South African ‘Eureka’ lemon fruit must be exposed to chilling temperatures (± 0.6°C) as a mandatory quarantine treatment against insect pests for all its overseas markets. Chilling lemon fruit at such temperatures may develop chilling injury (CI) symptoms on the flavedo. This negative effect on fruit quality reduces fruit marketability. This study evaluated postharvest factors influencing physiological, biochemical and ultra-structural mechanisms involved in alleviating CI in lemon fruit. It was hypothesised that treatment with methyl jasmonate (MJ) and salicylic acid (SA) may enhance chilling tolerance in lemon fruit by maintaining cellular integrity and inducing synthesis of enzymatic and non-enzymatic antioxidants. Furthermore, fruit susceptibility to CI was associated with the source of fruit. Lemon fruit were harvested from three locations representative of moderate subtropical, warm temperate and cool subtropical environments. Harvested fruit were treated either with 10 μM MJ, 2 mM SA or 10 μM MJ plus 2 mM SA, stored either at -0.5, 2 or 4.5°C for 0, 7, 14, 21, or 28 days and afterwards transferred to 23°C for a week as shelf-life simulation. Thereafter, fruit were evaluated for alterations in physiological, biochemical and ultra-structural features involved in the manifestation of CI symptoms. Chilling damage was more severe in untreated lemon fruit than in treated lemon fruit. Storing lemon fruit at 4.5°C accelerated the manifestation of CI symptoms more so than at 2°C while storage at -0.5°C delayed the manifestation of CI symptoms. Lemon fruit of moderate subtropical origin were more chilling-tolerant than lemon fruit of warm temperate and cool subtropical origin. Treatment with 10 μM MJ plus 2 mM SA significantly (P < 0.05) improved chilling tolerance in lemon fruit. This treatment effectively maintained membrane integrity, thereby retarding electrolyte leakage and membrane lipid peroxidation as well as mass loss and respiration rate. Treatment with 10 μM MJ plus 2 mM SA was also effective in enhancing the antioxidant concentrations of vitamin E and carotenoids. The production of these antioxidants could have been part of a defence system against chilling damage, reducing CI and maintaining fruit quality. Treatment with 10 μM MJ plus 2 mM SA enhanced the concentration of compounds involved in chilling resistance, such as proline, soluble sugars, ascorbic acid and total phenolics as well as the enzyme phenylalanine ammonia-lyase (PAL). The enhancement of the defence mechanisms may have played a role in enhancing chilling tolerance in lemon fruit. The treatment also inhibited certain enzymes involved in tissue browning, such as peroxidase (POD) which might have contributed to delaying manifestation of symptoms. Polyphenol oxidase (PPO) was found to not be a good biochemical marker of the occurrence of CI. Treatment with 10 μM MJ plus 2 mM SA appeared to be able to enhance chilling tolerance in lemon fruit by maintaining the ultra-structure of the cuticle, cell wall integrity, cell membrane of parenchyma cells of the flavedo. This treatment also preserved the mineral nutrients of the flavedo (carbon, oxygen, phosphorus, potassium, calcium, magnesium, sulphur, sodium, silicon and aluminium) during cold storage. This could have played a role in protecting the fruit against chilling stress and maintaining fruit quality. Treatment with 10 μM MJ plus 2 mM SA reduced ROS production, while the activity of enzymatic antioxidants such as catalyse (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), and accumulation of essential proteins was enhanced. This increase in activity of enzymatic antioxidants and the presence of stress-responsive proteins in the lemon flavedo could have been directly involved in enhancing chilling tolerance. The CI symptoms were accompanied by an increase in membrane permeability, membrane lipid peroxidation as well as phospholipase D (PLD) and lipoxygenase (LOX) activity; however, treatment with 10 μM MJ plus 2 mM SA effectively reduced the membrane permeability, membrane lipid peroxidation, and PLD and LOX activity induced by the cold treatment. This could have contributed to the efficacy of 10 μM MJ plus 2 mM SA in inhibiting the manifestation of CI symptoms. Treatment with 10 μM MJ plus 2 mM SA enhanced flavedo total antioxidant capacity measured by ferric reducing ability of plasma; 2,2-diphenyl-1-picrylhydrazyl; 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) and the oxygen radical absorption capacity assays. The enhancement of antioxidant capacity in lemon flavedo could have contributed to the fruit’s chilling tolerance. Therefore, the effect of 10 μM MJ plus 2 mM SA treatment, enhancing chilling tolerance, may be attributed to its ability to enhance enzymatic and non-enzymatic antioxidants; activate essential proteins and mitigate the effect of ROS accumulation. With the use of 10 μM MJ plus 2 mM SA treatments, the South African citrus industry will be able to meet the quarantine temperature requirements for exportation of lemon fruit whilst reducing economic losses, depending on the preharvest conditions experienced by the fruit in each shipment. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

South Africa is the second largest exporter of citrus fruit in the world. There has recently emerged a strong demand for lemons in the world market due to their nutritional value, culinary and non-culinary uses. During exportation, fruit are subjected to low temperature (-0.5°C) for varying periods of time as an obligatory quarantine treatment. However, lemons are sensitive to low temperatures and easily develop chilling injury during this obligatory quarantine treatment. This has become a major limitation to the expansion of South Africa’s lemon industry. Postharvest treatments with methyl jasmonate (MJ) and / or salicylic acid (SA) have been successfully used in horticultural crops to reduce chilling injury. A similar treatment was applied to ‘Eureka’ lemons. During the 2008 harvest season, postharvest fruit were either dipped in 10 or 50 μM MJ or 2 or 2.5 mM SA solutions. A control or no dip treatment was also applied. Three replicates of 15 fruits per treatment were used. During the 2009 harvest season the following postharvest treatments were applied as dips: 10 μM MJ, 2 mM SA, 10 μM MJ & 2 mM SA, 1 μM MJ & 0.2 mM SA, or 0.1 μM MJ & 0.02 mM SA solutions. A control or no dip treatment was also applied. Three replicates of 15 fruits per treatment were used. Subsequently fruit were stored at -0.5ºC for 0, 7, 14, 21, 28, 35, and 42 days, before being transferred to room temperature (25°C) for 7 days where after chilling injury was rated. Treatments with 10 μM MJ and / or 2 mM SA reduced chilling injury symptoms in lemons harvested during the 2009 season. Although no visual symptoms of chilling injury were observed during the 2008 harvest season, treatments with 10 μM MJ and / or 2 mM SA reduced fruit mass loss, delayed the occurrence of stress symptoms such as lipid peroxidation and suppressed accumulation of ROS in the rind. Treatments with 10 μM MJ and / or 2 mM SA were more effective in inducing antioxidant capacity and other defence compounds such as phenolics, ascorbic acid, carbohydrates and chilling injury responses such as accumulation of proline in the rind. This may have increased the chilling tolerance of fruit during the cold storage. Therefore, this study revealed that MJ and SA have the potential to reduce and delay symptoms of chilling injury in lemons. This lead to the suggestion that both, MJ and SA dips should be further tested as treatments to mitigate chilling injury in lemons. Future studies should focus more on preventing the injury itself or preventing the primary event of chilling injury. This could probably reduce the chances of secondary events to take place. / Thesis (M.Sc.Agric.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

New emerging markets such as Japan and the United States require cold sterilisation of South African citrus fruit as a phytosanitary standard against fruit fly. However, citrus fruit are chilling susceptible, with lemons being the second-most chilling susceptible after grapefruit. Chilling injury is a physiological rind disorder; the occurrence of which is despite its prevalence in horticultural commodities, not well understood. Therefore, the aim of this study was to investigate physiological compounds regulating chilling susceptibility or resistance in citrus fruit, with special emphasis on lemons. Furthermore, the potential of hot water dips or “molybdenum soaks” to maintain a certain level of physiological compounds which determine manifestation of chilling injury symptoms in citrus fruit was investigated. Moreover, it was attempted to create an understanding of the order in which physiological compounds mitigate chilling injury. Lemon fruit from different farms known to be chilling susceptible or resistant were obtained during the 2007 and 2008 harvest season. Thereafter, fruit were treated by soaking for 30 min in 1μM NaMo04.2H20 solution followed by a 2 min HWD 47 or 53°C. Treated fruit were waxed, weighed and stored at -0.5°C for up to 28 days and sampled for chilling injury evaluation 7, 14, 21, or 28 days into cold storage. A second evaluation was carried out five days after withdrawal from cold storage to allow development of chilling injury symptoms as a shelf-life simulation. After the second evaluation fruit were peeled, peel freeze-dried, milled using mortar and pestle and stored at -21°C for further physiological analysis. Freeze-dried peel was analysed for soluble sugars (glucose, fructose, sucrose), vitamin C (ascorbic acid), vitamin E (α-tocopherol), β-carotene, polyamines (putrescine, spermine, spermidine), specific flavanones (naringin and hesperidin) using HPLC-UV-Vis detector and proline, total antioxidant assays (FRAP, ABTS, DPPH), total phenolics, total flavonoids, lipid peroxidation using spectrophotometry, as well as for the heat shock protein (HSP70) using electrophoresis and silver-staining. Chilling susceptibility of lemon fruit varied with fruit source; those sourced from Ukulinga and Eston Estates were chilling resistant, while fruit from Sun Valley Estates showed chilling injury symptoms after 28 days of cold storage plus five days shelf-life. Furthermore, hot water dips (HW) 53°C, 1 μM Molybdenum (Mo) and 10 μM Mo plus HW 53°C significantly reduced chilling injury symptoms compared with the control and HW 47°C. In addition, Sun Valley Estates fruit also showed higher fruit weight loss compared with non-chilling resistant lemons. The alignment of higher fruit weight loss during storage with chilling susceptibility ascertains the use of weight loss as a non-destructive parameter for chilling susceptibility. With respect to flavedo sugars, glucose was found to be the dominant soluble sugar with multi-functional roles during cold storage. This plays a significant role in mitigating cellular stress. Chilling susceptible lemons from Sun Valley Estates had low flavedo glucose concentrations and, therefore, little conversion of glucose to ascorbic acid was possible resulting in a low antioxidant capacity. However, treatments with HW 53°C and Mo soaks seemed to enhance the enzymatic conversion of glucose to ascorbic acid leading to a higher antioxidant capacity in the flavedo of such treated fruit. Furthermore, glucose also feeds into the pentose phosphate pathway which is coupled with the shikimate pathway synthesizing secondary metabolites, especially of the phenolics group. The decrease in glucose was aligned to the levels of total phenolics, but not to that of β-carotene, naringin and hesperidin through 28 days into cold storage period. Moreover, as glucose also feeds into shikimate pathway, simultaneously an increase in proline flavedo concentration was observed. Proline is an antioxidant synthesized from glutamate; as cellular glucose decreases so does the total antioxidant capacity during cold storage. Ascorbic acid is a dominant and potent antioxidant in lemon flavedo as proven with the FRAP, ABTS and DPPH assays. Chilling resistant fruit have significantly higher ascorbic acid conversion. Furthermore, ascorbic acid also acts to generate the α-tocopheroxy radical to further important membrane-bound antioxidant, vitamin E (α-tocopherol equivalent). Furthermore, the DPPH assay was found to be effective in quantifying total antioxidants in lemon flavedo since it detects both lipophilic and hydrophilic antioxidants compared with the ABTS and FRAP assays which are bias to the estimation of liphophilic or hydrophilic antioxidants, respectively. The hot water and molybdenum treatments increased total antioxidants (DPPH assay) with reduced lipid peroxidation 7 days into cold storage and therefore, reduced chilling symptoms in fruit from Sun Valley Estates. The capacity of antioxidant to scavenge reactive oxygen species (ROS) was increased during cold storage and membrane stability significantly improved. Furthermore, putrescine as low valency polyamine was reduced as such compound acted as precursor to the synthesis of the high valency polyamines, spermine and spermidine. Chilling susceptible lemons from Sun Valley Estates showed increased soluble-conjugated polyamines as a response to stress. Furthermore, HW 53°C, 1 μM Mo and 10 μM Mo plus HW 53°C significantly increased the protein concentration and, therefore, likely also the occurrence of proteins with 70kDa (as estimator of HSP70). Additionally, the concentration of conjugated high valency polyamines was also increased, resulting in reduced chilling injury symptoms. The effect of ROS has only been viewed as damaging, while recently their role has also been viewed as stress acclamatory signalling compounds when produced concentrations below critical damaging threshold. Therefore, hot water dips seems to signals synthesis of total protein which include HSPs which then act throughout cold stress to protect other protein and channel other damaged proteins towards proteolysis. While molybdenum increased ROS production below damaging critical threshold, with ROS signalling stress acclimation by further signalling production of bioactive compound with antioxidant properties. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.