Doctoral Degrees (Horticultural Science)

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The cascade of physiological events leading to chilling injury : the effect of post-harvest hot water and molybdenum applications to lemon (citrus limon) fruit.
(2012) Mathaba, Nhlanhla.; Bertling, Isa.; Bower, John Patrick.
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.
Factors affecting mesocarp discolouration severity in 'Pinkerton' avocados (Persea americana MILL.)
(2005) Van Rooyen, Zelda.; Bower, John Patrick.
The susceptibility of the 'Pinkerton' avocado cultivar to mesocarp discolouration, after storage, has seriously threatened its export from South Africa. This disorder has proven to be complex, requiring a better understanding of the fruit's physiology. The purposes of this study were to identify the role of pre- and postharvest factors, or their interactions, in the development of the problem. This was done by obtaining fruit from several production areas of varying mesocarp discolouration histories (referred to as "high", "medium" or "Iow risk" areas) during the 2000 and 2001 seasons. Fruit were stored at 8, 5.5 and 2°C for 30 days, as well as ambient (20°C). Evaluations of fruit quality were made before and after storage, as well as after softening. Once removed from storage the weight loss (during storage) was determined, and fruit firmness and carbon dioxide (C02) production rates monitored daily. It was found that temperatures below the recommended shipping temperature of 5.5°C, i.e. 2°C, produced the best internal fruit quality. This was supported by the membrane integrity studies that showed less membrane stability at the warmer storage temperature of 8°C. Furthermore, remained hard during storage and subsequently had an extended shelf life. Fruit origin was also found to play a major role in browning potential, with discolouration being consistently more severe in fruit from "high risk" areas and increasing in severity as the season progressed. The rate of CO2 production was found to follow a similar trend, with rates increasing as the season progressed, and also being slightly higher in fruit from "high risk" areas. The higher CO2 production rates were thought to be related to a decrease in membrane integrity as the season progressed. While storage temperature was not found to have a significant effect on the rate of CO2 production after storage, it did affect the time taken to reach the maximum rate, with fruit stored at 2°C taking longer. Biochemical analyses to determine the concentration of total phenolics and the activity of the enzyme polyphenol oxidase (PPO) also showed that the potential for browning was initiated by preharvest conditions. While no significant differences were found between growers with regards to total phenol concentrations, the PPO activity was found to be higher in fruit from poor quality areas, and subsequently browning potential was expected to be higher in these fruit. It was, however, found that the potential for browning could be reduced by storing fruit at 2°C, as this decreased the total phenolics concentration. This evidence further emphasized the idea that storage at 2°C could be highly advantageous. Fruit mineral analysis showed that certain key elements played a significant role in the severity of mesocarp discolouration, with excessive fruit nitrogen and decreasing copper and manganese concentrations appearing to play major roles. The high fruit nitrogen concentrations were suspected to reflect fruit grown on very vigorous trees, resulting in shoots competing with fruit for available reserves. It is suggested that 'Pinkerton' of a quality acceptable to the market, can be produced by manipulating source:sink relationships, particularly through decreasing the availability of nitrogen, followed by low temperature (24° C) shipping. Future work should concentrate on manipulation of source:sink relationships, to take account of both climatic conditions and leaf to fruit ratios. The evaluation of chlorophyll fluorescence as a tool for predicting mesocarp discolouration potential in 'Pinkerton' proved to be unsuccessful in this study and future studies may require modifications to the current technique. It is suspected that differences in chlorophyll content, for example, between fruit from different origins, will have to be taken into account when interpreting results. The success of using 2°C storage to improve the internal quality on 'Pinkerton' fruit prompted further studies, during 2004, to ensure that the development of external chilling injury would not decrease the marketability of the cultivar. Low temperature conditioning treatments, prior to storage, proved to be highly successful in reducing the development of external chilling injury, thus further improving fruit quality as a whole. Preconditioning treatments consisted of fruit that were kept at either 10°C, 15°C or 20°C for 1 or 2 days before being placed into storage for 30 days at 2°C or 5.5°C. All preconditioning treatments were compared to fruit that were placed directly into storage. The effect of fruit packaging on moisture loss (as determined by weight loss) and chilling injury was also investigated using unwaxed fruit, commercially waxed and unwaxed fruit individually sealed in micro-perforated polypropylene bags with an anti-mist coating on the inside (polybags). Holding 'Pinkerton' fruit, regardless of packaging treatment, at 10°C for 2 days prior to storage at 2°C or 5.5°C significantly decreased the severity of external chilling injury. The use of polybags during preconditioning and storage showed potential in further reducing the development of external chilling injury, although the higher incidence of fungal infections in these fruit needs to be addressed. The determination of proline concentrations in fruit exocarp tissue after storage was helpful in determining the level of stress experienced by fruit that were subjected to different packaging and preconditioning treatments. In this study waxed fruit subjected to 1 d preconditioning at 10°C, 15°C or 20°C or placed directly into storage at 2°C showed very high proline concentrations and also displayed more severe external chilling injury, despite unwaxed fruit losing more weight during these treatments. The role of moisture loss thus needs further investigation. The thickness and method of wax application was thought to play an important role in the higher external chilling injury ratings in this study as waxed fruit often developed chilling injury symptoms around the lenticels and it was suspected that either the lenticels were damaged by the brushes used to apply the wax or that the lenticels became clogged thus resulting in reduced gaseous exchange. Nevertheless, the success of low temperature conditioning in reducing external chilling injury, while maintaining sound internal quality, may enable storage temperatures to be dropped even further, thus enabling South Africa to export avocados to countries that require a cold disinfestation period prior to entry to eliminate quarantine pests (e.g. fruit fly).
Gladiolus scabridus - the road to conservation and commercialisation.
(2005) Campbell, Tracey Barbara.; Bower, John Patrick.; Van Niekerk, Renate.
There is at present a growing concern and awareness of the endangered status of many indigenous South African plants in the wild, a number of which have potential for commercial production. One such example is Gladiolus scabridus, a vulnerable species endemic to the mountains of northern KwaZulu-Natal and southern Swaziland. It has considerable potential due to its floral characteristics. However, little is known about its horticultural requirements. Thus propagation and cultural practices were investigated with the aim of both conservation and commercialisation of the species. The ideal conditions for G. scabridus seed germination were determined. The presence or absence of light had no significant effect. Optimum germination was achieved at 20 QC of both winged and dewinged seeds. Higher temperatures appear to have a negative effect on germination and none of the winged seeds germinated at 30 QC. Under ideal conditions, fresh seed showed significantly higher vigour and viability than stored seed although the germination of stored seed was hampered by a higher internal fungal content. Successful tissue culture protocol was established for G. scabridus axillary bud and cormel halve explants. During the initial shoot initiation and proliferation stages (stage I and 11), the significantly higher shoot numbers occurred in the absence of growth regulators in both explant types. However, shoots with 1.0 mg 1 -1 6-benzyl-amino-purine (BAP) and 0.5 mg 1 -1 1-naphthalene-acetic-acid (NAA) were healthier in appearance. Higher levels of 5.0 mg 1 -1 NAA inhibited shoot production and encouraged root development in cormel halve explants. During stage 11, axillary bud explants showed root and cormlet development. More roots were initiated without growth regulators, whilst 5.0 mg 1 -1 NAA resulted in significantly better cormlet development. Shoot and cormlet growth of cormel halve explants during stage II was not significantly affected by the presence or absence of NAA and BAP. Significantly more roots were produced with 5.0 mg 1 -1 NAA. During stage III (rooting), the presence of activated charcoal (AC) was essential for the initiation and development of roots in vitro. Root and cormlet development in all explant types was significantly affected by the interaction between the previous treatments from stages I and 11 and the new treatments. During stage IV (hardening off) , most explants died down in the hardening off media leaving resting cormlets. There was a significant interaction between 1.0 mg 1 -1 NAA and 0.3 % AC from stage III, resulting in significantly more cormlets in both axillary bud and cormel halve explants. There was successful cormlet growth after cold storage which is advantageous in reducing the need for acclimatization. G. scabridus corms were successfully forced out of their normal flowering period. Temperatures of 10,5.5 and 2 QC showed successful corm initiation although the corms need to be stored for longer at warmer temperatures. It is suggested that a treatment of 2 QC for 6 weeks is the optimal condition for forcing G. scabridus corms. Although originating from a stressful environment, G. scabridus appears to show capacities for improved growth under controlled conditions. Fertilizer applications enhanced growth and reduced the time to flowering. Nitrogen (N) was found to be important for vegetative growth, flowering and daughter corm development, whereas potassium (K) influenced cormel production. Fertilizer with higher N and lower K is appropriate for the beginning and middle of the growing season and then adjusted to a lower N and higher K fertilizer to promote cormel formation. G. scabridus was found to produce prolific numbers of cormels which is an important source of plant material. Mineral leaf analysis showed that optimum levels for wild species are lower than those for hybrid gladioli with the optimum levels affected by physiological corm maturity and subsequent plant growth. Norms for postharvest handling of cut G. scabridus spikes have been developed. Spikes held in 2 % sucrose had a longer vase life and better floret opening and quality than those kept in distilled water, Prolong, Chrysal, 2 % sucrose and 2 % ethanol, 1 % sucrose, 4 % sucrose, 1 % sucrose and 0.5 % JIK, 2 % sucrose and 0.5 % JIK and 4 % sucrose and 0.5 % JIK, 2 % fructose and 2 % glucose. The use of commercially available solutions should be used with caution. Florets produced a climacteric-like CO2 peak, but levels of ethylene were unmeasurable. Cold storage and the use of polypropylene sleeves delays senescence. G. scabridus spikes secrete droplets of a sticky substance which was confirmed to be extra floral nectar through HPLC analysis. Market research revealed a positive response to the species from consumers and retailers alike with potential for cultivation as a cut flower and bedding plant. However, the cost will determine supply and demand. A field study conducted at Bivane Dam, northern KwaZulu-Natal, confirmed that G. scabridus colonies prevail in rocky, quartzite outcrops where they become wedged between the rocks. Plants were found at different stages of development with populations of not more than 108 plants per colony. Soil data of G. scabridus sites was compared to that of two sites nearby. It was found that G. scabridus soils are higher in phosphorus (P), zinc (Zn) and organic carbon. Leaf analysis confirmed that they have adapted their growth to low nutrient levels. The G. scabridus studies have clearly shown that the species can be successfully moved from a wild plant to a commercially viable one and in so doing its conservation status can also be improved.
Management of avocado postharvest physiology.
(2011) Blakey, Robert John.; Bower, John Patrick.; Bertling, Isa.
Avocados are an important horticultural crop in South Africa, especially in the provinces of KwaZulu-Natal, Mpumalanga and Limpopo. The distance to traditional export markets, phytosanitary restrictions to lucrative markets such as China, the USA and Japan and increased competition in the European market have challenged the South African avocado industry. The industry has responded with improved logistics and shipping, a co-ordinated market access program and a global system to co-ordinate exports of avocados to the European market. To remain competitive on the global market, further improvements and innovations are required to improve the efficiency of postharvest operations. These improvements and innovations should be guided by a greater understanding of postharvest physiology. Avocados are a relatively new export crop, so there is still much to be learnt about avocado postharvest physiology and the optimisation of postharvest management. In this regard, reduced temperature storage (1°C) and modified humidity packaging (MHP) were investigated for their effect on fruit physiology and quality, the effect of a water- and ABA-infusion on ripening was examined and the effect of a cold chain break on fruit physiology and quality determined; near-infrared spectroscopy was also examined for its potential for its use in the avocado industry. As an initial study, the relationships between individual sugars, protein and oil were studied to understand the changes in avocado fruit during ripening. It was found that mannoheptulose and perseitol were the predominant sugars at harvest, but declined to very low levels during the first 10 days postharvest. The concentrations of glucose and fructose increased, while sucrose declined slightly during ripening. The concentration of protein increased sigmoidally during ripening, reflecting the increase in the ripening enzymes, particularly cellulase and polygalacturonase. The oil content fluctuated slightly during ripening. It is suggested that mannoheptulose and perseitol are important carbon and energy sources during ripening. Glucose concentration was also found to increase earlier in fast ripening fruit compared to slow ripening fruit, which is related to increased cellulase activity and may be related to the ABA functioning. Thereafter, storage and ripening trials in two consecutive seasons showed that 1°C storage and the use of MHP for 28 days reduced mass loss, water loss, ethylene production, respiration, softening and heptose consumption, without appreciably affecting fungal rots, physiological disorders or external chilling injury, compared to fruit stored at 5.5°C and regular atmosphere respectively. Also, the storage of fruit in MHP delayed the rise in the activity of cellulase during ripening, compared to fruit not stored in MHP, but there was no significant difference in the peak activity of cellulase, polygalacturonase or pectin methylesterase. In a separate experiment, fruit ripening was significantly affected by the infusion of ABA in an aqueous solution. Water slightly reduced the variation in ripening while ABA reduced the time to ripening and the variation; it is suggested that water stress and ABA are intrinsically involved in the ripening processes and may act as a ripening trigger. The water concentration in fruit was measured non-destructively using reflectance NIR; this model was used to determine the maturity of fruit and the loss of water during cold storage. In the cold chain break experiment, it was found that although fruit recovered after a cold chain break, in terms of ethylene production and respiration, there was a loss in quality because of severe shrivelling as a result of increased water loss. Fruit that were stored at 1°C were generally of a better quality at ripeness, if the cold chain was broken, compared to fruit stored at 5.5°C. In a follow-up experiment, it was found that significant changes occurred in avocado physiology over a 6h period. The respiration rate of fruit significantly increased after 4h at room temperature and mannoheptulose declined by 32% in control fruit and by 16% in ethephon-treated fruit after 6h. This demonstrates the potential for quality loss in a short amount of time. Furthermore, a model of avocado ripening is proposed, outlining the role of water, ABA, ethylene, respiration, ripening enzymes and individual sugars. This study has contributed to the understanding of avocado postharvest physiology and should aid in better management of avocados for improved fruit quality and consumer satisfaction.
Some aspects of water relations on avocado Persea americana (Mill.) tree and fruit physiology.
(1985) Bower, John Patrick.; Wolstenholme, B. Nigel.
The effect of long-term irrigation on water relations of 'Fuerte' avocado trees, and the consequences for fruit ripening and physiology, particularly physiological disorders, were studied. Four irrigation regimes were used, namely dryland relying on rainfall, occasional (irrigation when soil moisture tension reached 80 kPa), regular (soil moisture tension 55 kPa) and frequent (soil moisture 35 kPa). Seasonal tree leaf water potential was studied. This became more negative during the dry season (winter and spring) and less negative during the period of summer rainfall. During the dry periods, the dryland and 80 kPa treatments had considerably more negative leaf water potential than the 55 kPa and 35 kPa regimes. These differences decreased during the summer rainfall period. Acclimation was studied by measuring leaf osmotic pressure and osmotic pressure at zero turgor. A pattern similar to seasonal leaf water potential emerged. Further, the dryland treatment showed higher osmotic pressure, particularly at zero turgor, during winter and spring. It was concluded that these trees may have acclimated. Diurnal cycles of stomatal resistance, transpiration and leaf water potential on a summer, two winter and a spring day were monitored. Dry land trees showed acclimation, with delayed reaction to environmental water demand and decreasing soil moisture. Trees of the 80 kPa treatment showed greatest stress. Fruit water potential became more negative between April and July, with fruit softening becoming more rapid. Treatment differences were inconclusive. Polyphenol oxidase activity (PPO), soluble and total, was measured. For fruit picked in April and July 1983, the 55 kPa treatment showed the lowest activity and the 80 kPa the highest. Storage at 5,5⁰C for 30 days increased the activity, while fruit softening decreased it. July activity was higher than the April-harvested fruit. The same pattern emerged for fruit harvested in April 1985, although treatment differences were not significant. Rainfall was considerably higher during the fruit development period of 1985 fruit as compared with that of 1983. A significant interaction between restricted container ventilation during ripening and irrigation history was obtained, the 80 kPa fruit showing higher PPO activity than 55 kPa fruit. Ethylene evolution during ripening showed a normal climacteric pattern for 55 kPa and dryland fruit, but a delayed peak for 80 kPa fruit. Fruit calcium concentrations showed rapid changes between 7 and 16 weeks after fruit set thereafter remaining constant to harvest. There were no clear treatment differences. Fruit abscisic acid levels at 50% soft (100% is eating soft) were lowest in 55 kPa fruit, and highest in 80 kPa. A significant correlation between these values and soft fruit PPO activity was found. A preliminary fruit quality prediction model is suggested.

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