars indicate SEM. TSS at harvest = 6.9%. ...... CJ) 01 ...-..-, -':= 1 2 ..-, 0> ...:::.:::: � 1 0 --- c 0 8 :.;:::. U :::J ""0 0 6 "- 0.. Q) c 4 Q) >. ..c .- 2 W 0 � Control uninoculated � Control inoculated � Low r.h . (34-80%) � Med. r.h . (75-90%) � High r.h . ( 1 00%) c c c 1 a ab 4 Weeks of incubation a b b 6 Figure 7-7. 1 992 : Ethylene production of inoculated kiwifru it over a six week period at O°C and one of three relative humidities. Letters a, b & c refer to Duncan's test (P < 0.05). Vertical bars indicate SEM. ... 0> 0> --.. T""" 35 I.e C> � 30 ('t) E u -...- C 25 o +=i U :::J 20 ""0 e c... 1 5 C\.I o () 1 bc 4 1m Control uninoculated � Control inoculated � Low r.h . (34-80%) IZI Med. r.h . (75-90%) � High r.h . ( 1 00%) b ab 6 Weeks of i ncubation c Figure 7-8. 1992: Rate of respiration of inoculated kiwifruit over a six week period at OOG and one of three relative humidities. Letters a, b & c refer to Duncan's test (P < 0.05) . Vertical bars indicate SEM. Chapter Seven 1 68 cinerea infection while fruit from the inoculated control increased this to over 40%. The general pattern was for a reduction in disease incidence with increasing treatment time for all relative humidities. Compared with the control fruit, the lowest infection levels were recorded from the medium (one week) and lowest relative humidity (fourth and sixth weeks) (Fig.7-9) . Experiment No.2 Relative humidity behaviour Except for the medium relative humidity during the third day of treatment at the first harvest and the lowest relative humidity during the first day of treatment at the second harvest, the input/output ranges of each relative humidity range obtained for each harvest date remained constant throughout the seven days treatment. The lowest relative humidity from the first harvest date (Fig. 7-1 0) varied from an input of 40 to 60%, that of the second maturity (Fig.7-1 1 ) from 42 to 57% and that of the third maturity (Fig.7- 12) from 41 to 50%. The medium input relative humidity from the first harvest was slightly different (63%) compared with the remainder two harvests (65%) and the output varied from 79% (second harvest) to 81% (first and third harvest). The highest relative humidity input varied from 90% (first harvest), 94% (second harvest) and 93% (third harvest) , with an output simi lar for all harvests of 97%. Fruit quality after initial relative humidity coo/storage period At the first harvest (May 1 2th) , the weight loss of fruit incubated at the lowest relative humidity was significantly greater than that of fruit from the medium humidity which in turn was significantly greater (P < 0.05) than that in fruit incubated at the high relative humidity (Fig .7-1 3) . This pattern of weight loss was repeated at both the second (May 27th) and third (June 1 0th) harvests although the differences between weight loss at the low and medium relative humidity were not significant at the third harvest. 90 80 c o 70 � u 2 60 c � 50 CO ...... c 40 Q) � Q) 30 0.- 20 1 0 Controls 1 � Control uninoculated ImI Control inoculated � Low r.h . (34-80%) � Med . r.h . (75-90%) � High r.h . ( 1 00%) 4 Weeks of i ncubation 6 Figure 7-9. 1 992 : Mean percentage infection of inoculated kiwifruit over a six week period at ooe and one of three relative humidities after 1 2 weeks coolstorage. . Chapter Seven 1 00 80 60 40 20 ...- � 0 --- >. 100 ..... "'0 E 80 :::J .c Q) 60 > ..... CO 40 Q) a: 20 100 80 60 40 20 • - Input • - _ . Output �-----�--�--�--�--� ----. ..---­�.-�!----.-�.�---. Low r.h . (40-60%) Med. r.h. (63-81%) �--�--�--�--�--�--� . . . . ------. . . High r.h . (90-97%) 2 3 4 5 6 7 Days of treatment 1 70 Figure 7-1 0. 1 993: Input and output relative humidity for fruit from the first harvest over a seven day period at ooe. Chapter Seven 100 80 60 40 20 - � 0 --- >, 100 ....... "0 E 80 ::J .!: Q) 60 > :;::; a:s 40 Q) II 20 100 80 60 40 20 • - Input • -- . Output r//�-----�-�-�--' . . . . . ... Low r.h . (42-57%) .... ...------...---..--- ---t--- �.--__ .--�I� __ !�---�t----.--�4 Med. r.h . (65-79%) -------------------....... I • • • • • High r.h . (94-97%) 2 3 4 5 6 7 Days of treatment 1 71 Figure 7-1 1 . 1 993: Input and output relative humidity for fruit from the second harvest over a seven day period at DOC. Chapter Seven 1 00 80 60 40 20 - � 0 -- >, 100 - "0 E 80 ::l .s:::. CD 60 > - CO 40 Q) a: 20 100 80 60 40 20 • - I nput • --. Output ------------------- • • I • • Low r.h . (41 -59%) �--�--�--�--�-----� I • • • - Med. r.h . (65-81 %) ------�-----------­...... . . . . . High r.h . (93-97%) 2 3 4 5 6 7 Days of treatment 1 72 Figure 7-1 2. 1 993: Input and output relative humidity for fruit from the third harvest over a seven day period at O°C. 1 .8 en 1 .6 a en 0 1 .4 ....... ..c .0> 1 .2 Q) 3: Q) 1 .0 0> CO C 0.8 b Q) i��� u 0.6 ..... Q) 2 � �« 0... 0.4 2 I� % /-%% 0.2 //- � � % May 1 2 c T a c May 27 Harvest date � Low r.h . (40-59%) E8 Med. r.h . (65-80%) I8B High r.h . (92-97) a a I� I� I� I� % �:� K� I % �id �< -;;;:.;: %..-; � % % % b � % � June 1 0 Figure 7-1 3. 1 993: Percentage weight loss of inocu lated kiwifruit incubated for seven days at DOG and one of three relative humidities. Letters a, b & c refer to Duncan's test (P < 0.05). Vertial bars indicate SEM. 1 4 1 2 .:;::::-- 1 0 t::1) � ---- en 8 (J) Q) c E 6 � iL 4 2 0 a ab May 1 2 In i tial f ir. 9 .0 b a b May 27 In itial f i r. 9.5 b Harvest date 1Zl Low r. h . (40-59%) o Med. r.h . (65-80%) � High r.h . (92-97%) June 1 0 In it ial fir. 6 .8 b Figure 7-1 4. 1 993: Fi rmness of inoculated kiwifru it incubated for seven days at aoc and one of th ree relative humidities. Letters a & b refer to Duncan's test (P < 0.05) . Vertical bars indicate SEM. .....- � � en en r- 1 2 1 0 8 6 4 2 � Low r.h . (40-59%) IZ] Med. r.h. (65-80%) � High r.h . (92-97%) a T a :+�:��i���:r.�·r-.l. llmi� ��:�:·:���2 ��;�-: �m�l · :�:�:��.:.;��::':�� . ;:��2::::·0::;��;·: �� ;���� . ��&���$t�� / . May 27 TSS 1 1 .2 Harvest date T a June 1 0 TSS 1 2.8 b Figure 7-1 5. 1 993: Total soluble solids of inocu lated kiwifruit incubated for seven days at DoC and one of three relative humidities. Letters a & b refer to Duncan's test (P < 0.05) . Vertical bars indicate SEM. 1 00 c 80 o ...... u Q) '+-C 60 m C'> ctj ...... c 40 Q) u � Q) a... 20 a May 1 2 a Control inocu lated. 8ill Low r.h . (40-59%) � Med. r.h . (65-80%) � High r.h . (92-97%) a a May 27 Harvest date a b June 1 0 Figure 7-1 6. 1 993: Percentage infection of inoculated kiwifruit incubated for seven days at aoc and one of three relative humidities after six weeks coolstorage. Letters a & b refer to Duncan's test (P < 0. 05). Vertical bars indicate SEM. 1 00 c 80 o += u Q) -- c 60 W 0> ro +-' C 40 Q) u � Q) a.... 20 a May 1 2 b Control inocu lated D Low r.h . (40-59%) (?LJ Med. r.h . (65-80%) � High r.h . (92-97%) a a May 27 Harvest date a b June 1 0 Figure 7-1 7. 1 993: Percentage infection of inoculated kiwifruit incubated for seven days at O°C and one of three relative humidities after 1 2 weeks coolstorage. Letters a & b refer to Duncan's test (P < 0. 05). Vertical bars indicate SEM. Chapter Seven 1 78 Fruit firmness at the first and third harvests was significantly greater (P < 0.05) in the high humidity treatment than in the low relative humidity. The contrary was found at the second harvest where the low firmness was found at the h ighest relative humidity (Fig.7-1 4) . Brix levels (TSS) were not significantly affected by treatment except at the third harvest where TSS in fruit from the medium and h igh relative hum idity treatments were significantly lower (P < 0.05) than fruit from the lowest relative humidity. The TSS increasHd with each successive harvest (Fig .7- 1 5) . Infection levels during coolstorage The percentage infection of fruit was similar after six and twelve weeks coolstorage although there were more infections in total after twelve weeks, particularly at the second harvest. Fruit from the first harvest incubated in h igh humidity had fewer infections after six weeks coolstorage than fruit incubated in the low relative humidity (Fig.7- 1 6) . By twelve weeks these differences significantly increased (P < 0.05). A simi lar pattern emerged at the second and thi rd harvests although the differences were not significant at 6 or 1 2 weeks after the second harvest but were significant at both six and 1 2 weeks for the third harvest (Fig.7-1 7) . DISCUSSION Fruit quality and physiological changes during incubation time In this present study, weight loss from the first experiment and the third harvest from the second was not significantly different between the medium and low relative humidity range:s. In general, moisture loss measured during incubation of both experiments increased with decreased relative humidity and with storage timH. A simi lar tendency among weight loss/relative humidity/storage time has been shown for various vegetable Chapter Seven 1 79 and fruit commodities. In vegetables such as carrots cv. Chantenay and Nantes, rutabagas, brussels sprouts, celery, chinese cabbage, leeks and to some extent parsnips, weight loss dE�creased when ambient storage relative humidity was almost saturated (98-1 00%) at 0 to 3°C for a period of several months. In these studies the low relative humidity was within the range 90- 95% and a higher moisture loss wa.s recorded in those vegetables held at this relative humidity compared with the saturated atmosphere (Van den Berg & Lentz 1 966; 1 974; 1 978) . Although in other studies a relative humidity range was not given, a similar trend in weight loss to that in the present study was observed. For example, in apples and pears cv. Bramley's seedling and Beurre Clairegeau respectively, water loss measured as a rate of evaporation (at 3°C for a period of up to five for apples and two months for pears) , was h igher in fruit held at relative humidities of approximately 75% compared to those fruit held at 95% relative humidity. In both commodities evaporation tended to increase with storage time (Smith 1 933). Similarly in grapes stored at ooe and relative humidities of 95, 90 and 85% for six and 1 3 days incubation, weight loss from the lowest relative humidity was almost three times greater at the end of the fi rst incubation period and three and a half times greater after the second incubation period compared with that at the highest relative humidity (Allen & Pentzer 1 935). I n the first experiment of this present study, fruit firmness decreased with incubation time for both controls. Regardless of the relative humidity in which fruit were incubated firmness greatly decreased between the first incubation period (one week) and the second one (four weeks) . Fruit firmness after four or six weeks of incubation at any relative humidity was reduced within the ranges of 4.7-2.6 kgf and 2.2-2.8 kgf respectively, remaining at these levels during the normal coolstorage period of 1 , 3 or 6 months. Reports from commercial coolstores showed that firmness decreased, about 2.5 kgf after Elight weeks of normal coolstorage with a further reduction to 0.5-1 .0 kgf at the end of the storage period (Reid & Chapter Seven 1 80 Harris 1 9n; Nicolas et al. 1 988; McDonald 1 990). For the second experiment, fruit firmness after the seven days incubation period for all relative humidities was maintained in levels similar to those recorded in fruit normally coolstoraged at O°C (McDonald 1 990) . Commercial storage of kiwifruit is recommended at a relative humidity between 90-95% for bin-stored fruit (Sale 1 990) , 80% (Reid & Harris 1 977; Sale 1 990) or 90-95% (McDonald 1 990) for packed fruit to avoid h igh moisture loss and consequent reduction in firmness. In this present study, differences in fruit firmness according to relative humidity were more evident at the second experiment than the first one. For example, at the second experiment firmness in fruit incubated at the medium (60-80%, 0.80-0. 1 2 VPD) and high (92-97%, 0.04-0.0'1 VPD) relative humidity ranges was significantly higher than that of fru it coolstored at the lowest relative humidity range (40-59%, 0.35-0.24 VPD). In other studies (Whitelock et al. 1 994) fruit were not incubated at QOC, but a simi lar pattem of results was reported on four different cultivars of peaches incubated at temperatures and relative humidities of 6 ± 1 °C, 97%, 5.6 ± 0. 1 °C, 88% and 4.3°C, 75% respectively and two different air flow rates (0.7 to 4.0 and 0.2 to 1 .5 m.s·1 ) . In that study firmness was reported to be reduced with the lowest relative humidity at both air flows and overal l firmness increased as VPD decreased. In this present study, fruit firmness of kiwifruit decreased with an increase in harvest maturity at the second e)(periment. Similar findings in kiwifruit and in other commodities such as apples, peaches and mangoes have been reported. In those studies of kiwifruit firmness decreased with harvest date (Crisosto et al. 1 984) and with storage time (Kempler et al. 1 992) . Further studies of kiwifruit firmness stored in temperature ranges of -0.5 to 2.5 for 1 0, 20 or 24 weeks showed a silmi lar pattern of firmness decrease with storage time (Lallu et al. 1 992) . Fteports on peaches harvested at various dates, showed a firmness decrease with increase in maturity (Whitelock et Chapter Seven 1 81 al. 1 994} . Similarly in other studies, although the storage temperature was not specified , in apple cultivars such as Golden delicious and Pink lady, it was reported that storage time had a significant effect on firmness. Mango harvested at three different maturity stages (different harvest dates) and stored at 1 2°C for 0, 1 4 and 21 days showed firmness reduction with storage time and with maturity stage for all harvest dates (Seymour et al. 1 990) . In general , in both experiments, thl3re was no effect of relative humidities on the TSS. Changes in brix levels during the incubation time and at each harvest date can be explained as the normal increase in maturity. Results of this present study, agreed with numerous investigations carried out not only with kiwifruit but with other fruit crops. A simi lar pattern of TSS increase with harvest and storagEl period has been reported in different cu ltivars of mangoes (Seymour et al. 1 990) and apples (Chvyl & Tugwell ( 1 993) . Crisosto et al. ( 1 984); Lawes & Sawanobori ( 1 984) ; N icolas et al. ( 1 988) ; Lallu ( 1 989b) ; Lal lu et cll. ( 1 989) and MacRae et al. ( 1 989b) reported an increase in TSS of kiwi'fruit ( 1 5% approximately) with harvesting season and 4-1 2 weeks coolstorage period. However it is important to mention that high levels of TSS were achieved at the second and third harvest (second experiment) after seven day incubation period compared with those levels recorded in fruit from the first experiment after a simi lar incubation time, a difference that was apparent in the values obtained from fruit at harvest. I n this study, differences among TSS in fruit from the first experiment at harvest (9.9%) compared with those obtained after the fi rst week of incubation could be explained by the variation in TSS concentrations in kiwifruit at harvest such as canopy density, position of the fruit within vine etc . . reported by Hopkirk et al. ( 1 986). No specific pattern in ethylenl3 production was observed in relative Chapter Seven 1 82 humid ity/incubation time combinat ions. In this study fruit from the uninoculated control at any incubation period and fruit from the highest and lowest relative humidity after a one or six weeks incubation period respectively did not produce ethylEme. Studies on uninoculated kiwifruit stored at O°C showed no autocatalytic ethylene production (Sfakiotakis et al. 1 989) . Similar findings were 1'ound in carnation flowers (Dianthus caryophyl/us) stored at 2°C for up to five days. In that study ethylene production completely ceased after the third and fourth days storage, but after the fifth days there was an increase in ethylene production (Field & Barrowclough 1 988) . Differences in ethylene production in this present study can be explained by the markHd differences during ripening of kiwifruit i .e . ethylene production among individual fruits already found by Pratt & Reid ( 1 974) and Hasegawa & Van!) ( 1 990) . Fruit from the remainder of the inoculated controls and the other treatments produced h igher ethylene levels than those considered as trace amounts (0. 1 J.l1) (Pratt & Reid 1 974) . It has been widely reported that plants infected by pathogens such as bacteria olr fungi can hasten ethylene production (Archer 1 976; Archer & Hislop 1 9?5; E lad 1 990; Boller 1 991 ) . Similarly in in vitro studies, l Iag & Curtis ( 1 968) , EI-Kazzaz et al. ( 1 983b) and Kepczynska (1 989) found that conidia of B. cinerea and 22 other fungi species produced ethylene. Kiwifruit inoculated with B. cinerea and stored at O°C for more than three montils, produced high amounts of ethylene compared with uninoculated fruit which produced a very low amount of ethylene after two months storage' (Niklis et al. 1 993). In other studies with kiwifruit, a close relation was found between soft rot extension caused by Botryosphaeria and Phomopsis spp. and ethylene production (Hasegawa & Yano 1 990) . Although in other studies the store temperatures were above O°C, a similar effect induced by other pathogens was observed. In tomatoes inoculated with Phytophthora infE�stans ethylene production increased 36- 40h after inoculation coinciding with visual symptoms of infection and disease and compared with small amounts of ethylene in uninoculated Chapter Seven 1 83 tomatoes (Spanu & Bol ler 1 988) . In oranges var. Valencia, incubated at 30°C, 94-96 relative humidity for four weeks, an increase in susceptibil ity to Ditiorella natalensis was reportEld with an increase in exogenous appl ications of ethylene. Doses of 0, 1 , 1 0, or 50 JlIII for 96 h increased infection about 4. 24, 47 or 65% respectively (EI-Kazzaz et al. 1 983a; Barmore 1 985) . In this study, rate of respiration did not show a specific pattem according to relative humidity and incubation time. Only fruit from the inoculated control, and the high relative humidnty showed a peak of respiration after the second incubation period declining after the third period of incubation . Fruit from the lowest relative humidity increases their respiration rate for all incubation times. Similarly, in othl�r studies, carried out at 20°C, kiwifruit showed the typical respiratory pattHm around five weeks after harvest (Pratt & Reid 1 974) . In this present stucly CO2 values after harvest were h igher than those reported by McDonald (1 990). Although further details of treatments were not given, CO2 production of kiwifruit was about 1 .3 ml ·kg 11 at O°C. Similar low CO2 values for kiwifruit respi ration were reported by Wright & Heatherbell ( 1 967) who found that respiration of kiwifruit stored at O°C was 2.0 ml .'kg ·h after six weeks. According to Kader (1 992), kiwifruit stored at 5°C is classified as a fruit with a low respiration rate (5-1 0 mg CO/kg·h) . Convers1ely, Turk ( 1 989) studied half mature and immature figs stored at 1 °C, 900/b relative humidity and found a range of initial respiration rates between 39-27 ml CO!kg ·h at harvest, with a cl imacteric peak after about two or four weeks storage for half mature and mature figs respectively. In 01her studies carried out on vegetable commodities such as cabbage, carrots, celery and others, the rate of respi ration was measured on the basis of respiratory heat production (010) (rates of heat production calculated from steady state rates of CO2 production) at 0, 5 or 1 6°C. It was found that the heat of respiration was dependant on temperature, storage time and to a lesser extent cultivar. In that study lower 010 values were reported in commodities incubated at O°C Chapter Seven 1 84 for longer periods (4-6 weeks) compared with higher temperatures of 5 or 1 6°C (Van den Berg & Lentz 1 972) . An explanation for the undefined pattern of CO2 production during storage in some treatments of this present study can be related to individual differences in the ripening process already explained by Pratt and Reid ( 1 974) . In this present study, ethylene production was not paralleled by increased flesh softening, the rise of respirat ion rate and soluble sol ids content as reported in other investigations in kiwifruit (Hyodo & Fukasawa 1 985; Cheng et al. 1 994). Contrary to other reports, the synergistic effect of rate of respiration versus ethylene production were not observed in this present study (Sfakiotakis et al. 1 989) . Likewise, related effects of relative humid ity, water loss and ripening were not apparent in this study, as reported for other commodities (Gac 1 956; Vaa.dia et al. 1 961 ; Littmann 1 972). In those studies although the storage time was not specified it was reported that weight loss hastened the ripening process i.e. the green l ife of pears were reduced five days when stored at !;OC, 57-86% relative humidity and four to ten days in avocados, bananas and pears when stored at 20°C, 1 3% relative humidity. Infection levels during coolstoragl3 In the second experiment there was a more marked increase of infection levels with a low relative humidity. An analogous tendency was observed in the fi rst experiment when fruit were incubated for one week. Similar findings have also been reported by Goodl iffe & Heale ( 1 977) who inoculated carrots with B. cinerea before incubation in a range of humidities at 5°C. They suggested that the abil ity of the secondary phloem parenchyma of the root to resist invasion by this pathogen was markedly reduced when the weight loss of carrots in storage was between 5 and 1 0%. Carrots kept at lower humid ities lose weight more quickly and become susceptible to Botrytis attack sooner. The kiwifruit stem scar is wound tissue and not strictly comparable with the secondary phloem parenchyma of the Chapter Seven 1 85 carrot root but the principle of the conditions being more favourable to the host defense mechanism than to the pathogen stil l apply. Thus at a low relative humidity the stem scar tissue could desiccate and die before active defense mechanisms are established thereby providing dead tissue for colonisation by necrotrophic patho!�ens such as B. cinerea in addition to weakening or inactivating the host defense. This argument is supported by the work of Van den Berg & Lentz ( ·1 974) who observed that decay in some vegetables was reduced when stored at 0-1 °C at a relative humidity between 98-1 00% than when stored at a relative humidity of 90-95%. In the current study, infection levels from both experiments at the highest relative humidities (1 00% and 92-97%) were reduced compared with those at the lower relative humidities. Conversely, Sharky & Peggie (1 984) studied the influence of three relative humidity ranges on storage decay of cherries, lemons and peaches and found that in the first crop, percentage decay in fruit held at 90-94% and 95- 99% relative humidities was simi lar. However, infections in lemons stored at 95-99% were fewer compared! with those at other relative humidities while storage rots were low in peaches stored at any relative humidity range. In this study, at the second expe!riment ( 1 992) , percentage infection also varied according to harvest maturity. High infection levels were recorded in fruit from the third harvest. On the other hand, infection levels in fruit from the inoculated control from the second experiment were lower compared with those infection levels recorded from the inoculated controls of the first year experiment. In epidemioloQ)f studies of B. cinerea in kiwifruit patterns of infection levels developing in coolstorage varied on a day-to-day basis of harvest and also year-to-year (Brook 1 990a) . He related these daily and seasonal variations to weather conditions to differences in the overall population of B. cinerea in the orchard and to numbers of mobile spores at the time of harvest. Brook ( 1 990a) also reviewed the contradictions in Chapter Seven 1 86 evidence for the concept that kiwifru it stem-end rot susceptibil ity is reduced with kiwifruit maturity. Data reported by others (Hopkirk et al. 1 990a; 1 990b) showed that increase in harvest date reduced B. cinerea rots from 1 4.9 to 1 .3%. CHAPTER EIGHT GENERAL DISCUSSION AND FUTURE RESEARCH Kiwifruit are one of New Zealand I s major export crops and any constraints on production and export of h igh quality produce will have an adverse effect not only on the kiwifruit industry but on New Zealand as a whole. The New Zealand Kiwifruit Marketing Board is the sole New Zealand exporter of this commodity worldwide and has suffered severe economic losses from the Botrytis storage rot problem. These losses involve a direct loss from diseased fruit during coolstorage plus a loss of other fruit which have softened because of the ethylene produced by diseased fruit, cost of transporting diseased fruit overseas and the high cost of manual inspection and repackaging of fruit from infected l ines. As recently as 1 994 there were several reports highlighting the high levels of · Botrytis infections observed in fruit sold to overseas markets (Anonymous 1 994b; 1 994c; Tapper 1 994) . Of greatest concern is the perception of New Zealand kiwifruit by our major markets. The New Zealand Kiwifruit Marketing Board makes great efforts to ensure that New Zealand fruit is of top quality and heavi ly promotes a top qual ity image in its advertising campaigns. The Botrytis storage rot problem has dented this image as evidenced by reference to it in some European countries such as Germany, Switzerland and Austria as the New Zealand disease (Tapper 1 994) . Before commencing a study of the effect of humidity and temperature on infection of kiwifruit by B. cinerea it was important to know more about the variables which could affect the results and to develop a standardised procedure for inoculation. Work. by Long and Wurms (1 993) had already established that high inoculum Ileveis were necessary to give a reasonable incidence of infection for exp13rimental work. Other workers (Yoder & Whalen 1 975) have found tha't at high concentrations the conidia of B. Chapter Eight 1 88 cinerea have a low percentage ge rmination. However, this self-inhibition found by those workers was not manifest in terms of percentage infection of kiwifruit in the work of Long and Wurms (1 993). Disease incidence of B. fabae on beans has been shown to be proportional to inoculum concentration (Last & Hamley 1 9513) and aggressiveness as measured by lesion size has been shown to be influenced by spore concentration of B. cinerea (Louis 1 963) , B. allii (Segall & Newhall 1 960) and B. tu/ipae (Price 1 970). A range of inoculum levels were used in this work to ensure that a reasonable number of infections were obtained without overwhelming the host defenses. Long & Wurms (1 993) found that infection levels of B. cinerea on kiwifruit could be increased by adding nutrients to the spore suspension - a phenomenon already wel l documented for B. cinerea infections of other crops (Chu-Chou & Preece 1 968; Fokkema 1 971 ; Kh61 & Fokkema 1 994) . This was not attempted here as the aim was to achieve a natural type of inoculum as far as possible. However there remains the question of whether the growing medium affects the nutrient status of the spores. The age of B. cinerea spores is known to affect both germination rate and infectivity, an effect that can be modified by nutrition, hence the in itial trials focused on the question of substrate and colony age on which the inoculum was produced. B. cinerea is a weak, facultative parasite which colonises wounds and dead tissues of a wide range of host plants. Despite this necrotrophic growth habit Will iamson & Hargreaves ( 1981 ) and Wil l iamson & Jennings ( 1 986) have found that there is some host specialisation on raspberries. The contribution of inoculum from neighbouring fields or orchards of other crops such as grapes is not known but there are definite differences in percentage infection of kiwifruit by B. cinerea between orchards in different regions, within orchards and with fruit maturity at harvest (Hopkirk et al. 1 990a; Manning & Pak 1 993; Pyke et al. 1 993). Such d ifferences were found in the Chapter Eight 1 89 present work where disease incidence of fruit harvested at Levin was greater than that of fruit at Palmerston North (Chapter 3). Similarly, conidia produced on autoclaved kiwifruit leaves caused more infections at Levin while those produced on MA caused more infections at Palmerston North. However, there was no evidence of any host specialisation of isolates obtained from kiwifruit compared w ith those from other crops. At the conclusion of this first set of trials it was decided to use the K3 Massey University isolate since there was no apparent host special isation, this isolate was known to be viru lEmt on kiwifruit and i t had already been used extensively on kiwifruit trials here. Inoculum was prepared from colonies grown on MA for 1 0- 14 days since MA was more consistent and easier to prepare than autoclaved It 92% relative humidity �vJ provides a basis for larger scale, semi-commercial applications which should give greater confidence to the kiwifruit industry in the curing process. REFERENCES Adams, M . J . and R. L. Griffith. 1 978. "The effect of harvest date and duration of wound healing conditions on the susceptibi l ity of damaged potato tubers to infection by Phoma exigua (gangrene). " Annals of Applied Biology 88:51 -55. Agrios, G . N. 1 988. Plant Pathology. 3rd. Ed. USA: Academic Press Inc. Pp. 703. Alderman, S. C. and M. L. Lacy. 1 983. 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USA: Springer-Verlag. Pp 1 43. APPENDIX Tissue Dehydration Procedure: (Feder & O'Brien 1 968) 1 .- Remove excess fixative in running water. 2 . - Transfer the fixed tissue to methyl cellosolve at about O°C. 3 . - Two changes of methyl cellosolve (24h ea.) . 4 .- Transfer the tissue to 1 00% ethanol at O°C (24h) . 5.- Transfer the tissue to xylene at O°C (24h) . Embedding Procedure: (Bautista-Banos 1 989) 1 .- Transfer dehydrated tissue to a mixture of xylene and paraffin in the following order: 75:25 (24h) , 50:50 (1 2h) and paraffin (24h) . 2 . - Vacuum infiltrate overnight in Paraplast. 3.- Place infiltrated tissue in embedding moulds and fi l l with paraffin. 4.- Trim and section the tissue. 5 . - Place sectioned tissue on glass slides. 6.- Keep microscope slides in oven at 35°C for 24 h . Removal of paraffin from the tissue: (Bautista-Banos 1 989) 1 .- Place sl ides in two changes of xylene (5 min. ea. ) . 2 . - Transfer to another solution of equal parts of xylene and absolute alcohol (5 min.) . 3 .- Transfer trough an ethanol series and xylene (1 00, 95, 75 , 50 and 25%; 5 min. ea.) . 4 .- Transfer to distilled water. 5 . - Stain. Staining procedures: a) Safranin-Fast Green: (Bautista-Banos 1 989) Appendix 1 .- Stain with safranin "0" (5 min.) . 2.- Wash with distilled water. 243 3.- Transfer through an ethanol series: 25, 50, 75, 95 & 1 00% (5 min ea.) . 4.- If overstained, transfer to acidulated alcohol (20 sec.) . 5 .- Stain with Fast-Green (20 sec.) . 6.- Transfer to xylene (Two changes: 4 min ea.) . 8.- Mount in D .P.X. and add coverslip. b) Differential staining for fungus and host: (Johansen 1 940) Stain the sl ides for 2 minutes in 1 % methyl violet in 50% ethyl alcohol, wash in 50% alcohol and stain for 45 minutes in 1 % eosin . Transfer to two changes of xylene (5 min. ea.) , mount in D.P.X. and add coverslip. c) Glycerine-ferricyadine: (Sherwood and Vance 1 976) Place the tissue in glycerine solution (5 min.) , rinse with water, dry and add one drop of 0.02 M FeCI3 solution and one drop of 0.02 M potassium ferricyanide solution. Preparation of solvents and stains: Formalin-Aceta-Alcohol (FAA): (Johansen 1 940) Absolute alcohol 500 ml Acid Glacial Acetic 50 ml Formaldehyde 1 00 ml Disti l led water 350 ml Safranin "0": (Johansen 1 940) Dissolve 4g of safranin in 200 ml Methyl Cellosolve. Add 1 00ml each of 95% alcohol and distil led water. Appendix Fast-Green: (Bautista-Banos 1 989) Saturated solution A Fast Green 1 part Alcohol absolute 1 part Methyl Cellosolve 1 part Solution B Alcohol Absolute 25 parts Clove oil 75 parts Mix solution A and B (1 : 1 ) Sudan IV: (Johansen 1 940) 244 Saturated alcoholic solution. Stain for about 1 0 minutes and wash in alcohol Sudan Black B: (Peacock & Bradbury 1 973) Sudan Black B Alcohol (70%) Reflux for 20 minutes cool and filter 5 g 1 00 ml Phloroglucinol - Hel: (Peacock & Bradbury 1 973) Phloroglucinol Alcohol (75%) HCL (concentrated) 5 g 1 00 ml 1 drop Toluidine Blue "0": (Peacock & Bradbury 1 973) Toluidine Blue Alcohol (70%) HCI (concentrated) 0.25 9 1 00 ml 0.5 ml PUBLICATIONS ABSTRACTS 6th International Congress of Plant Pathology Palais des Congres de Montreal Montreal, Canada July 28 - August 6, 1 993 Organized with the support of: Canadian Phytopathological Society National Research Counci l Canada RESUMES 6e Congres i nternational de phytopathologie Palais des Congres de Montreal Montreal (Quebec) Canada du 28 ju i l let au 6 aoOt 1993 Organise en collaboration avec: La Societe canadienne de phytopathologie Le Conseil national de recherches Canada Etiology and Control of Post· harvest Diseases 14.3.6 EiTIOLOGY OF PREHARVEST INFECTIONS OF CHERRY FRUITS . .E..M. Dugan and R . G . Roberts . USDA ARS Tree Fruit Research Lab , Wenatchee , washington . Samp les of 100 symptomless Bing cherry fruits were collected from each of J orchards for 10-11 weeks after petal fal l . Fruits were surface-dis infested and incubated . By the second week more than 10% of fruits contained infections ; at harvest 90-100% of fruits contained one or more species of fung i . Most infected fruits yielded species of Cladosporium, Al ternaria and/or Aureobasidium . Most infections originated at stylar or receptacular scars . Tests with representative isolates demonstrated pathogenicity to cherry fruits . Fungi in approximately 50 other genera were isolated , including Penicillium, Ulocladium, Botrytis, Aspergill us, Phoma , Stemphylium, Geotrichum and Arthrinium . 1 4.3.8 KIWIFRUIT PICKlNG SCAR TREATMENT AND DEVELOPMENT OF BOTRITIS CINEREA INFECTIONS DURING COOL STORAGE. 'K.V.wurms. 'P.G.Long. 'N.Pyke. 'G.Tale. 'S.Ganeshanandam. 'Depanment of Plant Science. 'Depanmenl of Statistics. Massey University. PalmerslOn North. 'HortResean:h. Riwaka R=h C�ntre. 'HortResean:h. Hawkes Bay Resean:h Centre. New a:uand. nje influence of age and condition of kiwifruit picking scars on susceptibility lO in�ection by B. cillu