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Item Open Access Genetic diversity in yield traits and kernel composition of selected Ethiopian sorghum landraces(University of the Free State, 2023) Engida, Chalachew Endalamaw; Labuschagne, Maryke T.; Herselman, Liezel; van Biljon, Angeline; Nida, HabteClimate-resilient and high-yielding sorghum cultivars must be developed for sustainable food systems and improved livelihoods in sub-Saharan Africa. Low grain protein, Fe and Zn concentrations are a frequent problem in food crops and they affect the crop's nutritional value, particularly in cereals. In this study, a total of 361 Ethiopian sorghum landraces with four commercial checks were grown under well-watered, wet intermediate and natural drought conditions for two consecutive seasons. The purpose of this research was to assess the genetic diversity and drought tolerance of this Ethiopian sorghum landrace collection using agronomic and grain quality traits and to select promising lines for production and/or breeding. The specific objectives were to (i) assess the level and pattern of genetic variation and drought adaptation in a sorghum landrace collection using phenotypic traits, (ii) evaluate chemical composition, genotype by environment interaction, drought effects and environmental influence and determine performance and stability of sorghum landraces, (iii) identify genomic regions associated with grain quality to determine the genetic basis for starch, protein and mineral concentrations of Ethiopian sorghum landraces. In stress environments, yield and yield components as well as grain quality traits had low means, range, genetic variance (σ²g) and heritability values. In terms of grain yield performance under different water regimes, Melkassa (irrigated) had a 54.5% higher mean grain yield than Miesso (drought stress) in 2020 and Melkassa (irrigated) had a 26.9% higher mean grain yield than Miesso (drought stress) in 2021. Therefor drought stress reduced mean grain yield by 44.6% and grain weight by 14%. In all environments, ten genotypes (G189, G210, G125, G324, G325, G353, G319, G219, G187 and G229) had the highest stress tolerance index (STI), geometric mean productivity (GMP), mean productivity (MP) and yield in non-stress conditions (Yp) and stress conditions (Ys). In stress environments, starch content decreased by 5%, while protein content increased by 21% when compared to wet intermediate conditions. The Zn content was 60% and 55% higher in wet intermediate environments than in irrigated and stress environments, respectively. The Fe content was 21% and 13% higher in wet intermediate environments than in irrigated and stress environments, respectively. A genome-wide association study (GWAS) on 365 diverse Ethiopian sorghum landraces identified 209,572 single nucleotide polymorphisms (SNPs) (HB 𝘱 ≤ 0.05). Several candidate genes were identified that were significantly associated with grain Zn, Fe, starch, protein and ash concentrations, such as LEA, CHI, bZIP, ARF12 and WOX6. The Late Embryogenesis Abundant (LEA) and basic leucine zipper (bZIP) family of proteins are involved in various stress responses in plants. The ABC transporter and sugar carrier proteins, Auxin Response Factors 12 (ARF12), Alpha-glucan phosphorylase and Zinc finger CCHC gene, are known for starch synthesis and storage in growing seeds. The WUSCHEL-related homeobox 6 (WOX6) and TatD family of hydrolases genes, acts as a transcription factor and is known to regulate stem cell maintenance and differentiation. Chitinase (CHI), flavonoid glycosides, architecture1 (tga1) and Class III peroxidase 70 and 69 precursors are all engaged in biological processes including plant defence against pathogens. The Zinc finger proteins (ZNFs) family is involved in Zn and Fe ion uptake and transport. These genes are involved in various biological processes such as stress response, starch synthesis, plant development and defence. The identification of loci associated with grain quality provides new insight into the genetic control of the traits, while sorghum landraces with nutritious grains can serve as sources of genes for breeding for good nutritional value.Item Open Access Pharmacological screening and isolation of bioactive compounds from plants used against elephantiasis in the Eastern Cape, South Africa(University of the Free State, 2023) Adams, Zanele; Mojau, Pheello Jeremia; Komoreng, L. V.; Thekisoe, M. M. O.Elephantiasis, also known as lymphatic filariasis, is a medical condition brought on by parasitic worms that invade the lymphatic system causing obstructions of lymph fluid in the passaged that is associated with excessive swelling of the lower and upper limbs resulting in disability, swelling of genitalia and breasts. The condition is also associated with non-filarial causes including certain sexually transmitted diseases, tuberculosis, leishmaniasis, leprosy and podoconsiasis. The World Health Organization lists the illness as one of the neglected tropical diseases that primarily affects developing nations. Although no precise figures have been provided, South Africa is one of the African nations where incidences of the illness have been reported. Plants are reported in treating several medical conditions that affect human kind. Traditional medicine has been considered as an alternative to Western medicine as it is easily accessible and the latter being more expansive. The continued use of plant medicine creates the need to identify those substances that are responsible for the biological activity or the healing properties found in plant extracts through scientific validation. Plants are used by indigenous people and traditional healers in different areas to treat the similar or different conditions. South Africa has a wide variety of plants, and traditional plant studies is reported in literature. In three municipalities of the OR Tambo District Municipality in the Eastern Cape of South Africa, an ethnobotanical survey on medicinal plants used in the management and treatment of lymphatic filariasis was carried out. A total of 29 therapeutic plants from 25 different Angiosperm families were recorded. Acokanthera oblongifolia Curtisia dentata, Dioscorea sylvatica, Elephantorrhiza elephantina, Gunnera perpensa, Hypoxis hemerocallidea, Pentanisia prunellloides and R. melanophloeos were reported as the most used plants in the study to treat elephantiasis and various ailments. The roots followed by the leaves and stem bark were the most used plant parts with infusions and decoctions being reported as the most frequently methods of administration. In vitro studies such as antimicrobial activity, antioxidant activity, phytochemical analysis, anti-inflammatory activity and cytotoxicity activity on mammalian cells were investigated. Bioactive compounds were also isolated and identified from K. drepanophylla.Item Open Access Pathogen variation and genetic control of Puccinia triticina in Zimbabwe(University of the Free State, 2022) Chiuraise, Nyashadzashe; Boshoff, W. H. P.; Visser, B.; Maré, A.Genetic resistance is the most cost-effective approach to manage wheat leaf rust caused by Puccinia triticina Eriks. (Pt). However, the continuous emergence of more virulent races can deplete monogenic sources of resistance. The aim of this study was to determine the distribution, race and genetic diversity of Pt isolates in Zimbabwe and to characterise the sources of resistance in selected wheat accessions. In total, 104 single pustule isolates of Pt were established from infected wheat samples that were collected from the main wheat production regions of Zimbabwe during surveys from 2019 to 2021. Results from phenotyping a set of 46 differential and additional wheat lines revealed Pt race MCDS as dominant in Zimbabwe. Genotyping of 48 Pt isolates with 19 microsatellite markers, followed by DARwin and STRUCTURE analyses, confirmed a high genetic similarity between the Zimbabwean isolates and representative isolates of the South African Pt races MCDS, MCPS and MFPS. However, five isolates (19_1_2019, 24_3_2019, 5_1_2020, 20_1_2020, 23_2_2020) with genetic similarity to South African races SDDN and SCDS were detected. The detection of the five genetically distinct Pt isolates among the Zimbabwean isolates indicates genetic variation that could have arisen from foreign introductions. The infection type (IT) data from screening the 39 differential lines and 72 Zimbabwean wheat accessions with nine Pt races were not informative in postulating the presence of any all-stage resistance genes (ASR). Forty-nine Zimbabwean varieties showed low (resistant) seedling ITs to all nine Pt races tested in the greenhouse and at least 53 varieties were strongly resistant with immune responses to races CFPS+Lr20 and MFPS in the field. From these, 25 wheat lines with ASR to all Pt race isolates were crossed with an MCDS susceptible variety. Twenty-three varieties displayed an F2 segregation ratio of 3:1, indicating the inheritance of a single dominant leaf rust (Lr) resistance gene. Molecular markers detected Lr19 in 20 of these varieties. Five adult plant resistance genes (APR) namely Lr27, Lr34, Lr37, Lr46 and Lr68 were detected in the Zimbabwean germplasm, with Lr46 being the most common and Lr34 the least common. A multi-environmental trial (MET) conducted over two seasons in Zimbabwe identified wheat varieties SC001, SC002, SC004, SC027 and SC W9101 as widely adapted with stable yields, acceptable leaf rust resistance while meeting the quality traits required in the wheat value chain. Overall, the outcomes of this study make a valuable contribution to shaping longer term strategies to control wheat leaf rust in Zimbabwe.Item Open Access Priming effect of leaf rust and salicylic acid in Russian wheat aphid resistance(University of the Free State, 2022) Bilal, Huzaifa; Mohase, L.; Boshoff, W. H. P.Wheat (Triticum aestivum L.) is one of the primary sources of carbohydrates for humans and livestock (Karakas et al., 2021). It is an essential cereal for the human diet and contributes to global food security. Almost 50% of calories for human consumption come from grains; out of this, about a quarter comes from wheat (González-Esteban, 2017). Wheat grain is a rich source of carbohydrates, dietary fibre, vitamins (B-vitamins) and phytochemicals (Shewry and Hey, 2015). In addition to this, it has 13-17% bran, 2-3% germ and 80-85% mealy endosperm (Šramková et al., 2009). Wheat is a significant source of globulin, albumin, and amphiphilic protein content (Dubreil et al., 1998). Furthermore, wheat provides lipids and essential minerals like calcium, copper, iron, phosphorus, potassium, manganese, magnesium, and zinc (Rachon et al., 2015). The current global wheat production is 642 million tons, and the future (2050) demand is about 840 million tons. This demand may be attained on limited resources (water, land) if new agronomic, physiological and genetic research strategies and practices are introduced (Sharma et al., 2015). Domestication of wheat occurred 10,000 years ago, and wheat spread worldwide as a major cereal crop. Its diverse adaptability to different environments makes it easy to domesticate. Genetic miscellany (ploidy level) of wheat and its progenitors reward novel diversity quickly in different climatic zones (Dubcovsky and Dvorak, 2007). Commercial wheat cultivation started in South Africa in the early 1910s in Cape Town, with seeds introduced earlier by the Dutch traders (Nhemachena and Kirsten, 2017), and has become the second most crucial grain crop cultivated in South Africa after maize (Anonymous, 2021; Bester, 2014). Both tetraploid and hexaploid wheat cultivars are produced in approximately 90% of the available agro-climatic regions of South Africa (Lantican et al., 2005). The dominant wheat-producing areas are the Western Cape (winter rainfall, mainly dryland), Free State (summer rainfall, both dryland and irrigated), Northern Cape (irrigated) and North West (mainly irrigated) provinces. Even though cultivation occurs in winter and summer rainfall regions, between 1983 and 2008, wheat was cultivated predominantly under dryland conditions where annual production averaged 1.5 to 3 million tonnes (2-2.5 tons/ha) (Nhemachena and Kirsten, 2017). However, about 30% of harvested wheat is produced under irrigation, where the yield potential varies between 6 to 12 tons/ha, with higher winter temperatures being the main limitation in the lower-yielding areas (Anonymous, 2021). The major companies or institutions supplying improved wheat cultivars in South Africa are Sensako (now part of Syngenta), Pannar Seed (Corteva AgrisciencesTM) and the Agricultural Research Council-Small Grains (ARC-SG) (Nhemachena and Kirsten, 2017). The wheat varieties are constantly improved for high yield and tolerance or resistance to prevailing drought, salinity, heat, pests and diseases. In South Africa, the wheat industry contributes about USD 40 billion to the gross value of agricultural production (Jankielsohn, 2016) and 28 000 jobs (Bester, 2014). Some pathogens (causing diseases like rust and powdery mildew) and pests similar to the Russian wheat aphid (RWA) significantly reduce yield and flour quality (Kazi et al., 2013). Russian wheat aphid infestations significantly challenge successful wheat production (Njom et al., 2017) because they reduce wheat yield and deteriorate flour quality (Girma et al., 1993). The emergence of RWA biotypes with increased virulence threatens wheat production and reduces the desired targets to meet the South African demand for high-quality wheat grain.Item Open Access Abiotic stress tolerance and nutritional traits of newly developed quality protein maize hybrids in sub-Saharan Africa(University of the Free State, 2022) Engida, Bitew Tilahun; Labuschagne, M. T.; Terekegn, A.; Van Biljon, A.; Wegary, D.Drought and poor soil fertility are some of the most serious maize production challenges in sub-Saharan Africa (SSA). Identification and development of quality protein maize (QPM) cultivars that have high yield potential and tolerance to these stresses is a reliable and affordable option to improve food security and malnutrition problems in the region, especially for small scale farming communities. Although several stress tolerant maize varieties have been released and disseminated for commercial production in SSA so far, limited development and release of stress tolerant and high yielding QPM varieties compared to normal maize varieties is evident. Limited attention has also been given to the development of nutritionally enriched varieties compared to grain yield improvement. Therefore, the main goal of this study was to study 40 newly developed QPM hybrids obtained from the International Maize and Wheat Improvement Center (CIMMYT) – Zimbabwe, under stressed and non-stressed environments to allow selection of QPM hybrids that could outperform the existing commercial QPM and normal maize cultivars with respect to grain yield and concentrations of tryptophan, iron (Fe), zinc (Zn) and molar ratios of Fe and Zn to phytic acid. The specific objectives were: (1) to determine variability and performance of QPM hybrids for grain yield and agronomic traits under stressed and non-stressed environments, (2) to determine tryptophan, Zn and Fe concentrations, and molar ratios of Zn and Fe to phytic acid in QPM hybrids grown under stressed and non-stressed environments, (3) to analyse genotype by environment interaction and grain yield stability of QPM hybrids and (4) to determine correlations among grain yield, agronomic and nutritional traits in QPM hybrids evaluated under stressed and non-stressed environments. Significant variation was seen for grain yield, and almost all studied agronomic and nutritional traits under stressed and non-stressed environments. Phenotypic coefficient of variation (PCV) was higher than genotypic coefficient of variation (GCV) for grain yield and all other agronomic and nutritional traits under all conditions, indicating that environment effect was higher than genotype effect on the expression of the traits under stressed and non-stressed environments. Broad sense heritability of grain yield was higher than 0.6 across all environments, with the exception of managed drought conditions. Anthesis silking interval (ASI) had relatively high GCV estimates and genetic advance, as a percentage of the mean, across all conditions. This indicated that the presence of sufficient genetic variability among genotypes can improve synchronization under different management conditions through selection. Grain yield was reduced by 47% under random stress, 68% under managed drought and 71% under low N conditions. Protein and tryptophan concentrations in the grain were decreased by 36.0% and 21% respectively under low N conditions and Fe and Zn concentration also decreased by 48% and 36% under low N stress and 63% and 9% under random stress, respectively. Some QPM hybrids showed better or comparable performance in terms of grain yield potential and nutritional quality traits compared with the best QPM and normal maize checks under different management conditions, indicating the genetic gain that has been made in the QPM breeding programme. Based on Additive Main effect and Multiplicative Interaction (AMMI) analysis and Genotype and Genotype by Environment interaction (GGE) biplot analysis entries 10 (CZH142238Q) and 14 (CZH15142Q) under optimum; 23 (CZH17192Q) under random stress; 19 (CZH17188Q) and 40 (CZH17209Q) under managed drought and 14 (CZH15142Q) under low N were the most stable and the highest yielding hybrids. Environments Kwekwe (KW), Bindura (BIN), Chokwe (CHO) and Bako (BK2) were identified as discriminating and representative sites for optimum conditions, random stress, managed drought and low N stress conditions, respectively, therefore these environments are promising for selecting well adapted genotypes in the respective management conditions. Grain yield was significant and positive correlated with number of ears per plant and negatively with days to anthesis and silking under low N stress. This confirmed the importance of these secondary traits in developing high yielding and early maturing genotypes. Grain yield was not significantly correlated with most of the nutritional quality traits under all management conditions, indicating a lack of common genes for simultaneous improvement of grain yield and these nutritional traits. Significant and positive correlations were observed between Fe and Zn under low N and random stress conditions.Item Open Access Determining the trajectory of graminoid invasions in Southern Africa's mountains: The case of Nassella(University of the Free State, 2022) Mapaura, Anthony; Steenhuisen, S.; Canavan, K.; Clark, V. R.; Richardson, D. M.Nassella neesiana (Trin. & Rupr.) Barkworth, N. tenuissima (Trin.) Barkworth and N. trichotoma (Nees) Hack. & Arechav are three alien grass species that have become naturalised in South African montane grassland – mostly known historically from remote mountainous areas in the Eastern Cape. Currently, only N. trichotoma and N. tenuissima are recognised as serious invasive species in category 1b of the National Environmental Management: Biodiversity Act (NEM:BA, 10/2004): Alien and Invasive Species (AIS) Regulations of 1 October 2014. However, N. neesiana, which is currently not listed in NEM:BA, is a prolific invader in Australia and New Zealand, where it is causing serious damage to both natural and planted areas, and costing thousands of Australian dollars in control measures and lost land potential. In South Africa, some research and central government-facilitated management measures were undertaken from the late 1970s to the year 2000. Since then, very little data have been generated about the status of any of these species in South Africa. Literature pointed out, inter alia, the difficulty of identifying the species in the field, especially when plants were not fertile; this may be a reason for sparse distribution records including limited records from citizen science projects that record invasive species occurrences. Chapters 1 and 2 provide a synthesis of global knowledge on Nassella available at the start of this project. The principal aims of the research were to (i) establish the current invasive status of the three Nassella species in South Africa, (ii) determine the impacts of N. trichotoma and N. neesiana on native flora, and (iii) to predict how their invasive status might change in the future under projected climate change scenarios. A combination of fieldwork, laboratory analysis, and modelling were used to answer these questions. The impact of N. trichotoma and N. neesiana on co-occurring plant species and chemical properties of the soil was investigated using field surveys and modelling techniques (Chapter 3 and 4). The research focused on vascular plant biodiversity differences between dense patches of Nassella and matched nearby uninvaded areas – space-for-time substitution. Nassella trichotoma was found to reduce the diversity of native species in montane rangelands while increasing the concentrations of minerals in topsoil. Nassella neesiana, on the other hand, has little impact on native plants in a disturbed urban setting. However, the presence of both Nassella species is associated with increased concentrations of minerals in topsoils and an increase in ruderal vascular plant species. This study is the first known research on N. neesiana in South Africa. The possibility of using DNA markers to distinguish the three Nassella species and separate them from co-occurring morphologically similar species was investigated (Chapter 5). Two candidate gene loci, petL–psaJ (petL) and ETS, were evaluated to determine their ability to discriminate the three Nassella species from each other and from three co-occurring native grass species (Aristida diffusa Trin., Festuca caprina Nees and Koeleria capensis (Steud.) Nees), and the exotic species Jarava plumosa (Spreng.) S.W.L. Jacobs & J. Everett in South Africa. The three native grass species have been confused with Nassella species before. Jarava plumosa and the three Nassella species used to be in the same genus, Stipa, before the recent taxonomic revisions. The other minor objective was to find out if this method can be used as a viable option for field practitioners. The results indicate that the petL locus has no power to discriminate the three species, but can discriminate the different genera; in contrast, ETS has sufficient power to discriminate all the tested species, and can be outsourced at a reasonable cost, making it a viable identification method even for people without technical knowledge. The current and future (2081 to 2100 period) potential distribution of the three Nassella species was investigated (Chapter 6). The bioclimatic variables were downloaded from the WorldClim database (http://www.worldclim.org), at a spatial resolution of 2.5 arc minutes. The distribution of all three species is mainly driven by temperature-based bioclimatic variables, especially Isothermality. The research found that – under current climatic conditions – all three species can potentially occupy more area than they currently do. Compared to currently suitable envelopes, future projected envelopes will be much smaller for the three species. The Maloti-Drakensberg mountains (including Lesotho) will remain at high risk of invasion across all future climate-change scenarios, and it should be a priority for keeping N. trichotoma out of the Maloti-Drakensberg, along with managing N. tenuissima and N. neesiana invasions in the Maloti-Drakensberg. The overall finding of this research is that the three Nassella species remain very high-risk species for continued invasion of montane grasslands in South Africa, particularly in the most important regional water tower: the Maloti-Drakensberg. Nassella neesiana should be formally placed on an invasive “watch-list”, for potential addition to the AIS Regulations (Category 1b), as it appears to have a much wider distribution and be much more widespread in the landscape than records show. Considering their impact on vascular plant diversity, rangeland quality and difficulty of management, they should be cause for major concern by landowners and government. This research provides a platform for resurrecting a research and management focus on Nassella in South Africa (and Lesotho), with the aims of securing montane rangeland-based livelihoods, ensure conservation of endemic mountain biodiversity, and safeguard mountain catchment potential under climate change.Item Open Access Biological and pharmacological activities of root extracts and isolated compounds of hermann/a geniculata Adeniran(University of the Free State, 2017) Ariyo, Adeniran Lateef; Ashafa, A. O. T.Hermannia geniculala is of the genus of flowering plant from the subfamily Byttnerioideae in the family Malnaceae. H. geniculata has been used in treatment of several diseases like colic, diabetes mellitus and other oxidative stress induced illnesses. The dry root material is chopped, boiled in water and taken three times daily to ameliorate blood sugar disorders, management of diarrhoea, heartburn, stomach disorder and flatulency called "leletha" in pregnant Basotho women. Phytochemical analysis revealed the presence of saponin, phenols, flavonoids, alkaloids, tannin, phytosterol, triterpenes and anthraquinone. Ethanolic extract exhibited the highest free radical scavenging capability with the lowest ICso value (0.52, 0.38, 0.59, 0.63, 0.39 mg/mL) for I, ldiphenyl- 2-picrylhydrazyl (DPPH), 2,2-Azino-bis(3 ethylbenzothiazoline-6-Sulphonic acid (ABTS), hydroxyl radical, superoxide anion radical, metal chelating abi lity which is significantly lower (p<0.05) than the standard si lymarin while hydro-ethanol has the highest reducing power showing a significant (p<0.05) ICso value of 0.24 mg/mL compared to citrate (!Cso value; 0.5 mg/mL). In antidiabetic studies, ethanolic extract was a potent inhibitor of a-glucosidase (!Cso; 0.0 I mg/mL) which is significantly lower (p<0.05) than standard acarbose ICso value (0.52 mg/mL) and hydro-ethanol decoction and aqueous extracts. It also has a milder percentage inhibition of a-amylase enzyme with lC.'io (0.57mg /mL) which is significantly higher (p<0.05) than the standard acarbose ICso (0.047 mg/mL). The mode of inhibition of a-amylase is by competitive inhibition and uncompetitive inhibition of the o.-glucosidase enzyme was observed in ethanolic extract. These find ings provide an empirical rationalization for the use of the root extract of Hermannia geniculata in the management of diabetes mellitus and other oxidative stress induced ai lments. The Vero, HepG2 and RAW 264.7 macrophage cell lines were used to determine the toxicity of the extracts on cells. Similarly, the capabilities of the extract to inhibit 5-lipoxygenase enzyme activities and overproduction of nitric oxide from LPS-activated RAW 264. 7 macrophages were evaluated. Results showed selective toxicity of the extracts with LCso values of Vero cells ranges from (0.40-0.57 mg/mL) while the LCso value of HepG2 cells varies from (0.0 16-0. 136 mg/mL). The selectivity index (SI) were (3 1.87, 18.87, 33.33 and 3.52) for ethanol, hydro-ethanol, decoction and aqueous extracts respectively. The ethanolic extract inhibited NO production in a concentration dependent manner. There was a decrease of 82% at concentration of0. I mg/mL and the LCso:3.64 mg/mL is lower and significantly different (p<0.05) compared to the reference compound quercetin with LCso value of 8.28 mg/mL. Similarly, the ethanolic extract exhibited potent inhibition of 5-lipoxygenase enzyme with the lowest ICso value of 0. 14 mg/mL which is significantly different (p<0.05) compared to all other extracts and indomethacin. The GCMS chromatograms revealed five compound (2-keto-butyric-acid, 2, 2-Bis ( 4-nitrobenzyl)-l - phenylbutane-1 ,3-dione, n-Undecane, 1,4,5,8-tetrathiadelin and imidazo-1,5-pyrimidine) which has been reported to have antioxidant, anti-inflammatory and antifungal properties. This result suggested that Hermannia geniculata roots extract is not toxic and possesses antioxidant, antinflammatory and anticancer activities which could be exploited in development of new safe and effective drugs. The chemical profiling and in vitro biological activities of flavonoids of Hermannia geniculate (FHG) roots was also investigated using High Pressure Thin Layer Chromatography (HPTLC) finger print analysis. Antioxidant, antidiabetic anti-inflammatory activities and the ability of FHG extract to inhibit the production of nitric oxide (NO) in lipopolysaccharide (LPS) activated RA W264.7 Macrophage were investigated using standard methods. The selective cytotoxicity of the extract on Vero and HepG2 cells was also determined. Kaempferol (Rr0.81) was detected in the extract, its Rr value is similar and comparable with the kaempferol standard used (Rr 0.80). Other flavonoids were also present in the extracts with their Rr values of 0.08-0.95. The FHG extract showed commendable antioxidant properties with !Cso values (3.07± 0.12, 2.13± 0.67) for DPPH and ABTS radicals which was lower and significantly different (p<0.05) compared to standard silymarin with !Cso: (3.55± 0.10, 2.77± 0.75) for DPPH and ABTS respectively. The results indicated milder inhibition of a-amylase with ICso: (5.55± 0.37) which was higher and significantly different from the standard acarbose with ICso: (3.81± 0.29) Nevertheless, the extract exhibited 73% inhibition of a-glucosidase which exerted better inhibitory effect on 5- lipoxygenase enzyme than indomethacin with their respective !Cso: ( I 0.15± 0.02 and 12.03± 0.02). Inhibition of NO production was observed in LPS activated RAW 264. 7 Macrophages with the highest concentration of 0.1 mg/mL decreasing NO production by 87%. Selective toxicity of Vero and HepG2 cells with their respective LC so value of(> I and 0.02 mg/mL) was also observed. The antipro liferative potentials of the extract was confirmed with Selectivity Index of 50. This study indicated for the first time that FHG extract was non-toxic to normal cells and possess antioxidant, antidiabetic, anti-inflammatory and antiproliferative activities. The bioactive constituent and pharmaco logical activ ities of pheno ls extracted from Hermcmnia geniculata (PoHG) roots was investigated using in vitro methods. The chemical profile was determined by HPTLC analysis. Antioxidant, antidiabetic, anti-inflammatory and cytotoxic effect of PoHG on Vero and HepG2 cells was carried out using standard procedures. Phenolic compounds were detected in the sample at Rr (0.14, 0.81 and 0.95). PoHG radical scavenging capabilities on DPPH, ABTS+ and superoxide anion radicals were similar to the standard (silymarin). The ICso values were DPPH (0.12± 0.00), ABTS (0. 13± 0.01) and superoxide anion (0.20± 0.00). The values of the metal chelating activity of Po HG extract is lower and significantly different from the standard (silymarin) their respective ICso values were (0.06± 0.00 and 0.18±0.0 I). The antidiabetic effect was determined by its ability to mildly inhibit a -amylase and strongly inhibit a -glucosidase enzymes, the respective ICso values obtained were (7.52± 0.23 and 1.76 ± 0. 14). PoHG extract exhibited a commendable inhibition of 5-lipoxygenase enzyme with ICso value of (0.15 ± 0 .03) which is similar to the ICso: (011 ± 0.0 I) value for the standard (indomethacin). However, the extract was non-toxic to Vero cells with LCso value of> 1.00 mg/mL but highly toxic to HepG2 cells with LCso: 0.08 mg/mL. The selectivity index of 12.50 was recorded. The presence of phenolics/ carboxylic acids were a lso confirmed in the extract, the result of the antiox, antidiabetic and antinflammatory activities of Po HG suggested that the phenols extract may be useful in the management of oxidative stress induce diseases, type 2 diabetes and asthma. It is also safe for use and its antiproliferative activities can be exploited in search for anticancer agents. A new xanthene derivative Hermannol (9-(7-methyloctyl)-9H- xanthene-2,3-diol) was isolated from the roots of Hermannia geniculata. The structure was e lucidated by ana lysis of their ID, 2D NMR, MS and IR spectroscopic data. The compound displayed good antioxidant and antidiabetic activities. In conclusion, it is evident from the study that d ifferent crude extracts of H geniculata roots and its bioactive constituents (tlavono ids and phenols), the isolated compound (Hermanno l) is nontoxic and possess varied degree of antiox idant, antidiabetic, antiiflammatory and antiproliferative activities which can be exploited for new drug development.Item Open Access Genetic variability of carotenoids and polypoid induction towards vitamin A biofortification in plantain (Musa spp.)(University of the Free State, 2018-07) Amah, Delphine; Labuschagne, Maryke Tine; Swennen, Rony; Van Biljon, AngelineVitamin A deficiency (VAD) is one of the most prevalent nutrient deficiencies affecting the health of resource-poor populations in developing countries. Plantains (Musa spp. AAB) are a specific group of bananas, which serve as a staple for millions of people in the humid lowlands of West-Central Africa and high provitamin A carotenoid (pVAC) plantain varieties, could have significant impact on VAD in these regions. In this regard, the International Institute of Tropical Agriculture (IITA) based in Nigeria, is developing a breeding pipeline aiming to generate and deploy plantain hybrids combining high pVAC content with other consumer-preferred traits. The overall objective of this study was therefore to assess the variability of fruit pVAC content in banana cultivars and hybrids present in the collection of IITA, Nigeria, and investigate the potential of induced polyploidization as a breeding approach towards plantain biofortification. A wide collection of 204 genotypes of bananas (AAB-plantains, M. acuminata cultivars and bred hybrids) was screened to determine variability in fruit pVAC content using high- performance liquid chromatography (HPLC) and spectrophotometry. Mean total carotenoids (TC) measured by spectrophotometry ranged from 1.28 to 32.03 with a mean of 8.88 μg g-1 fresh weight (FW) indicating a high variability of carotenoids in bananas. There was a strong correlation between TC measured by spectrophotometry and that estimated from HPLC, confirming the potential of spectrophotometry as a useful, inexpensive method for rapid screening for pVACs in banana. Predominant carotenoids isolated were α-carotene (38.67%), trans β-carotene (22.08%), lutein (22.08%), 13-cis-β- carotene (14.45%) and 9-cis β-carotene (2.92%), demonstrating that about 78% of the carotenoids in bananas are pVACs. Provitamin A content estimated in terms of β-carotene equivalents (BCE) ranged from 0.24 to 21.06 μg g-1 FW with a mean value of 4.42 μg g-1 FW across all genotypes. Importantly, 10 plantain cultivars, three M. acuminata diploids and four hybrids with relatively high pVAC contents were selected for integration in banana biofortification efforts to tackle VAD. To assess the effect of ripening on pVACs in plantain fruits, nine cultivars across the three main plantain types (French, False Horn and Horn) were screened at the unripe, ripe and overripe stage. Mean TC measured by spectrophotometry for plantain cultivars at the unripe, ripe and overripe stage was 16.94, 11.98 and 10.11 μg g-1 FW while mean BCE was 13.65, 6.95 and 5.05 μg g-1 FW, respectively. Notably over 80% of carotenoids in plantain cultivars were pVACs α-carotene and β-carotene across all ripening stages. French plantains had slightly higher BCE contents than False Horn and Horn types but this difference was only significant (P<0.05) at the unripe stage. Overall, ripening led to a decrease in pVACs content from the unripe to the ripe to the overripe stages accompanied by a corresponding increase in lutein content indicating that unripe fruits could yield more provitamin A than ripe and overripe fruits. To explore the application of induced polyploidization as a breeding strategy for pVAC enhancement in plantains, 10 induced tetraploids derived from six diploid cultivars were evaluated for their agronomic attributes, carotenoid content and fertility. Tetraploids had distinct plant morphology but generally displayed inferior vegetative and yield traits from their corresponding diploids. Similarly, a 50% decrease in pVACs accompanied by a corresponding increase in lutein was recorded in induced tetraploids in comparison to their original diploids. Nevertheless, preliminary fertility assessments indicated over 70% pollen viability for induced tetraploids from four diploid cultivars. These findings demonstrated the use of induced polyploidization to generate useful genetic material that could be incorporated in hybridization programmes aiming to produce high pVAC triploids.Item Open Access Characterisation of the physiological, biochemical and molecular responses of sorghum to drought stress(University of the Free State, 2018-11) Goche, Tatenda; Ngara, Rudo; Chivasa, StephenDrought is a major threat to global food security due to its detrimental effects on plant growth, productivity and yield quality. Many climatic models are predicting the increasing duration and severity of drought episodes. Therefore, understanding plant adaptive responses to drought stress is important in developing new biotechnological solutions to avert crop yield losses to drought. Sorghum (Sorghum bicolor) is an African indigenous crop that is well-adapted to thrive on marginal lands. This makes the crop a suitable model plant for studying adaptive responses to drought. In this study, the physiological and biochemical responses of two sorghum varieties with contrasting phenotypic traits to drought stress was analysed under drought stress. The two sorghum varieties used were the drought susceptible ICSB 338 and the drought tolerant SA 1441. The sorghum plants were grown in soil until the V3 growth stage before withholding water for 8 days, re-watering and then assessing the physiological changes following the drought stress treatment. Physiological analyses of the plants revealed striking differences between the sorghum varieties. The growth parameters of both roots and shoots exhibited more tolerance related responses in the drought-tolerant variety, while the susceptible variety was adversely affected and had poor recovery after re- watering. The leaf relative water content, stomatal conductance and chlorophyll content, supported the observed physiological adaptations. The analysis of proline and glycine betaine showed that there was an increase in the accumulation of the two omolytes in response to drought stress. The drought tolerant variety showed significantly higher osmolyte accumulation earlier than the drought susceptible variety in both root and shoot tissue. The isobaric tags for relative and absolute quantitation (iTRAQ) analysis was used to identify drought-stress responsive root proteins in the two sorghum varieties. In the root proteome, 1169 and 1043 proteins were positively identified for ICSB 338 and SA 1441 sorghum varieties, respectively. Of these proteins, 237 and 184 were drought responsive for ICSB 338 and SA 1441, respectively. A large proportion of the proteins are involved in disease/defence (26% for ICSB 338 and 23% for SA 1441) followed by metabolism (25% for ICSB 338 and 21% for SA 1441). To validate gene function, eight proteins with the highest fold-change in response to drought were selected for gene expression analysis using quantitative real time- polymerase chain reaction (qRT-PCR). The results showed that all the genes evaluated were drought stress responsive. In order to develop the eight target genes as drought markers, their expression was analysed in cell suspension cultures of White sorghum and ICSB 338 with and without sorbitol treatment. The gene expression analysis showed that seven of the eight drought responsive genes could distinguish between White sorghum and ICSB 338 in the cell suspension culture system without sorbitol treatment. In addition, all the eight genes could distinguish between White sorghum and ICSB 338 in response to the sorbitol-induced osmotic stress. Following this, the responses of the genes to heat stress was analysed in the White sorghum cell suspension cultures. The results showed that seven of the genes were also heat responsive. These genes are recommended for use as drought markers in marker assisted selection for drought tolerance. As proof-of-concept and to develop a workflow for the use of the drought markers in other crops, three published genes from our research group were used. Four Arabidopsis (Arabidopsis thaliana) homologues of each sorghum gene were selected for gene expression analysis. The results showed that there is differential gene expression between homologues of the same gene in response to osmotic stress. In conclusion, the comparative sorghum physiological, biochemical, protein and gene expression data generated in this study forms a foundation for further sorghum molecular studies. Furthermore, the drought marker genes toolkit developed in this study can be utilised by plant breeders in marker assisted selection for the improvement of agriculturally important crops against drought.Item Open Access The influence of sorghum physiology on rhizosphere interactions and their effect on root disease(University of the Free State, 2018) Chung, Hung Yu; Swart, W. J.; Mclaren, N. W.This study evaluated and investigated sorghum allelopathy and its various effects on microbial populations in the rhizosphere and effect of soil-borne pathogens. The effect of sorghum allelopathic extracts on rhizosphere soil microbial activity and diversity as well as the growth and incidence of soil-borne pathogens was investigated. The effect of different sorghum genotypes on the rhizobiome, root rot and yield were also investigated. Evaluation of the allelochemical contents of 22 sorghum genotypes showed variation between the concentration of their total phenolics and sorgoleone. The highest phenolic content was genotypes BTX ARG-1 (tan plant), RTam 2566 (purple plant), RTx 436 (tan plant) and BTx 635 (tan plant) and the lowest phenolic content was genotypes S5C719-11E (purple plant) and SCAY 21 (tan plant). The highest amount of sorgoleone was produced by genotype BTx 3197 (purple plant) and the lowest was genotype Tx 2911 (red plant). Phenolic extracts of RTx 430, RTx 436, Rtam 428, SCAY 21 and sorgoleone extracts of genotype BTx 3197 were selected for further experiments. The effect of sorghum phenolic and sorgoleone extracts on eight soil-borne pathogens in vitro showed that the extracts significantly stimulated the colony diameter of most of the pathogens. Phenolic extracts of RTx 430 and RTx 436 was found to be the most stimulating against the eight soil-borne pathogens. Sorgoleone extracts was found to significantly inhibit the colony diameter of C. capsici and F. equiseti compared to the control. Microcosms investigating the direct effects of sorghum phenolic and sorgoleone extracts on soil microbial populations showed differences (< 55 % similarity) between the functional diversity of rhizosphere soils treated with phenolics and sorgoleone compared to the untreated control. Microcosms investigating effect of the extracts on soil-borne pathogens showed significant differences in leaf length, root length and root rot rating of the treated plants compared to the control. The root rot of sorghum plants inoculated with A. alternata, C. trifolii and F. thapsinum were found to be the most inhibited by the extracts and plants inoculated with A. strictum and C. capsici were found to be the least inhibited. Extracts of SCAY 21 0.5x, RTam 428 1x and sorgoleone 1x were the most inhibiting and extracts of RTx 436 0.5x and SCAY 21 1x, the least. Microcosms investigating the indirect effects of the extracts on soil- borne pathogens showed that soils previously treated with extracts were most suppressive against A. alternata and F. thapsinum and least against P. macrostoma. Soils previously treated with RTx 436 0.5x and SCAY 21 0.5x extracts were most effective in suppressing the pathogens. The rotation trial with two sorghum genotypes showed significantly higher microbial activity in soils undergoing rotation compared to monoculture and fallow. The trial also showed differences (< 84 % similarity) between soils previously planted with soybean compared to other treatments. In the 1st season, a significantly higher root mass was recorded in sorghum undergoing cowpea and dry bean rotation compared to monoculture. In the 2nd season, significantly higher root mass was recorded in sorghum undergoing soybean and dry bean rotation compared to fallow. A significantly lower (P < 0.05) yield was recorded in sorghum plants undergoing fallow and monoculture compared to other rotations. The results of this study revealed various effects of sorghum allelochemical extracts on microbial populations in the rhizosphere and soil-borne pathogens. Further research should be conducted to clarify these effects by using molecular techniques such as next generation sequencing (NGS).Item Open Access Genetic diversity analysis of linseed (linum usitatissimum L.) in different environments(University of the Free State, 2002-11) Gemelal, Adugna Wakjira; Labuschagne, M. T.; Osthoff, G.; Viljoen, C. D.English: The study was carried out to assess the genetic diversity of 60 linseed accessions mainly collected from Ethiopia in different environments using morphological and amplified fragment length polymorphism (AFLP) markers from 2000 to 2002. AFLP and morphological characterization were conducted under glasshouse conditions at the University of the Free State in South Africa. Similarly, morphological evaluations were undertaken under field conditions at Holetta Research Centre in Ethiopia during 2000 and 2001 cropping seasons. The main objectives of the study were to determine the levels and patterns of genetic diversity along with other genetic parameters using both morphological descriptors and the AFLP markers, and to compare the usefulness and relationships of these two methods in discriminating the accessions of linseed by applying univariate, bivariate and multivariate statistical analyses. The analysis of variance for the glasshouse experiment showed highly significant difference (P < 0.01) among the accessions for Il quantitative traits measured, indicating the presence of high genetic diversity. This result was also confirmed by the principal component analysis (PCA) and cluster analysis (CA). PCA displayed that days to flowering, maturity and seeds/boil accounted for 20% of the total variability. CA grouped the accessions into Il main clusters, consisting one to 24 accessions each. No correspondence was found between clustering and geographic origins of the accessions. Estimation of broad sense heritability and predicted genetic gains (as percent of mean) were also computed to forecast the possible genetic advance in the future. Heritability ranged from 15.60% for seed yield/plant to 85.82% for initial days to flowering, whereas predicted genetic gains varied from 3.16% for days to maturity to 24.26% for plant height. The analysis of variance for the field experiments revealed highly significant differences (P < 0.01) among the accessions for Il characters. PCA and CA also denoted the same thing, showing the presence of a wide range of genetic variations between the accessions studied. CA clustered the accessions into nine classes, each consisting one to 39 accessions. The clustering was independent of collection areas. Phenotypic coefficient of variation ranged from 6.26% (days to maturity) to 54.97% (seed yield/plant), while genotypic coefficient of variation differed from 5.46% to 50.18% for the same characters. Heritability (broad sense) was in the range of 67.38% to 91.38%, whereas the predicted genetic gain varied from 9.38% to 94.37%. In both cases, the highest value was estimated for seeds/ball, while the lowest was for days to maturity. A combined analysis of variance of 10 quantative traits across four environments (two localities and two years) displayed highly significant differences (P < 0.01) among the accessions for all characters. There was also significant difference between the localities, years and their interactions for most characters, indicating the differential responses of the accessions across environments. PCA and CA also confirmed the differences between accessions and the prevalence of diversity among the accessions. PCA displayed that secondary branches/plant, plant height, days to flowering and seed yield/plant played major roles in differentiating the accessions. CA grouped the accessions into nine major clusters based on their mean performance rather than their geographic origins. Broad sense heritability and expected genetic advance were found higher across multi-environments than for the single one, indicating the importance of evaluating germplasm under different and appropriate environments. Both parameters were highest for plant height and days to flowering, indicating the effectiveness of selection for these traits. Diversity analyses for oil content, oil yield and fatty acid profiles generally indicated significant (P < 0.01) variations among the studied accessions. Oil content varied from about 29 to 36%, while oil yield ranged between 1443 and 3276 g/m2. The highest oil yield was obtained from Belay-96, followed by CDCVG (3212 g/m2), an introduction from Canada, which was identified as one the most promising genotypes in this study. Fatty acids that are principally grouped into two main groups (saturated and unsaturated) showed wide ranges of variation. Saturated fatty acids (palmitic and stearic) ranged from about 8 to 12%, whereas the unsaturated (oleic, linoleic and linolenic) ones significantly (P < 0.01) varied from 85 to 91%. The highest variation was recorded for oleic acid (14-29%), followed by linolenic (47-59%) and linoleic (10-16%) fatty acids. The accessions were grouped into six cluster based on their oil content, oil yield and linolenic acid, but into 10classes based on all their fatty acid profiles. Associations of characters were analysed across different environments to determine the extent and consistency of correlations and to identify major yield attributes of linseed. The degrees of correlation varied across years and locations due mainly to climatic factors, such as temperature, moisture regime and disease incidences. Seed yield was significantly (P < 0.01) and positively correlated with yield/plant, boils/plant and 1000-seed weight. These three traits were also strongly and positively related with plant height, branches/plant and days to maturity. Oil yield was significantly and positively associated with polyunsaturated (linoleic and linolenic) fatty acids, while it was negatively correlated with saturated (palmitic and stearic) fatty acids. The results generally showed tremendous variations in correlations of characters in response to the growing environments. The current accessions of linseed were assessed for their genetic variation by using AFLP markers. The estimated genetic distance for the entire pairwise accessions varied from 0.29 to 0.71. The top three pairs of accessions with the highest genetic distance were 22 and 57, 25 and 57 and 20 and 45. High levels of polymorphism were also observed for the analysed accessions, indicating the prevalence of genetic diversity in both exotic and local collections. Collections from central and northwest regions of Ethiopia revealed considerable variations, implying further explorations in these areas. Cluster analysis grouped the accessions into 13 classes, each consisting one to 14 accessions. AFLP was found effective in discriminating and grouping the accessions for more predictable breeding and efficient management of genetic resource in the future. Combining phenotypic and genotypic assessment of genetic diversity could help in acquiring more reliable genetic information for discriminating germplasm and cultivars. To this end, morphological and AFLP data were employed to investigate the levels and patterns of variation existing in linseed accessions and to compare the two methods. Analyses of ANOVA, PCA and CA revealed the existence of wide range of genetic diversity among the accessions. The average genetic distance of all pairwise accessions was 0.6684 for morphology and 0.5734 for AFLP markers. Higher level of variation was noted for morphology than for AFLP. Correlation between the two distances was very weak and their clustering patterns were also different. In fact, clustering patterns were variable based number of traits and environments tested. In spite of this disparity, however, both methods were found independently adequate and useful in germplasm characterization and/ or cultivar identification based on circumstances, like the availability of research facilities, fund and other essential resources.Item Open Access Studies on the survival of Verticillium dahliae in soil(University of the Free State, 1979-12) Baard, Schalk Willem; Pauer, G. D. C.Various aspects concerning the survival of Verticillium dahliae Kleb. in soil, in the field, green-house, and laboratory were studied. 'With biological control in mind, attention was paid to factors affecting antagonists of V. dahliae and to colonization of microsclerotia (MS) in soil. Possible activation of antagonists during the period of absence of host plants was considered. Regression analysis of survival data indicated that individual MS are capable of surviving up to 43 months in soil in the absence of host plants. Soil moisture and temperature could not be related to the attrition of the pathogen. However, microbial colonization of the MS could have had an effect. Pathogen propagules were released into rhizosphere soil after the plants had been killed. Antagonistic fungi did not appreciably increase in the rhizosphere soil after the release of pathogen propagules. Bacteria and actinomycetes antagonistic to V. dahliae could be stimulated to increase in high pH soil by the addition of MS and fertilizers containing phosphate. In low pH soil, which favoured fungal antagonists, these tendencies were less obvious. The largest numbers of bacterial and actinomycetous antagonists occurred in high pH soil in which V. dahliae survived best. The attrition rate of MS was fastest in low pH (c. pH 4,5) soil. However, it was established that fungal antagonists were not mainly responsible for the attrition. Active microbial invasion of MS in soil was established by electron microscopic studies. Apparently lysed cell walls and eroded areas in the immediate vicinity of bacteria indicated that enzymes may be involved in the deterioration of the cell walls. It is concluded that antagonists are capable of actively destroying MS in soil, but this probably is a much slower process than that which was observed in acidified soil. Various techniques were used to establish the fact that attrition was much faster in acidified than in alkaline soil. The use of several techniques demonstrated that the effect of ·low pH on the attrition of the pathogen was real and not a reflection of the inadequacy of a single technique. It was also evident that the effect of low pH was fungitoxic and not fungistatic to the pathogen. Experiments to test the validity of the claim that the Al-ion is toxic to V. dahliae at very low concentrations indicated that the attrition of V. dahliae was as fast in low pH soil devoid of aluminium salts as in aluminium-amended soil. Soil acidification may be considered as a control measure. However, practical and economic considerations will prohibit its implementation. Apart from the cost factor, most plants do not tolerate such a low soil pH. Liming of the soil would be necessary, with the result that favourable conditions are again created for renewed increase in pathogen numbers. In a green-house study it was found that varying the moisture content of the soil and incorporating N and P, were ineffective as measures to reduce Verticillium populations. However, in flooded and air-dried soils, significant decreases occurred. The addition of urea at 0,25% or higher to the soil, reduced Verticillium populations appreciably. Various organic soil amendments gave diverse results. Maize residues, followed by soyabean pods caused the fastest attrition of NS in the soil. The addition of urea to soil at high rates would not be practical as a control measure. In situations where cotton can be produced in rotation with paddy rice, flooding may be of practical use.,Air-drying of the soil will depend on weather conditions, but it may be possible to devise agronomic practices to speed up the drying-out process and thereby reduce pathogen populations. This 'finding should, however, be studied under field conditions to verify the results obtained in the laboratory. The reduction of MS in the soil after amendment with plant residues holds promise as a control measure. Various crops could effectively be used in rotation with cotton and when the residues are incorporated into the soil, a significant attrition rate could be expected. However, the large quantity (1%) of residues required to effectively reduce the population of V. dahliae suggests that attrition would not be as fast under field conditions as it was under laboratory conditions. A long term rotation might thus be necessary. Electron microscopic studies on the fine structure of the MS indicated that they are composed of numerous thin- and thickwalled cells. The cell walls varied in thickness and were impregnated with melanin which also occurred in the matrix between the individual cells. These properties confer resistance to attrition to the MS. In the soil they apparently germinate over extended periods and give rise to limited hyphal growth which exhausts the reserves of the less resistant cells. However, some of the more resistant cells may remain· dormant and retain the viability of the MS. This may explain the survival of some propagules even under the adverse conditions to which they were subjected in the present study. It also explains why V. dahliae is such a difficult pathogen to eradicate under normal agronomic conditions.Item Open Access A phylogenetic study of the South African representatives of the tribe Andropogoneae (Poaceae)(University of the Free State, 2003-11) Holder, Francisca; Spies, J. J.English: The tribe Andropogoneae makes up half of the grass subfamily Panicoideae, with approximately 85 genera and 960 species (Hartley 1958, Clayton and Renvoize 1986). The genera of the tribe are typically tropical with only a few species extending beyond the tropics into warm temperate regions. From information available it is clear that the African representatives form an integral part of the tribe. In this study we only concentrated on the South African representatives of the tribe. The tribe Andropogoneae has been studied extensively over the last millennium, but there is still an uncertainty about the true basic chromosome number. Previous molecular studies include sequencing of the ndhF, GBSSI and phytochrome B. The morphological variation in the tribe provides an interesting context to examine. This study focused on the sequencing of plastid chloroplast gene trnL-F and nuclear ribosomal DNA the Internal Transcribed Spacer (ITS) to determine the phylogenetic relationships within the tribe. In this study the chromosome numbers of 58 specimens were determined. The genetic chromosome numbers varied with n = 5, 9, 10, 10, 11, 20, 30. For the first time the basic diploid number of n = 5 was observed in the genus Andropogon. Urelythrum aggropyroides was studied for the first time with a somatic chromosome number of 2n = 20. The absence of multivalent and prevalence of bivalents in this study indicate that the genomes of the specimens studied are homologous. This lead us to the conclusion that the tribe consists of allopolyploid species, derived from interspecific hybridization and chromosome doubling. Both the trnL intron and trnL-F intergenic spacer was sequenced. Analysis of the trnL-F gene included 56 accessions and 61 accessions for the ITS gene. Combined analysis of both ITS and trnL-F included 59 accessions. Sequencing data indicated Cymbopogon to be a diploid progenitor of Hyparrhenia and Andropogon of Bothriochloa bladhii. Reticulate evolution was demonstrated in nrITS alleles. ITS and trnL-F data supports the inclusion of Arundinella in Andropogoneae and rejects the subtribal classification of Clayton and Renvoize (1986). This data also does not support the division of the tribe into awned and awnless genera and does not support the “core Andropogoneae” lineage as previously been observed. Most genera in the tribe are polyphyletic, suggesting a much more complex nature for the South African representatives. Molecular data could not prove the basic chromosome number but supported the maturity of the polyploid complex for the South African specimens.Item Open Access Quality prediction and improvement in Lesotho commercial wheat (Triticum aestivum L.) cultivars(University of the Free State, 2005) Morojele, Motlatsi Eric; Labuschagne, M. T.English: • The objectives of this study were to characterize wheat cultivars grown in Lesotho according to their glutenin and gliadin banding patterns with the use of SOS-PAGE, estimate GCA, SCA, h2, heterosis and correlation of wheat quality traits by crossing poor, medium and good breadmaking qualities in a full diallel design to create F 1 and F2 progeny, and determine the potential of SE-HPLC in predicting quality of wheat cultivars. • Glutenin and gliadin proteins were separated using SOS-PAGE. Cultivars used for estimating combining ability were grown in pots and crossed in the greenhouse at Bloemfontein. The F 1 progeny were grown in Lesotho and Bloemfontein while the F2 progeny were grown in Bloemfontein. Protein extracted from the wheat flour of all this material were analysed by SEHPLC. • Seven wheat quality characteristics were analysed at the Agricultural Research Council, Bethlehem for parents, F1 and F2 progeny. • Analysis of variance and correlations were employed as statistical tools to analyse the data generated from storage protein, combining ability trials and SE-HPLC. • Results revealed that gliadins and LMW-GS were able to distinguish 30 cultivars of wheat grown in Lesotho, while HMW-GS enabled cultivars to be grouped. • The ANO VA indcated significant differences between parents and F 1 progeny and parents and F 2 progeny for all characteristics studied. The mean squares for GCA, SCA and reciprocals were significant for all measured characteristics in the F 1 generation, while in the F 2, the mean squares for GCA were significant for all but one characterisics. Mean squares for SCA and reciprocals were significant for all characteristics in the F2 generation. • To improve or enhance quality, Wanda, SST 124 and Sceptre could be incorporated into the breeding programme. Crosses such as Sceptre x Kariega, SST 124 x Nata, Wanda x Nata, Sceptre x Wanda, Kariega x Wanda, Kariega x Sceptre, SST 124 x Sceptre, Wanda x SST 124, Nata x Kariega and Nata x SST 124 should also be incorporated into the programme. • F 1 progeny showed non-additive gene action in all characteristics while F2 progeny showed four characteristics to be controlled by non-additive gene action while the others were controlled by additive gene action. • Heritability in the broad sense was high for all characteristics in the F 1 and F2 progeny, whereas heritability in the narrow sense was higher for F2thanF1 progeny. • Midparent heterosis and best parent heterosis was expressed in four characteristics. • Highly significantly positive and negative correlations were observed between quality characteristics in F1 and F2 progeny. • Results ofSE-HPLC showed that large polymeric and large monomeric proteins can be used as predictors of good and poor quality respectively, in the parents and F 1 and F2 progeny.Item Open Access The influence of abiotic stress on CIMMYT provitamin A elite maize germplasm(University of the Free State, 2017-01) Manjeru, Pepukai; Labuschagne, Maryke Tine; MacRobert, John Peter; Van Biljon, Angeline; Setimela, PeterMicronutrient malnutrition, including vitamin A deficiency, affects more than half of the world population, having a major effect on children less than five years old, pregnant and lactating women. The problem is significant in sub-Saharan Africa (SSA) where people subsist mostly on white maize which lacks vitamin A. Vitamin A deficiency is responsible for a number of health disorders that include poor vision and reproduction, and supressed growth and immunity. Biofortification of staple food crops such as maize with β-carotene can be a sustainable approach to address dietary vitamin A deficiency. Orange maize contains high levels of β-carotene, making it an important crop for combating vitamin A deficiency. The SSA region is also prone to various abiotic stresses that impact negatively on maize productivity. To ensure food security in the region, there is a need to breed highly nutritious maize cultivars adapted to the major abiotic stresses experienced in the region. To breed increased provitamin A hybrids, it is important to understand the mode of gene action affecting grain yield and β-carotene expression, and the heritability of β-carotene concentration under the prevailing stresses. There is also a need to determine the stability of provitamin A germplasm for grain yield and nutritional traits such as β-carotene under these stress conditions. In this study, 22 elite provitamin A inbred lines and five yellow drought tolerant inbred testers were crossed following a line × tester crossing design. Thirty hybrids had sufficient seed for replicated trials out of a potential of 110. The 30 hybrids and five checks were evaluated in Zimbabwe under optimum conditions, random drought stress, managed drought stress, combined drought and heat stress, low N stress and low P stress in 2014 and again in 2015. There was significant variation between hybrids for grain yield for all environments, except grain yield under low nitrogen stress. There was a significant interaction between year, environment and genotype for grain yield but no interaction was observed for grain texture. Inbred lines were highly heterotic for grain yield, especially under stress conditions. Narrow sense heritability for grain yield was more than 50% under optimal conditions, managed drought stress, combined and drought and heat stress and low P stress. AMMI and GGE analyses showed that genotype by environment interaction (GEI) was a very important source of maize grain yield variability. The environments were grouped into one mega-environment. The highly significant correlations between the environments suggest that testing can be done in only one environment. Hybrid environment, year and GEI effects for β-carotene were highly significant. Beta-carotene concentration was higher under optimum than under stress conditions and was highly significantly correlated with grain yield. Heritability for β-carotene was very high; 97% and 90% under optimum and 70% and 94% under managed drought stress in 2014 and 2015 respectively. General combining ability for β-carotene was significant and specific combining ability was not, emphasising the importance of additive gene action in the expression of the trait. Provitamin A hybrids had β-carotene concentration in the expected range (5-12 μg g-1) for first generation medium to high provitamin A maize genotypes. Lines 6, 7 and 8 can be used for breeding hybrids suitable for all environments except for managed stress conditions. Testers 1 and 2 were ideal for breeding for optimum conditions, managed drought stress, tester 2 for random drought stress and tester 3 for low P stress. Line 8 contributed consistently positively to grain yield, line 3 was favourable under managed drought stress and combined drought and heat stress, lines 6, 7, 8 and 9 were desirable under low N, 6, 7 and 8 under optimum conditions, 4, 6, 7, 8, and 10 under random drought stress, and 3, 8 and 10 under managed drought. The best performing and most stable genotypes for both grain yield and β-carotene can be distributed to SSA farmers for production. These hybrids will go a long way to alleviate vitamin A malnutrition among resource poor households in the region.Item Open Access Development of wheat lines with complex resistance to rusts and Fusarium head blight(University of the Free State, 2017-06) Maré, Ansori; Herselman, Liezel; Boshoff, WillemWheat is the second most important food crop worldwide. The world human population is increasing daily. Sustainable food production is necessary to ensure sufficient future food supply. Wheat production is negatively affected by various pathogens that challenge breeders to develop more disease resistant wheat cultivars. Leaf-, stem- and stripe rust as well as Fusarium head blight (FHB) are considered some of the most destructive fungal pathogens of wheat. Resistance breeding is considered the most economic and eco-friendly solution to control wheat diseases in the long-term. Combining of several effective resistance genes/quantitative trait loci (QTL) into a single wheat line is necessary to increase the probability of obtaining durable resistance. Molecular markers linked to resistance genes/QTL can assist breeders in selecting the best offspring throughout their breeding scheme. The main aim of this study was to develop a wheat line with combined resistance to the rusts and FHB. Both wheat rust and FHB resistant lines used in this study were developed in previous studies. The best resistant lines developed in these studies were identified using marker-assisted selection (MAS) and were then used in a breeding scheme to combine wheat rust and FHB resistance genes/QTL. Offspring from crosses was evaluated for the presence of six rust resistance genes/QTL: Lr19, Lr34/Yr18/Sr57, Sr2/Yr30, Sr26, Sr39 and QYr.sgi.2B-1, as well as for three FHB resistance genes/QTL: Fhb1, Qfhs.ifa-5A-1 and Qfhs.ifa-5A-2. The best identified lines obtained from a series of crosses were selected to develop two double-cross populations as well as self-pollinated populations. Selected pre-breeding lines were also used to optimise and apply the doubled haploid (DH) technique to develop lines with fixed genotypes. Molecular markers linked to the high-molecular-weight glutenin subunits (HMW-GS) and the 1BL.1RS translocation were used for screening the final populations developed because the primary focus for selection was rust and FHB resistance genes/QTL and not HMWGS and the 1BL.1RS translocation. Validation of the phenotypic expression of the rust and FHB resistance genes/QTL was done through greenhouse inoculation of the parental, control and experimental lines/cultivars containing different resistance gene/QTL combinations. Seedling and adult plants were inoculated with the three rusts respectively, firstly for the timeline study to determine the optimal day’s post inoculation (dpi) to collect sample material for quantitative-polymerase chain reaction (qPCR) analysis and secondly to determine fungal gene expression in the experimental lines. Both point and spray inoculations for FHB were used and disease development was evaluated at 4, 7, 10, 14, 18 and 21 dpi. Sample collection for qPCR analysis was done at 21 dpi. Good correlations between the data obtained from the phenotypic evaluations, qPCR and MAS were obtained for both rust and FHB resistance evaluations. Therefore, markers linked to rust and FHB resistance genes were phenotypically expressed. Lines with combined wheat rust and FHB resistance were obtained from the selfpollinated populations but are still segregating for some of the genes/QTL. Lines with combined rust and/or FHB resistance genes/QTL were also developed through the DH technique. No markers linked to stripe rust resistance were used in this study except for genes with multiple disease resistance such as Lr34/Yr18/Sr57 and Sr2/Yr30. Additional stripe rust resistance genes are however present in the genetic background of the experimental lines as they were present in the parental cultivars/lines originally used to develop these lines like Kariega, AvocetYrSP, 2S#/163 and CM-82036. Valuable prebreeding wheat lines with combined rust and FHB resistance have been developed which can be used in future disease resistance breeding programmes.Item Open Access Genetic variability for forage yield and nutritive quality characteristics in selected inbred x Triticosecale genotypes(University of the Free State, 2009-12) Venter, Willem Daniel; Van Deventer, C. S.English: The objective of this study was to study the combining ability, heritability and additive genetic correlation of various nutritive quality, forage yield and animal production characteristics in a selected fixed population of triticale genotypes. The vegetative matter obtained from two sequential cuts per plot was used for chemical analyses and the calculation of plant production and animal production. The trial was a split-plot in time experiment planted in a randomised block design. Three replications were planted and the plots were made up of three rows each. Characters measured for forage quality in both cuts were NDF, ADF, ADL, hemicellulose, crude cellulose, NDFadj, EE, NSC, IVDOM, ME and MRT. Characters used for plant production were kg DM/ha, crude cellulose yield/ha, EE yield/ha, NSC yield/ha, IVDOM yield/ha and ME yield/ha. Characters used for animal production were DM Intake/steer/day, ME Intake/steer/day, LWG/steer/day and Live weight gain/ha. Each one of the animal production characters were calculated according to three different feed intake models. Significant differences were shown in cut 1 between genotypes for NSC, EE and all fibre characters except hemicellulose and crude cellulose. Significant differences were shown in cut 2 for NSC, ME and MRT. Significant differences were shown in both cuts for all the plant production characters. Highly significant differences were shown in cut 1 for Live weight gain/ha according to all three different intake models. The F1 progeny of a 6 x 6 half diallel cross were evaluated in the combining ability analyses using the Method 4, mixed model B analysis of Griffing (1956b), because only one reading per plot was obtainable for each of the characteristics measured. In the combining ability analyses of the F1 progeny, significant differences were shown in cut 1 for gca effects of EE and all fibre characters except hemicellulose. Significant differences were shown in cut 2 for EE, IVDOM, ME and MRT. Significant differences were only shown in cut 1 for all the plant production characters. Highly significant differences were shown in cut 1 for Live weight gain/ha according to all three different intake models. The levels of relative high parent heterosis did not warrant the forming of hybrids in an effort to increase Live weight gain/ha. The σ2 gca/σ2 sca and h2n were moderately high for NDFadj, kg DM/ha and Live weight gain/ha in cut 1 and moderate for MRT in cut 2. Highly significant additive correlations were found between IVDOM yield/ha and Live weight gain/ha according to all three different intake models in cut 1. A significant negative additive genetic correlation was found between kg DM/ha in cut 1 and IVDOM in cut 2. Recommendations were given for the identification of breeding parents, selection in the early generations as well as the final evaluation in order to select a new cultivar for registration.Item Open Access Antioxidant, antidiabetic and cardioprotective activities of Dicoma anomala (sond.) used in the Basotho traditional medicine(University of the Free State, 2016) Balogun, Fatai Oladunni; Ashafa, A. O. T.𝑬𝒏𝒈𝒍𝒊𝒔𝒉 Dicoma anomala (Sond.) belongs to the Asteraceae family and locally called Hloenya (South Sotho), fever or stomach bush (Afrikaans). The plant is used in the management of various diseases, particularly diabetes mellitus among the Basotho tribe of eastern Free State Province, South Africa. The study evaluates the antioxidant, antidiabetic and cardioprotective potentials of the plant as a way of validating the folkloric usage. The result of in vitro antioxidant assays [2, 2- azino-bis (3-ethylbenzothiazoline-6-) sulfonic acid (ABTS), reducing power, superoxide anion, hydroxyl radicals, 1,1-diphenyl-2-picryl hydrazyl (DPPH) radicals, etc.] as well as phytochemicals (such as total phenol, total flavonoids and total antioxidant capacity) in various concentrations (1.56-25 µg/ml) tested using water, ethanol, hydro-ethanol and methanol extracts of the plant’s root revealed that the water extract exhibited the best activity with half maximal inhibitory concentration (IC50: 15.20, 11.70, and 0.84 µg/mL) in DPPH, hydroxyl radical, and superoxide anion radicals respectively. The four extracts also possessed high phenolic contents, total antioxidant capacity with lower total flavonoids content. The effect of treatment with 125, 250 and 250 mg/kg body weight (b.w.) aqueous roots extract of Dicoma anomala (AQRED) was investigated in vivo in CCl4- induced hepatotoxic rats in a 15-day curative and prophylactic study. The result revealed that pre-treatment and treatment with AQRED lowers the elevated serum activities of aspartate transaminase (AST), alanine aminotransferase (ALT) and the level of thiobarbituric acid reactive species (TBARS) while restoring the activities of liver antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) towards normal control in a dose-dependent manner. This result proved the antioxidant and hepatoprotective activity of the plant The in vitro antidiabetic potential of D. anomala was investigated via the inhibition of αamylase and α-glucosidase using same extracts (as above) at the range of 1.56 - 25.00 µg/mL concentrations. All the tested extracts of the plant were active against both enzymes, although, the most potent against α-amylase and α -glucosidase was hydro-ethanol (IC50: 9.00 µg/mL) and water (IC50: 27.41 µg/mL) respectively. Similarly, aqueous extract of the D. anomala displayed competitive and non-competitive inhibition of α -amylase and α - glucosidase respectively using Lineweaver-Burk plot. Treatment with AQRED at concentration 125, 250 and 500 mg/kg b.w. in Wistar rats reversed towards control the elevated blood glucose levels, lipid peroxidation, lipid profile, glycosylated haemoglobin and activities of gluconeogenesis enzymes, with concomitant reduction in the activities of enzymatic antioxidants, glycolytic enzymes as well as the high-density lipoprotein – cholesterol (HDL-c) brought about by streptozotocin induction. Thus, the study proved the antihyperglycaemic activity of the plant. Additionally, AQRED at 125, 250 and 500 mg/kg b.w. was evaluated for its ameliorative activity against isoproterenol (ISP) –induced cardiotoxicity in an animal model. The results from the evaluated biochemical parameters revealed significant (p< 0.05) in these parameters was observed. The data obtained indicate that the lethal dose (LD50) of AQRED is in excess of 2000 mg/kg and its oral administration for 90 days is unlikely to cause any toxic effects. In conclusion, the results from this study proved the antioxidant, antihyperglycaemic and cardioprotective potentials of AQRED. The results further validate the folkloric usage of the plant in the management of diabetes mellitus among the Basotho tribe of Eastern Free State Province, South Africa. ___________________________________________________________________Item Open Access 'n Morfologiese studie van die genus Acacia Miller in Suid-Afrika(University of the Free State, 1971-07) Robbertse, Petrus Johannes; Van der Schijff, H. P.English: The genus Acacia is an extensive one with representatives in Africa, America, Asia and Australia. When Bentham revised the subfamily Mimosoideae in 1875 he divided the taxon into six subgenera. All the South African representatives of the genus were placed in the two subgenera Vulgares and Gummiferae. Bentham distinguished between the two subgenera by the differences in morphology of the stipules. Bentham's classification of the subgenera differs from that of Oliver (1871), whose grouping of the South African Acacia species is based on the morphology of the inflorescence. Species which are affected by this difference are A.albida, A.schweinfurthii and A.kraussiana. Because of this problem and the fact that, judging from the literature, there is some uncertainty regarding the application of taxonomic criteria, it is necessary that the whole genus be revised. Due to the extent of such a project it was decided to limit this investigation to the South African species with the main emphasis on characters which could be of taxonomic importance. Where it was deemed necessary a few species from other geographical areas were included in the investigation. Various characters were found which were valuable for considerations of phylozenetic history of the genus and for delimitation of taxa. On the basis of the presence of starch grains in the cotyledons and the morphology of the seedlings, A.albida, A.kraussiana and A.schweinfurthii were placed in a new subgenus, Farinosae. On the basis of these characters the subgenus Farinosae shows affinity with the subgenera Phyllodinae, Bothryocephalae and Pulchellae of Australia and certain Vulgares species of America. The subgenus Farinosae can possibly be phylogenetically regarded as the oldest extant taxon of the genus Acacia. With the species A.albida, A.krau8siana and A.schweinfurthii in a separate subgenus, the remaining South African members of the subgenera Vulgares and Gummiferae can be separated on the basis of classical features such as the morphology of the stipules and inflorescences. Other clear differences which were found between these two subgenera are as follows: (a)The flower of the Vulgares (and Farinosae) species contains a cup-like disc to which the filaments are adnate, and the ovary has a prominent pedicel (gynophore). In the flower of the Gummiferae species the disc is lacking and the ovary is almost sessile. A disc and pedicelled ovary are also found in the flower of A.albida and the name Feidherbia albida which Chevalier proposed for this taxon in 1934 is therefore not justified. The origin of the cup-like disc on which the stamens are found can probably be regarded as a relict of an ancient branched system on which stamens were borne and which later became reduced. (b) In the pods of the Vulgares (and Farinosae) species the fibre zone consists of both cross and longtitudinally arranged fibres, while the pods of the Gummiferae species have only longtitudinally arranged fibres, or none at all. (c) In the pinnules of the Vulgares (and Farinosae) species a few layers of spongy parenchymatous cells are found between the abaxial and adaxial palisade parenchyma cells. In the pinnules of the Gummiferae species the abaxial and adaxial palisade cells are continuous. (d) The seedlings of the Vulgares species have membranous or leaf-like stipules while the stipules of the Gummiferae species are spinescent. Hooked spines are absent on the Gummiferae seedlings but appear on the Vulgares seedlings either dispersed on the internodes or arranged in groups of two or three directly below the nodes. On the basis of the morphology of the flower, the anatomy of the pod and the morphology of the seedlings, the Vulgares and Gummiferae species can be divided into smaller groups and, in certain cases, into definite species. Keys have been compiled to facilitate the classification. Interesting differences between the Vulgares and Gummiferae species were noted with regard to the nocturnal movements of the leaves. These differences were, however, not very clear in all cases. In the case of certain species such as A.polyacantha and A.robusta the nocturnal movements are very conspicuous and specific (see Fig. 125). A hypothesis was postulated concerning the evolution of the inflorescence systems and inflorescences of the genus Acacia. The hypothesis includes a theory concerning the development of the involucel (cupula). Flowering dates of the different Acacia species are determined to a large extent by the morphology of the inflorescence system. Date of flowering can therefore be considered as a genetic character which nevertheless can be influenced to a certain extent by ecological conditions. The development of the ovule and the ontogeny of the seed was investigated. Ideas were put forward as to the possible homology of the ovule. A study of seed ontogeny produced information concerning the presence of endosperm in mature seeds of A.kirkii, A.tortilis and A.xanthophloea. This characteristic cannot be used as a criterium to remove these three species, as well as other exotic species containing endosperm in the seed, out of the genus Acacia (cf. Vassal, 1968). The arrangement of vascular tissue in the seedling, leaves and young shoots are discussed. Judging from the arrangement of the primary vascular bundles in the seedling there is probably a relationship between a monolacunar node with two leaf traces, a trilacunar node and a node which is called the "fifth type" by Takhtajan (1969). It may be concluded that the extant South African taxa of the genus Acacia can be distinguished from each other on a specific level. By using variable characters such as degree of pubescence and numerical taxonomic characters such as seed and leaf size it is possible to carry the subdivision of species too far. It is therefore recommended that the taxa with an extensive distribution, manifesting considerable variation, such as A.karroo Hayne, A.caffra Willd., A.reficiena Wawra (= A.luederitzii Engl.) and A.robusta Burch., should not be divided into smaller taxa until more information is available concerning the abovementioned characteristics. As a final summary, a key has been composed in which as many characters as possible have been used to distinguish between the South African Acacia species.Item Open Access Etiology of Fusarium crown and root rot of grain sorghum in South Africa(University of the Free State, 2007-02) Ditshipi, Phoebe Mbochwa; Swart, W. J.; McLaren, N. W.English: This study investigated the interaction between grain sorghum and Fusarium spp. associated with the crop in field soils and on sorghum roots as causal agents of crown and root rot. The effect of disease on plant growth and development was investigated as was the efficiency of various inoculation techniques on disease severity. Factors affecting disease susceptibility such as plant age, soil type, soil moisture, soil pH, soil fertility and possible chemical and bio-control control tactics were also investigated. Fusarium spp. isolated from sorghum roots and field soils at Cedara (Kwazulu- Natal province), Bethlehem (Free State province) and Potchefstroom (North West province) indicated that F. oxysporum, F. solani, F. verticillioides and F. thapsinum were the most frequently recovered species. Other Fusarium spp. recovered were F. equiseti, F. nygamai, F. pseudonygamai, F. proliferatum, F. subglutinans and F. polyphialidicum. The most aggressive spp. were F. equiseti, F. thapsinum and F. solani while F. proliferatum, F. subglutinans, F. Verticillioides and F. nygamai were moderately aggressive and F. oxysporum, F. polyphialidicum and F. pseudonygamai least aggressive. Population densities of Fusarium spp. in field soils and on sorghum roots were affected by genotype resistance and initial inoculum. Population densities were higher for susceptible than for resistant sorghum genotypes. A study on the effect of Fusarium spp. on plant growth and development indicated that shoot and root mass did not always correspond with the severity of the disease. Various inoculation techniques for determining susceptibility of sorghum genotypes to crown and root rot and the virulence of Fusarium species were investigated. Wounding the crown and roots and inoculating them with ground colonized oat seeds and drenching soil with a conidial suspension were both very effective. An inoculum concentration of 1 x 106conidia per ml consistently reproduced the disease on inoculated sorghum plants. Crown and root rot severity increased and plant mass decreased with an increase in inoculum concentration. Sorghum genotypes differed in their level of resistance in accordance with the inoculation techniques used. Plant age was shown to affect resistance with two and four-week-old plants being more susceptible than six-week-old plants. Sources of partial resistance to Fusarium crown and root rot were present in some genotypes. Although immunity to Fusarium crown and root rot was not found, Trichoderma harzianum induced systemic resistance in sorghum through the reduction of crown and root rot severity. Fusarium spp. can survive in a wide range of soil types. Certain soils were suppressive while others were more conducive to crown and root rot development. Soil moisture studies indicated that Fusarium spp. causing crown and root rot can survive over a wide range of soil moisture levels ranging between 25 and 100 percent. Low 25% and high 100% moisture levels were suppressive to crown and root rot severity compared to conducive at 50% and 75%. Soil fertility studies indicated that nitrogen applied at normal and high rates significantly and differentially (by genotype) increased crown and root rot severity. Soil amendments also significantly reduced crown and root rot severity with chicken manure being most effective. Studies on the efficacy of fungicides indicated that four fungicides (carboxin / thiram (Vitavax ®Plus FS), tebuconazole/triflumuron (Raxil® 015 ES), tebuconazole, (Ingwe® 6FS), difenoconazole / metalaxyl-m (Dividend ® 030 FS) and a bio-control agent (Trichoderma harzianum) significantly reduced colonization of seedlings. Fungicides also improved plant growth and development by increasing the shoot mass, root mass and enhancing root health. Major Fusarium spp. responsible for crown and root rot were F. equiseti, F. nygamai, F. oxysporum, F. proliferatum, F. pseudonygamai, F. thapsinum, F. solani, F. subglutinans, and F. verticillioides based on the isolation frequencies and pathogenicity tests results. The present study revealed the wide occurance and distribution of Fusarium spp. associated with crown and root rot of sorghum in South Africa. It is hoped that these findings may motivate more research on variation in virulence of these Fusarium spp. Secondly work on the defense mechanisms present in the sorghum genotypes widely grown in in South Africa need to be evaluated in relation to crown and root rot development. The most useful parameters for assessing the disease would be crown and root rot severity and root mass.