Anaerobic digestion (AD) is becoming a widely adopted technology for conversion of organic waste and nutrient-rich fertiliser production due to its cost-effectiveness and sustainability. In this study, a batch experiment was conducted using five different types of food waste and cow dung (CD). No significant difference was observed among the four substrates that produced the highest methane (P<0.05). Based on the batch experiment results, two substrates were selected for semi-continuous digestion and the highest methane yield (67%) was obtained from co-digestion (CO). PCR-DGGE results revealed higher bacterial and archaeal diversity indices in CO as compared to mono-digestion of CD and mixed food waste. The high-throughput sequence analyses revealed that the Operational Taxonomic Units (OTUs) belonging to the phyla Bacteroidetes, followed by Firmicutes, Actinobacteria and Proteobacteria, were dominant in all treatments. The enhanced methane production in CO could be attributed to the neutral pH and partial shift of archaea from Methanosaeta to Methanosarcina. The digestate and fresh CD were screened for plant growth promoting bacteria (PGPB), nutrient and heavy metal content. The dung contained higher concentrations of heavy metals (P<0.05) and potential pathogens in comparison to the digestate. The use of digestate may, therefore, enhance soil fertility with minimal negative environmental effects. / School of Agriculture and Life Sciences / M. Sc. (Life Sciences)

African leafy vegetables have been shown and suggested to have potential to contribute to human diets and alleviate malnutrition; however, their levels of utilisation are currently low especially in South Africa. This is because there is limited access to these crops due to low availability in the market. Limited access is attributed, in part, to the lack of commercialisation as a result of limited agronomic information describing optimum management options for these leafy vegetables. Availability of such information would contribute to successful commercialisation of these crops. The primary objective of this study was to establish optimum agronomic management factors for Amaranthus cruentus, Corchorus olitorius, Vigna unguiculata and Brassica juncea for irrigated commercial production in South Africa. Seeds of Amaranthus cruentus, Corchorus olitorius were obtained from the Agricultural Research Council seed bank; Vigna unguiculata were obtained from Hydrotech and Brassica juncea seeds were obtained from Stark Ayres. The project consisted of three field studies whose overall objective was to evaluate growth and yield responses of the selected African leafy vegetables to agronomic factors under irrigated commercial production. These field studies comprised of two single factors; summer trials (planting density and nitrogen on three selected crops) and a combined winter trial (nitrogen, irrigation, plant density and planting date on a winter crop). Chapter three (3) investigated the effect of plant density on growth, physiology and yield responses of Amaranthus cruentus, Corchorus olitorius and Vigna unguiculata to three plant densities under drip irrigated commercial production. The plant density levels of 100 000, 66 666 and 50 000 plants/ha were used in the 2011/12 and 2012/13 summer seasons. Parameters measured included chlorophyll content index (CCI), chlorophyll fluorescence (CF), stomatal conductance (SC), leaf number, leaf area index (LAI) and biomass. Amaranthus cruentus and Corchorus olitorius showed better leaf quality at lower plant density of 50 000 plants ha-1 than at 66 666 plants ha-1 and 100 000 plants ha-1. These results are based on bigger leaves expressed as leaf area index (LAI), better colour expressed as chlorophyll (CCI) and higher biomass per plant observed in these crops at 50 000 plants ha-1 in comparison to 66 666 plants ha-1 and 100 000 plants ha-1. In Vigna unguiculata there were no responses observed in LAI and CCI. In Amaranthus cruentus, Corchorus olitorius and Vigna unguiculata fresh and dry mass yield of leaves were higher at 100 000 plants ha-1 compared to other treatments. In A. cruentus and C. olitorius, higher leaf quality parameters (CCI, plant height, leaf number, biomass per plant and LAI) indicated that these crops can perform better at lower densities of 50 000 than at 66 666 plants ha-1 and 100 000 plants ha-1 Therefore, using 50 000 plants ha- 1 is suitable for commercial production of A. cruentus and C. olitorius. In Vigna unguiculata, a plant density of 100 000 plants ha-1 produced the highest fresh and dry mass per unit area without compromising quality in terms of the leaf size (LAI) and colour (CCI). Therefore 100 000 plants ha-1 is a density recommended for commercial production in V. unguiculata.Chapter four (4) was conducted to investigate growth, physiology and yield responses of A. cruentus, C. olitorius and V unguiculata to nitrogen application under drip irrigated commercial production. Three nitrogen treatments levels were used viz. 0, 44 and 88 kg N ha- 1 in 2011/12 season and four nitrogen treatments levels viz. 0, 50, 100 and 125 kg N ha-1 were used in 2012/13 summer season. The nitrogen levels selected for each season were based on recommendations for Amaranthaceae species, Swiss chard (Beta vulgaris L.var cicla) derived from soil analysis of the trial (field) site. Parameters measured included chlorophyll content index (CCI), chlorophyll fluorescence (CF), stomatal conductance (SC), leaf number, leaf area index (LAI) and biomass. Results showed that application of nitrogen at 44 kg N ha- 1 in 2011/12 summer season and 100 kg N ha-1 in 2012/13 summer season improved LAI, CCI, biomass per plants and yield in A. cruentus. A similar trend was observed in C. olitorius except that 44 kg N ha-1 improved stem fresh yield. Further increase in nitrogen fertiliser above 44 kg N ha-1 during the 2011/12 season and above 100 kg N ha-1 in 2012/13 summer season reduced leaf quality and yield in both crops. In V. unguiculata, nitrogen application showed a slight increase in yield values from 0 to 44 kg N ha-1 followed by decrease at 88 kg N ha-1 in 2011/12 summer season; however, this increase in yield was not significant. During the 2012/13 summer season, yield in terms of fresh weight was significantly (P<.001) reduced by applying nitrogen at various levels. However, leaf dry matter content increased significantly (P<.001) with increase in nitrogen from 0 kg up to 100 kg N ha-1, then remained unchanged at 125 kg N ha-1. Therefore, the current study recommends that C. olitorius and A. cruentus could be commercialised at 44 kg N ha-1 and 100 kg N ha-1 which were lower nitrogen application rates than those recommended for Amaranthaceae species. In V. unguiculata, 50 kg N ha-1 improved leaf number; however, this did not translate to any fresh yield advantage, implying that the optimum rate for nitrogen application might be lower than 50 kg N ha-1. Therefore, nitrogen rates less than the ones used in the current study are recommended for V. unguiculata. Chapter five (5) was conducted in winter and it was necessitated by observations made primarily in the previous studies which focused on the effects of single factors such as plant density, planting date and nitrogen deficits. Therefore, there was a need to address interactions between irrigation, nitrogen, spacing and planting date. The objective of this study was to evaluate growth, physiology and yield responses of Brassica juncea to different agronomic and management factors in the 2012 and 2013 seasons. The treatments were as follows: two planting dates in main plot (1 June and 18 July, 2012); two irrigation frequency in sub main plot (once and three times a week); three nitrogen levels (0, 50, 100 kg N ha-1) and three plant densities (133 333, 80 000, 50 000 plants ha-1) as subplots. Parameters measured included chlorophyll content index (CCI), chlorophyll fluorescence (CF), stomatal conductance (SC), leaf number, leaf area index (LAI) and biomass. Results from this study showed a significant interaction effect on plant height, LAI, CCI and CF. Crops irrigated thrice or once a week with 50 kg N ha-1 combined with 50 000 plants ha-1 produced tall plants and bigger leaves (LAI) in the early planting date (1 June) compared to other combinations. Irrigating three times a week combined with nitrogen application at 100 or 50 kg N ha-1 improved CF for late planting date (18 July) in comparison to other combinations. Irrigating once a week combined with nitrogen application at 100 kg N ha-1 increased CCI. There was no significant interaction effect on yield. Application of nitrogen at 50 and 100 kg N ha-1 significantly (P>0.05) increased yield in early and late planting dates compared to the control (0 kg N ha-1), in 2012 and 2013 winter season. Irrigating three times a week led to a significant (P<0.05) increase in yield in the late planting date (18th July) and early planting date (1st June) in 2013 season. Higher plant density of 133 333 plants ha-1 resulted in significantly (P<0.05) higher yield in terms of fresh mass and leaf number in the late planting date 18 July in 2012 and 2013 seasons. However, leaf quality parameters such as leaf size and colour was compromised at 133 333 plants ha-1 relative to 50 000 plants ha-1. Therefore, farmers are recommended to plant early, apply 50 kg N ha-1, irrigate thrice a week and utilise a spacing of 50 000 plants ha-1. The current study indicates that growth and yield of traditional leaf vegetables can be optimised through improved agronomic practise. / Agriculture and Life Sciences / D. Litt. et. Phil. (Agriculture)

Indiana University-Purdue University Indianapolis (IUPUI) / Drought and cold conditions are among the major factors affecting plant growth and crop production globally. Dehydrins are group II late embryogenesis abundant (LEA) proteins characterized by a conserved K-region (EKKGIMDKIKEKLPG consensus sequence) that accumulate in many plants during drought, low temperature, and high salinity to confer stress tolerance. While it has been demonstrated that overexpression of dehydrins improves cold tolerance in various crop plants, the mechanism leading to cold tolerance is still unclear. Previous studies reported phosphorylation of AtERD14 dehydrin by casein kinase II (CKII) led to an increase in calcium binding activity. Mass spectroscopy analysis determined that the phosphorylation was localized to a poly-serine (S) region. To further characterize the S-region, GST fused ERD14 mutants were created via site-directed mutagenesis and deletion of either the amino or carboxyl ends of ERD14 via the QuickChange® Multi Site-Directed Mutagenesis Kit. Phosphorylation of purified mutant proteins by CKII was analyzed via gel shift and direct phosphorylation assays. The effect of phosphorylation on calcium binding activity was also analyzed. Results showed the serine (S) residue at position 83 was crucial to phosphorylation-dependent molecular mass shift and Ca2+-binding activities followed by the serine residue at position 85 in importance. Mutation of serines at positions 83, 84, and 85 completely eliminated the phosphorylation-dependent gel shift and calcium binding. Examination of truncation mutants determined the N-terminal was an important region for protein structure modification and phosphorylation ability leading to Ca2+ activation. Calcium binding activity of the truncated mutants indicated the calcium binding site was localized in the region between the S-region and the K-region near the C-terminal end. To characterize the acidic dehydrins contribution to cold tolerance in vivo, three single (erd10, erd14, cor47) knockouts (KOs) were characterized. Single KOs produced no cold sensitive phenotype indicating the need for multiple dehydrin KOs in Arabidopsis in order to potentially produce a cold sensitive phenotype.

Commercial AM fungi isolates, Rhizophagus clarus, Gigaspora gigantea, Funneliformis mosseae, Claroideoglomus etunicatum and Paraglomus occulum were tested on four seasonal ornamentals, Dianthus chinensis x barbatus, Impatiens wallerana, Petunia x hybrid and Viola x wittrockiana planted in peat-base medium. The experiment was conducted in a glasshouse with three replicates in a completely randomised design. Various vegetative (height, width, length, number of leaves, leaf area and dry biomass) and reproductive (number of flowers and buds) plant parts were measured in the course of three months. AM fungi was found to increase seedling growth and reduced seedling mortality rate of all the plants studied. Inoculated plants produced more leaves (16-33%) and grew taller (12-28%). Dry biomass of inoculated Dianthus, Impatiens and Viola plants were significantly increased by 25-53%. All plants under low colonisation rates displayed mycotrophic qualities and net growth output thereof were found to be similar to plants with equal or higher colonisation rate. Mortality were less frequent in inoculated plants and they were also less susceptible to transplant shock. / Agriculture, Animal Health and Human Ecology / M. Sc. (Ornamental Horticulture)

Indiana University-Purdue University Indianapolis (IUPUI) / The phototropin photoreceptors are important regulators of plant growth and development and can therefore affect the photosynthetic activity of plants. Phototropin1 and Phototropin2 are versatile protein kinases that become activated when exposed to blue light. Their photobiological actions are best understood in the model plant Arabidopsis thaliana, where they are known to trigger several responses to blue light, one of which is phototropism, the bending of plant organs towards light. Additionally, phot1 and phot2 drive stomatal opening, chloroplast arrangement in leaf cells, leaf expansion, and leaf orientation. The phot1-specific response is rapid inhibition of hypocotyl growth, leaf positioning and mRNA stability whereas phot2 mediates the chloroplast avoidance response to high light. These responses impact a plant’s ability to capture light for photosynthesis, therefore the phototropins play important roles in optimizing a plant’s photosynthetic activity. Soybean (Glycine max) is a very important crop plant in Indiana known for its nutritional versatility and is also utilized for biodiesel production.In spite of soybean being a key crop, there is currently no information about the functionality of soybean phototropins. Also, being a legume, soybean has many structural and functional features that are not present in Arabidopsis. Interestingly, PsPHOT1A (a photoreceptor from garden pea) was found to be a functional phototropin as it was able to complement the phot1 mutation in Arabidopsis. The roles of these proteins in soybean will be elucidated based on the hypothesis that soybean phototropins play essential roles in regulating photosynthetic activity as do the Arabidopsis phototropins. To date, five soybean phototropins, 3 PHOT1s and 2 PHOT2s, are believed to exist. These GmPHOT protein coding regions were amplified by RT-PCR and cloned into pCR8/TOPO or pENTR-D/TOPO vectors via TOPO cloning to utilize Gateway cloning technology to create plant transformation constructs subsequently. The cloned GmPHOT cDNAs from each of the 5 GmPHOTs were sequenced and compared to the GmPHOT sequences from the Phytozome database to assess the accuracy of the gene models. The gene models of all the GmPHOTs were found to be accurate except that of GmPHOT1B-2. The high level of sequence identity between the GmPHOTs and AtPHOTs and the conservation of LOV domains and catalytic domains indicate structural resemblance between them. This suggests that soybean phototropins should encode active photoreceptors. The cloned protein coding regions from soybean were then recombined into a plant expression vector via Gateway technology,which were then used for transformation of Agrobacterium tumefaciens. These plant expression constructs will be utilized in the future to determine the functionality of soybean phototropins in Arabidopsis.