Doctoral Degrees (DBMB)

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Browsing Doctoral Degrees (DBMB) by Subject "Beans, Namibia"

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    Diversity and distribution of Tylosema esculentum (Marama bean) endophytic bacteria communities in Omitara, Harnas and Otjinene, eastern Namibia
    (University of Namibia, 2016) Uzabakiriho, Jean D.
    Tylosema esculentum is a nutritious drought avoiding plant endemic to the Kalahari Desert. Our study assessed the density, diversity and distribution of endophytic microbial community structures associated with leaves, stems and tuberous roots of T. esculentum in Eastern Namibia. Culture-dependent and PCR-based 454 pyrosequencing methods were used. ANOVA with pairwise comparison revealed a significant difference in bacterial density between below and above ground. Endophytic bacterial isolates (605) were identified, grouped into 24 genera and three phyla. Proteobacteria was the most represented (67.4%) followed by Firmicutes (23.7%) and Actinobacteria (4.3%). Shannon diversity index, revealed a significant difference between the tuberous roots and leaves (p = 0.005) and stems (p = 0.006) microbial communities. The cluster analysis revealed a separation between the above and below ground microbial communities. The PCA and the Jaccard diversity indices confirmed these findings. Our results suggested that the microbial community composition was mainly governed by the plant parts rather than the location or sampling time. The phylogenetic analysis showed that all these microbial communities fell into two clades distinct from known cultivated bacteria from NCBI. All isolates associated with T. esculentum were positive for the nifH gene amplification. Only 42% nested with known strain in the NCBI GeneBank Database. This finding showed the presence of putative novel nitrogen-fixing bacteria associated with T. esculentum. Ten samples (leaves, stems and tuberous roots) from Omitara were examined using 16S rRNA gene pyrosequencing method. The presence of the three phyla Firmicutes (50.3%), Proteobacteria (38.32%) and Actinobacteria (4.46%) was confirmed. Two more phyla Fusobacteria (5.7%) and Bacteriodetes (1.368%) were revealed. Strikingly, 2 phyla (Firmicutes and Proteobacteria) of the five phyla represented 89 % of the total sequences. Similarly, four families (Enterobacteriaceae, Bacillaceae, Pasteurellaceae and Fusobacteriaceae) of the 18 recorded, represented nearly 91% of the total T. esculentum bacterial community assemblage. The genus Bacillus was predominantly found in the tuberous roots though shared across all samples. The class Clostridiale was exclusively found in leaves and stems. Within the Gammaproteobacteria class, the sequence grouping showed the Enterobacteriaceae family and the yet to be identified Enterobacteriaceae dominated. Unlike the Enterobactericeae that are mostly found in the tuberous roots, the Pasteurellaceae family was preferably found in the leaves and stems. Actinobacteria have shown a ubiquitous colonization compared with the Bacteriodetes that colonized the above ground part of the plant. T. esculentum organs exerted selective pressures on their associated bacterial communities. Only 68% of all reads could be classified at the phylum level. Firmicutes are the most dominant phylum in the current study with 46.9% sequences that have not yet been classified to any existing family, order or genus. Also, the rarefaction curves predicted that additional sampling will lead to significantly increased estimates of diversity. Sequences in this study, have shown similarities with sequences occurring in water-stressed environments with plant growth promoting traits. In conclusion, T. esculentum bean lives in community with a large diversity of potentially plant growth promoting bacteria
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    Domestication in Marama bean (Tylosema Esculentum): Agronomy, phenotypic and molecular characterization for its improvement
    (University of Namibia, 2014) Takundwa, Mutsa M.
    Marama bean [Tylosema esculentum (Burchell) Schreiber] belongs to the family Fabaceae and is a candidate for domestication in arid zones. It is indigenous to the Kalahari regions of Southern Africa thriving in low nutrient and low moisture soils. Marama bean seeds exhibit high oil (up to 48%) and protein (up to 42%) content comparable to peanut and soybean respectively. The main purpose of this study was to determine the usefulness of previously developed microsatellite markers in distinguishing phenotypically characterized marama plants and plants treated with gamma irradiation for improvement. The chromosome number was determined to lay a foundation for molecular mapping. Further to this, the study sought to establish the effect of improved nutrients, moisture and hormone treatments on vegetative growth of marama bean. Grafting was explored as a propagation method to side step juvenility and molecular identification of potential fungal pathogens of leaf tissue was achieved. SSR primers were screened using DNA isolated from phenotypically characterized individuals representing 13 marama bean ecotypes. Two microsatellite markers MARA 039 and MARA077 were found to be candidates for use in detecting differences in internode length as well as distinguishing seeds treated with gamma radiation from untreated seeds. The chromosome number in T. esculentum was found to be n=22 (2n=44) and this will be useful in future mapping efforts. A completely randomized block design was used in assessing the possibilities of enhancing vegetative growth with fertilizer, hormones and water: Lucky plant fertilizer (LS004990-00-00) (1g/L), 100μg/L of hormone Gibberellin (GA3), 200mL water for the control, 400mL water for the high water treatments were applied. The results obtained were analysed by one way ANOVA and showed no significant difference in internode length (p=0.362>0.05), stem length (p=0.256>0.05) and number of leaves (p=0.466>0.05) suggesting the treatments had no effect on the vegetative growth of T. esculentum. Marama bean was observed to be non-responsive to grafting in the trials carried out prompting the exploration of tissue culture methods for future studies. The overall inoculations of PDA and PDB with leaf tissue showing signs of necrosis and DNA isolation together with the internal transcribed spacer (ITS) region amplification of the total 8 single spore cultures plus sequencing followed by a comparison of the DNA sequences with GenBank revealed the presence of a complex with 8 known species: Penicillium brevicompactum, Epicoccum sorghi, Rhizopus stolonifer, Alternari solani, Fusarium equiseti, Penicillium olsonii, Fusarium chlamydosporum and Fusarium incarnatum. This study has made several contributions to knowledge and current understanding of plant sciences. To our knowledge, this is the first report describing the presence of these fungi on marama bean seedling leaf tissue. This study has made a major contribution to mapping efforts as it identified regions of the genome that can be used in these studies. This study has also confirmed the marama bean chromosome number. Molecular markers linked to internode length or germination rate or any other trait that may be of agronomic importance had not been reported previously. The results from the work on grafting and plant growth regulators will prevent farmers from using high cost agricultural practices in cultivation of marama bean. Based on the findings of this study, the recommended next steps would be to explore Next Generation Sequence data with genome walking to identify genes in marama, particularly genes controlling flower formation, as well as establishing F1 populations for mapping studies.
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    Isolation and characterization of starch, starch biosynthetic genes and protease inhibitors from Marama beab (Tylosema Esculentum)
    (2014) Nepolo, Emmanuel
    Marama bean (Tylosema esculentum) is a highly nutritious plant and is currently regarded as a prospective crop for the future in arid zone agri-ecologies of the world. Starch is a major storage component in higher plants and in marama bean it’s mostly found stored in the tuber. Starch is used in both food and non-food industries. Starch biosynthesis involves groups of committed enzymes. Aims of the present study were to determine the physicochemical and pasting properties of native marama bean starch isolate and characterize marama starch biosynthesis genes and detect serine protease inhibitor activities in green and mature marama seeds. The total starch content of marama bean tubers was determined by amyloglucosidase/α-amylase enzymatic digestion and amylose content by Concanavalin A precipitation. A complementary Deoxyribonucleic acid (cDNA) library was constructed from marama tuber for the screening and isolation of Soluble Starch Synthase I (SSSI) and a Polymersae Chain Reaction (PCR) based strategy was used to isolate Adenosine diphosphate-glucose pyrophosphorylase (AGPase) and Starch Branching Enzymes (SBEs) using degenerative primers designed at the conserved motif of corresponding cloned plant starch synthesizing genes. Detection of serine protease inhibitor activities in green and mature marama seeds was established using the reverse zymogram technique and fluorogenic substrate N-alpha-benzoyl-l-arginine 7-amido-4-methylcoumarin hydrochloride and cDNA clone encoding a serine protease gene from marama was isolated using newly developed degenerate PCR primers. Native marama starch content was 87.38 mg starch/gram fresh weight and the total amylose content was 35 %. Phosphate at the C-6 position determined as Glucose-6-Phosphate was 0.788 nmol G6P/mg. The starch granules were round to elliptical with smooth surfaces and their sizes ranged from 8 -20 μm. The pasting properties of pasting temperature, host paste, peak, final viscosity, breakdown and set back showed higher values for marama starch in contrast to commercial potato starch. A cDNA clone encoding a SSSI from T. esculentum was isolated and identified by cDNA screening. The cDNA clone is 684 bp in length and encodes 228 amino acid residues. Sequencing of cloned cDNA showed 100% identity with potato SSSI.The phylogenetic tree indicated the divergence of SSI in higher plants proceed in line with evolutions of monocots from dicots. AGPase and SBEs genomic clones from T.esculentum were isolated and their sequence features revealed. AGPase small and large subunit clones both showed 96% identities with Glycine max, while SBEI and SBEII clones showed 91 and 93% identities to Cicer arietinum and Medicago truncatula. Phylogenetic trees for both AGPase and SBEs clones exhibited higher sequence similarity to the AGPase and SBEs of dicotyledons. Reverse zymogram analysis revealed four putative serine inhibitor activities in mature seeds and not in green seeds. Fluorogenic substrate analysis showed significantly higher (P <0.05) trypsin activity in mature marama bean (2326±356 Fluorescence Units mg 1 protein) than green seeds (362±73 FU mg 1 protein).The analyses showed lower (P <0.05) serine protease activity in both cowpea (877±138 FU mg 1 protein) and soybean (381±36 FU mg 1 protein). A partial cDNA clone encoding a serine protease gene from marama bean showed 100% identity to trypsin inhibitor for cowpea. Physicochemical properties of marama starch revealed considerable high amylose content and pasting properties when compared to other tuberous starch producing crops. Molecular features of starch synthesizing genes in marama were similar to that of the same genes characterized in other plant species. Serine inhibitors activities in marama seed may as well be an indicative that marama seed may have potent serine protease inhibitors which could be utilized in pharmaceuticals and in human health. Physicochemical properties of marama starch would suggest that it could find applications in foods and non-food uses that require higher amylose content and high pasting properties. Isolation and characterization of marama starch biosynthetic genes provide theoretical foundation in understanding the structure and functions of SSSI, AGPase and SBEs. Serine protease inhibitor gene isolation and detection of serine protease inhibitor of marama seeds will broaden the pool for plant serine protease genes and might be an ideal choice for developing pharmaceutical products. Future studies need to establish the potential uses of marama starch, characterize marama starch biosynthetic gene structures, functions, regulation and expression. It would be interesting to study in the future any reason for the higher serine protease activity in mature seeds than in green seeds as established recently.This study has clearly contributed to starch biology by making known for the first time the physicochemical and functional properties of marama tuber starch. It also established for the first time the genetic signature of genes involved in starch biosynthesis of marama bean, which may confer unique properties to marama tuber starch. At the same time, the presence of serine protease inhibitors activities and serine protease inhibitor gene in marama seeds with possible application in pharamaceutical products were investigated for the first time making this investigation novel.