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Bibliography: leaves 137-156. / xiv, 166 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--Dept. of Horticulture, Viticulture and Oenology, University of Adelaide, 1993

Bibliography: leaves 137-156.

The seed storage fungi (xerotolerant) species of the genera Aspergillus and Penicillium} are renowned for their devastating effects on stored grain and grain products. In view of the fact that most of these fungi Iiberate toxins which can be harmful to both man and his livestock this problem is becoming increasingly relevant, particularly in developing countries. The seed storage fungi are said to be saprophytes and opportunistic invaders of dead or naturally dried organic matter, and as such no direct host-pathogen relationship has been ascribed to them. This dissertation reports aspects of an investigation into the modes/pathways utilised by these fungi in their infection of maize caryopses (seeds) and plants. The work involved studies on: the effects of protracted storage on maize seeds; the morphology of storage fungi; extra-cellular enzymes of storage fungi; the pathways utiIised by the storage fungi in invasion of seed tissues; and the effects of the storage fungi on the seeds. Correlations have been made on a species basis between the extent of seed deterioration and fungal aggressiveness. The results of these investigations indicated that apart from affecting seed vigour and viability, these fungi can also affect plant vigour. This latter aspect was further investigated to determine whether a seed storage fungus could infect germinating maize seeds, and remain an internal contaminant of the tissues during plant growth and development. These latter studies revealed that Aspergillus flavus var. columnaris is capable of systemic transmission from one seed generation to the next. This hitherto unrecognised phenomenon apart from indicating that the fungal species is in fact a biotroph as well as a saprophyte, also has implications In control measures. / Thesis (Ph.D.)-University of Natal, Durban, 1990.

The seeds of Trichilia dregeana, Trichilia emetica, Podocarpus henkelii and Syzygium cuminii display the characteristics typical of recalcitrant seeds. It is the phenomena of ongoing metabolic activity and desiccation sensitivity that render them unsuitable for storage by the conventional methods used for orthodox seeds. Investigations on the storage responses of 'sub-imbibed' (partially dried) and fully hydrated seeds of all four species were carried out to study the effects of partial drying on viability and subsequent storage lifespan; i.e. to assess whether 'sub-imbibed' storage is feasible for these species. The outcome of this investigation was proposed to contribute to the resolution of the argument that storing recalcitrant seeds at lowered water contents might extend their longevity; i.e. storage at a relatively high water content but below the fully hydrated level, might prevent germination but would not be sufficient to be injurious to the seed. Seeds of T. dregeana, T. emetica, P. henkelii and S. cuminii were dried to various target moisture contents (which were determined for each species in the initial drying experiment) and then subjected to storage for 3-22 weeks at 6, 16 and 25°C (in sealed containers). In parallel, seeds of each species were stored at the shedding water content. The seeds were periodically removed for sampling, and assessed for water content, germination, respiration, electrolyte leakage and microscopical features. Storage temperature appeared to affect viability of seeds of T. emetica and T. dregeana which displayed characteristics of chilling sensitivity. Storage at 6°C was detrimental (when compared with seeds stored under the same conditions at 16 and 25°C), but regardless of whether the seeds were undried or partially dried prior to storage. The seeds of P. henkelii did not demonstrate chilling sensitivity, the viability not being compromised at 6°C compared with those seeds stored at 16 and 25°C. Syzygium cuminii seeds were not subject to storage at 6°C because previous work indicated that they would be chilling-sensitive. Storage of 'sub-imbibed' seeds of T. dregeana, T. emetica, P. henkelii and Syzygium cuminii does not to confer any benefit over seeds stored in the fully hydrated state; rather it appears to be deleterious to seed survival during storage. This was apparent from the assessment of viability, electrolyte leakage and respiration. Vigour and viability of the 'sub-imbibed' seeds of all species declined more rapidly than the fully hydrated seeds. The only exception was P. henkelii seeds stored at 25°C, the fully hydrated seeds showed no survival after 11 weeks in storage, while 88% of the 'sub-imbibed' seeds survived this period. These results were, however, attributed to the proliferation of fungi on the fully hydrated seeds at 25°C. Although ultrastructural observations were made only on the T. emetica seeds, it was apparent that the cells from the 'sub-imbibed' seeds (after storage at 16 and 25°C) showed extensive degradation, with the intra-cellular components being largely unrecognisable. The cells from the seeds stored in the fully hydrated condition at 16 and 25°C maintained integrity and appeared metabolically active. In keeping with the suggestion that T. emetica seeds are chilling sensitive, the ultrastructure of the cells from both the 'sub-imbibed' and fully hydrated seeds showed deteriorative changes. All the results of the present study indicated that storage in the 'sub-imbibed' state is deleterious to seed survival. It is apparent that the removal of water, however small a proportion, accelerates seed deterioration during storage. Thus 'sub-imbibed' storage has no practical application for the storage of recalcitrant seeds. / Thesis (M.Sc)-University of KwaZulu-Natal, 2007.

published_or_final_version / Ecology and Biodiversity / Master / Master of Philosophy

One of the factors that affect the post-harvest behavior of seeds is provenance. Different geographical locations are characterised by different environmental conditions, hence, plant materials harvested from different locations may respond differently to laboratoryinduced stresses. The aim of the present study was to assess the role that provenance plays on the degree of recalcitrance of a single species – a factor that needs to be taken into consideration when choices are to be made for plant germplasm conservation. In this study, seeds of Trichilia dregeana, which have been shown to display recalcitrant post-harvest behavior, were harvested from four locations that were slightly different in climatic conditions: Mtunzini (MTZN), Durban (DBN), Pietermaritzburg (PMB) and Port Edward (P.ED), all within the KwaZulu-Natal province of South Africa. Clean seeds, whose aril and seed coat have been removed, were subjected to different drying and chilling stresses and their responses to the stresses are examined. Excised embryonic axes of the seeds were also subjected to flash (very rapid) drying and their responses to the stress are shown. Although the vegetation of these locations can be categorized as being sub-tropical, the present study suggests that there may be ‘degrees’ in the sub-tropical nature based on the rainfall and maximum temperature data. The ‘degree of sub-tropicality’ is suggested to be in the order MTZN > DBN > P.ED > PMB. Mean seed size assessed as seed length, width and fresh mass varied significantly across the four regions, with seeds from MTZN being the smallest and those from P.ED being the largest. The shedding water content of the embryonic axes of the seeds for 150 min. However, shoot production, root and shoot length and dry mass accumulation showed that axes of seeds from MTZN were the most adversely affected by rapid drying while those from PMB were the least sensitive. When clean whole seeds were dried slowly by burying in silica gel for 36 h, all seeds of the four provenances still retained more than 50% of their shedding water. MTZN seeds lost viability completely from the 24 h of slow drying while seeds from the other three locations retained some viability after 36 h. The order of survival of slow drying was MTZN < DBN < P.ED = PMB. Storage of cleaned whole seeds at chilling temperatures (3oC, 6oC and 16oC) caused increase in the water content of the embryonic axes of the seeds across the four locations. All seeds of the four provenances had lost more than 50% survival after they had been stored at 3oC for 10 weeks. Survival was completely lost from the 12th week for MTZN seeds, from the 14th week for DBN and P.ED seeds and from the 16th week for PMB seeds. There was no survival recorded for seeds across the four regions at the 18th week. The seeds were able to tolerate storage at 6oC for a little longer than those stored at 3oC. Only MTZN seeds completely lost viability at the 18th week; seeds from the other three locations retained some viability throughout the 18 weeks of storage. Storage at 16oC was the most tolerable, as all seeds from the four provenances maintained some viability until the 18th week in storage, with MTZN having the least survival and those from P.ED having the highest survival. Phylogenetic analysis of the internal transcribed spacer (ITS) region of the DBN and PMB samples showed intraspecific levels of genetic variation, and were separated by a genetic distance of 0.9%, an indicator that differences in storage behavior, chilling and desiccation sensitivity between seeds obtained from different collection localities may be genetically based. This study showed that MTZN seeds were the most sensitive, while seeds from PMB and P.ED were the most tolerant, to drying and chilling treatments, respectively. The prevailing climatic conditions may have a bearing on the responses observed in this study. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

Desiccation-sensitive seeds show differential viability characteristics during drying at different rates. A number of studies have demonstrated that rapid dehydration permits survival to lower water contents than does slower desiccation. The aim and objective of the present study was to test the hypothesis which states that rapid drying of desiccation-sensitive seeds removes water sufficiently fast to reduce the accumulation of metabolic damage. In addition, the hypothesis that wet storage subjects desiccation-sensitive seeds to mild, but increasingly severe, water stress causing oxidative damage if additional water is not supplied, was tested. In the present study, axes of germinating orthodox seeds of Pisum sativum and newlyshed recalcitrant counterparts of Quercus robur, Strychnos madagascariensis, Trichilia emetica, Trichilia dregeana and Avicennia marina were subjected to rapid or slow drying or wet storage. For those species where more than one harvest was investigated, differences were observed in water contents at shedding. For all the species studied, the dehydration rate could be described by an exponential and a modified inverse function for both desiccation regimes, and the water content remained constant with wet storage. The level of tetrazolium staining and germination percentage of axes decreased sharply drying and hydrated storage such that the marked decline took place at lower water contents upon rapid than slow dehydration. The conductivity of electrolyte leachate increased progressively during desiccation and moist storage of axes of all species investigated. Greater membrane leakage occurred upon slow, than rapid dehydration in axes of all species studied. Activities of respiratory enzymes which have a potentially regulatory role in glycolysis, phosphofructokinase (PFK), or the tricarboxylic acid cycle, malate dehydrogenase (MDH), and levels of the oxidized form of the coenzyme, nicotinamide adenine dinucleotide (NAD), of the enzymes of the electron transport chain, NADH dehydrogenases ofNADH-ubiquinone (coenzyme Q) reductase (complex I) and NADHcytochrome c reductase (complex IV), were monitored in the present investigation. v In addition, the role of free radical activity in the form of lipid peroxidation, which has been implicated in loss of viability in seeds, was examined by assaying the levels of hydroperoxides. The involvement of the free radical processing enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR), and the antioxidant, ascorbic acid (AsA), was also ascertained. The activity of PFK in axes of P. sativum remained constant during drying and wet storage. However, PFK activity increased as rapid dehydration and hydrated storage of Q. robur axes proceeded. In contrast, the activity of PFK in axes of Q. robur decreased during slow desiccation. Similarly, PFK activity was reduced upon drying, and moist storage, of T. dregeana axes such that higher activity of PFK was seen during rapid than slow dehydration. The activity ofPFK inA. marina axes also declined upon desiccation. The activity ofMDH in axes of P. sativum was also unchanged during drying and wet storage. However, an increase in MDH activity was recorded in Q. robur axes during dehydration and hydrated storage such that the activity of MDH was higher upon slow than rapid desiccation. In contrast, MDH activity in axes of T. dregeana decreased as drying proceeded. Similarly, the activity of J\.1DH declined during dehydration and moist storage of A. marina axes. An increase in the level of NAD occurred in axes of P. sativum during drying. In contrast, a decrease in NAD levels was seen upon dehydration and wet storage of Q. robur axes such that the level of NAD was higher upon rapid than slow desiccation. There was an enhancement of the level of NAD in axes of T. dregeana during hydrated storage. Conversely, NAD levels declined during drying ofA. marina axes. A decrease in the level of hydroperoxides in axes of P. sativum was seen as rapid drying proceeded. In contrast, hydroperoxide levels increased during wet storage of P. sativum axes. Similarly, the levels of hydroperoxides were enhanced upon dehydration and hydrated storage of Q. robur axes such that they were higher in axes during slow desiccation compared to those dried rapidly. Conversely, the hydroperoxide level in axes of T. dregeana was reduced upon rapid dehydration. In contrast, an elevation of the level of hydroperoxides was observed during moist storage. The levels of hydroperoxides remained constant as desiccation and wet storage ofA. marina axes proceeded. vi The activity of SOD in axes of P. sativum decreased during rapid drying. In contrast, SOD activity increased upon slow dehydration and wet storage ofP. sativum axes. There was a decline in the activity of SOD in Q. robur axes during slow desiccation. Similarly, SOD activity was diminished upon drying of axes of T. dregeana. The activity ofSOD in T. dregeana axes was enhanced during hydrated storage. An elevation in SOD activity also took place during rapid dehydration and moist storage of axes ofA. marina. The activity of CAT did not change during drying of axes of P. sativum. However, a decrease in CAT activity in Q. robur axes was seen upon slow dehydration and wet storage. Similarly, the activity of CAT declined as desiccation of axes of T. dregeana proceeded. In contrast, CAT activity inA. marina axes increased during slow drying. Whereas the activity of GR in axes of P. sativum increased during drying and wet storage, GR activity decreased in A. marina axes upon all treatments such that the activity ofGR was higher during rapid than slow dehydration. GR activity also declined upon slow desiccation and hydrated storage ofaxes of Q. robur. Similarly, the activity of GR in T. dregeana axes was reduced during moist storage. Finally, a decrease in the level of AsA in axes of P. sativum took place during drying. Nonetheless, dehydration and wet storage of Q. robur axes were associated with no siginificant change in AsA levels. There was also a decline in the level of AsA in axes of T. dregeana as rapid desiccation proceeded. Similarly, a reduction in AsA level occurred upon slow drying ofaxes ofA. marina. The results presented here are consistent with the observation that drying and wet storage adversely affected the respiratory enzymes, PFK, MDH and NADH dehydrogenase. It is suggested that the resultant metabolic imbalance led to more leakage of electrons from the mitochondrial electron transport chain than normal, and through lipid peroxidation increased levels of hydroperoxides. In addition, dehydration and hydrated storage may depress the activities of free radical processing enzymes, SOD, CAT and GR and levels of antioxidant, AsA. This phenomenon was less pronounced during rapid, in comparison to slow, desiccation and moist storage. However, it appears that the above biochemical events are overtaken by physical damage at higher water contents in the highly recalcitrant seeds. It was concluded that the differential effects of VII the drying rate and wet storage on responses of desiccation-sensitive seeds varies with tissue, harvest, species and the degree of desiccation sensitivity. / Thesis (Ph.D.)-University of KwaZulu-Natal, 2004.

Under storage conditions of ambient temperature and relative humidity in South Africa, seed-associated mycoflora proliferates. Fusarium moniliforme is ubiquitous in newly-harvested maize, persisting for variable periods in storage, while Aspergillus flavus may represent the final group of species in the succession of aspergilli after grain storage under high temperature and/or high humidity. Many strains of these fungi produce toxigenic secondary metabolites (mycotoxins) under local storage conditions. Since pathogenic fungi may be present within the tissues of stored seeds, these contaminants will not be eradicated by external fungicide treatment, therefore a possible alternative is biological control. The aim of the present investigation was to ascertain whether certain strains and/or species of Trichoderma have potential as biocontrol agents against the seed-associated pathogenic fungi, Aspergillus flavus and Fusarium moniliforme. A study of the fungal growth in dual cultures revealed that from nine isolates of Trichoderma spp. (T harzianum and T viride), four had a noticeable inhibitory effect on the growth of the pathogenic fungi. Scanning electron microscopical investigation of fungal interaction demonstrated no obvious hyphal penetration by - Trichoderma spp. In addition, significant alteration of Fusarium hyphae, with pronounced collapse and loss of turgor, and production of aberrant conidial heads and microheads by A. flavus were observed. Evidence derived from some biochemical studies revealed that antibiosis (by production of extracellular enzymes, volatile compounds and possible antibiotics) is probably the mechanism involved in the antagonistic effect of the four aggressive Trichoderma spp. The in vitro studies demonstrated that the use of Trichoderma spp. as biocontrol agents against A. flavus and F. moniliforme appears promising. / Thesis (M.Sc.)-University of Natal, 1995.