Doctoral Degrees (Plant Sciences)

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Open Access
A taxonomic study of the genus cryptolepis (periplocoideae: apocynaceae)
(University of the Free State, 2013) Joubert, Lize; Venter, A. M.; Venter, H. J. T.; Bruyns, P. V.
Cryptolepis R.Br. (Apocynaceae, Periplocoideae) was taxonomically revised. Detailed descriptions of macro and micro-morphology, palynology, geographic distribution and ecological characteristics were presented. An identification key to the species was compiled and the nomenclature of all species was revised while all available type material was studied and lectotypes and neotypes were designated where necessary. Molecular phylogenetic analyses, based on the gene regions ITS, trnD–T and trnT–F, of representative species of 28 periplocoid genera and 22 Cryptolepis species were presented and the monophyly of Cryptolepis was evaluated. Historically a total of 81 species names and four subspecies names were published for Cryptolepis. However, a large number of species names were later placed in synonymy or transferred to other genera, while several new combinations were published. This resulted in a total of 29 accepted Cryptolepis species at the commencement of this study. Three new species, C. ibayana, C. thulinii and C. villosa, resulted from this study and the latter two were described in this thesis. One species, C. producta, was synonymised with C. oblongifolia. Cryptolepis, therefore, comprises a total of 31 species at present. In terms of species diversity, distribution and potential pharmaceutical and economic value, Cryptolepis is one of the most significant genera in the Periplocoideae. Cryptolepis grows throughout sub-Saharan Africa, the southern parts of Yemen, the island archipelago of Socotra, and southern Asia ranging from India to southern China, Taiwan, the Philippines and Indonesia. Most of the species grow in tropical forests or savannah, but 13 species are also adapted to arid environments. The majority of Cryptolepis species are concentrated in four centres of diversity along the east coast of Africa and on Socotra. These hotspots are associated with both arid and forest refugia in areas which have been regarded as local centres of endemism for a number of other plant taxa. The phylogenetic analysis of Cryptolepis indicates that most of these hotspots were colonized repeatedly by different Cryptolepis groups. In addition to the influence of climate shifts, edaphic conditions and also fire had a significant influence on species diversity and distribution in Cryptolepis. Macro and micro-morphological investigations indicated that numerous characters, including growth form, leaf shape and size, leaf epidermal characters, venation, inflorescence structure, floral structure and seed coat surface characters, are of diagnostic value at species level in Cryptolepis. However, the species can only be accurately identified by using a combination of these characters. The molecular phylogenetic analyses revealed that Cryptolepis is paraphyletic and, in order to establish a monophyletic genus, it was proposed that the circumscription of the genus be broadened to include Parquetina as a synonym of Cryptolepis. Several vegetative and reproductive characters showed a high degree of homoplasy, suggesting a high degree of morphological plasticity. This plasticity was also found at species level in C. oblongifolia, which showed significant variation in vegetative and reproductive features. This, together with a high tolerance for disturbance, has resulted in C. oblongifolia becoming the most widely distributed of all Cryptolepis species.
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.
Open Access
Agronomic performance, consumer acceptability and nutrient content of new sweet potato varieties in South Africa
(University of the Free State, 2010-05) Laurie, Sunette Marlize; Labuschagne, M. T.; Faber, Mieke; Van Jaarsveld, Paul
English: Malnutrition, including vitamin A deficiency, and food insecurity are national priorities in South Africa. Sweet potato has several agronomic advantages giving it potential to address both priorities. The crop is popular amongst resource-poor farmers as it is easy-to-grow and hardy. Orange-fleshed genotypes (β-carotene-rich) are used internationally in crop-based approaches to address vitamin A deficiency. Due to a shortage of varieties with desirable traits for resourcepoor farmers, several new varieties have been developed. The overall objective of this study was to evaluate the agronomic performance, sensory quality and nutrient content of new South African varieties. The ANOVA of new varieties in MET, indicated significant differences in dry matter content, marketable yield, number of marketable roots and total yield. AMMI and GGE biplot analysis showed that cream-fleshed varieties Ndou and Lethlabula produced yields comparable to the commercial variety Blesbok. Ndou (unstable high yield), Mokone and Monate (stable, average yields) and Amasi (moderately unstable, average yield) had dry matter content similar to the control variety Mafutha (known for sweet and dry taste). Recommending these new varieties will have a considerable yield advantage over Mafutha, with a possible impact on food security. From MET with nine orange-fleshed varieties the ANOVA and GGE SREG analysis indicated that Impilo produced stable, high yield and 2001-5-2 unstable, high yield. Both had average dry matter content. The two varieties offer a significant yield advantage above USA imports, previously recommended to address vitamin A deficiency. Orange-fleshed varieties produced significantly lower yield than cream-fleshed ones. Raw roots of nine orange-fleshed varieties, harvested from MET at four agro-geographical production sites, had an overall mean trans-β-carotene content of 5091 to 16456 μg/100 g. Resisto, Khano, 2001-5-2, W-119, Beauregard and 1999-1-7 exceeded 5500 μg/100 g β-carotene (breeding target). Dry matter content ranged from 23.1% (Impilo) to 28.7% (W-119). The content of six minerals was determined. Consumption of a boiled portion of 100 g raw root of all nine varieties can potentially contribute to ≥100% of the recommended intake for vitamin A, 21% of magnesium, 12% of zinc and 9% of iron for 4 to 8 year old children. CIE L* and a* color measurement of nine varieties with varying orange flesh color had the best correlations with total β-carotene content, and is a faster selection method. A mathematical function with natural logarithm (ln) transformation derived to predict total β-carotene content from color L*, predicted 91% of the values with >75% accuracy. The color and taste acceptability of boiled sweet potato of new varieties were evaluated in two experiments by grade 1 – 7 learners and adults in a BIBD using a 5-point hedonic scale. A trained panel found clear differences in the sensory attributes of the varieties. Cream to yelloworange varieties, Mafutha, Lethlabula and Phala had the highest consumer panel (n=190) scores, associated most with wateriness, discolor, graininess, gumminess, sweet potato-like flavor and instronforce. Orange-fleshed varieties Impilo, Excel, Resisto, 2001-5-2, Serolane and W-119, and cream-fleshed varieties Monate and Ndou were acceptable to the second consumer panel (n=216), mostly related to dry matter content, maltose content, sweet flavor, wateriness, discoloration and dark edges. High maltose and starch content were found in Ndou, Serolane, Resisto and Monate; while Blesbok, Impilo and 2001-5-2 had high fructose and glucose content. Orange-fleshed varieties were as acceptable as cream-fleshed varieties. The present study provides novel results on G x E analysis, comprehensive and systematic determination of nutrient content, consumer and sensory assessment of a considerable number of new cream to orange-flesh sweet potato varieties promoted in South Africa. Varieties Ndou, Mokone, Monate and Amasi were recommended to address food security; and Impilo and 2001- 5-2 to address vitamin A deficiency.
Open Access
Analyses of drought tolerance in durum wheat (Triticum turgidum var. durum) genotypes
(University of the Free State, 2003-03) Fekybelu, Solomon Kebede; Labuschagne, M. T.; Viljoen, C. D.
English: In order to identify yield components associated with drought tolerance in durum wheat and evaluate the performace of Ethiopian durum wheat genotypes, 26 durum wheat genotypes, from different agro-ecologies of Ethiopia were evaluated under simulated moisture stress conditions. Stress caused dramatic reductions in grain yield and harvest index. Yield was significantly correlated primarily with number of kernels per spike and 100 kernel weight. Further decomposition of simple correlation coefficients into direct and indirect effects showed that number of kernels per spike and 100 kernel weight had the largest direct effects on grain yield, under both stressed and non-stressed conditions' The expression of drought tolerance in the F1generation obtained from all possible combinations among genotypes differing in their responses to moisture deficit stress was studied. Analysis of variance revealed significantly high variability among genotypes for yield, yield components and drought tolerance measurements due to the effects of treatments. Drought tolerance was expressed in the crosses involving tolerant parents. The diallel analysis showed that mean squares for both GCA and SCA were significantly high at both moisture regimes. GCA:SCA ratios indicated predominance of additive gene action for all characteristics positively correlated with grain yield under stress or negatively correlated with drought susceptibility index. Differences in water use and transpiration efficiencies and interrelationships among water use and transpiration efficiencies and associated traits were investigated among durum wheat genotypes with differing responses to moisture stress. Significantly high genotypic variability in the amount of cumulative water used before (ETba) and after (ETpa) anthesis was observed. Susceptible genotypes used higher amounts of water before anthesis and lower amounts after anthesis. In contrast, tolerant genotypes used a higher proportion of water during the post-anthesis period. Significantly high variability among the genotypes was observed for various measures of water use and transpiration efficiencies, total dry matter and harvest index. Ranking of cultivars for water use efficiency based on grain yield (WUEG) and transpiration efficiency based on grain yield, was consistent with ranking of cultivars for drought susceptibility indices. Drought susceptibility index was significantly but negatively correlated with harvest index, WUEG and grain yield. However, it was positively and significantly correlated with the ETba:ETpa ratio. A high positive correlation of WUEG with harvest index and grain yield with harvest index was found. Differences in flag leaf water potential were significant only for genotypes under stress treatments. Water potential declined with age under both treatment conditions, however, the fall was faster in stress sensitive types than in tolerant types in more advanced growth stages. Inheritance of water use and transpiration efficiencies was studied in a hybrid population obtained from six parents, selected for their different responses to moisture stress. GCA and SCA effects were significant at both the moisture levels. The interactions of GCA and SCA with moisture levels were also highly significant. Analysis of the genetic components of variation demonstrated that WU E and T were under the control of additive and dominance type of genes. Narrow sense heritability estimates for water use and transpiration efficiencies based on grain yield (WUEG) were higher at the moisture stress level. Measures of water use and transpiration efficiencies showed significantly high and positive genotypic and phenotypic correlations among them as well as with grain yield and harvest index. The impact of the effect of moisture stress on growth and phenological development was examined among durum wheat genotypes differing in their tolerance to moisture stress. Drought stress was found to delay major growth stages and shorten the grain filling period. Drought tolerant genotypes had fast early growth, whereas susceptible ones had slow RGR initially. Variation in RGR was associated with NAR and LAR. Differences in hybrid performance were due to significant GCA and SCA effects. Interactions of GCA and SCA with moisture level were also highly significant. Moderate to high levels of broad sense heritability estimates were found for most of the traits. Significantly high genetic and phenotypic correlations between NAR and RGR, and LAR and RGR were found. The genetic and phenotypic correlations of grain yield with total dry matter, harvest index, RGR and LAR were significant. The effect of moisture stress on the content of water soluble carbohydrate (sucrose and D-glucose) was investigated in durum wheat (Triticum turgidum L. var. durum) above ground organs to assess and characterise the responses of genotypes with differing responses to drought stress. In all organs examined, drought tolerant genotypes accumulated more glucose and sucrose, particularly, at an early age. Stress caused an overall increase in the level of these carbohydrates, but the levels were highest in the stems compared to other organs. Level of drought (drought susceptibility index, 'S') was strongly related with the level of carbohydrates in the various plant organs Genetic differences among genotypes with differing responses to drought stress and their progenies obtained from all possible cross combinations were assessed using AFLP markers. Moderate to high level polymorph isms were obtained with the primer combinations evaluated. Euclidean genetic distance estimates showed that there was considerable genetic diversity among the materials studied. The correlation coefficient for pair wise genetic distance estimates based on AFLP data and yield, yield components, and morphophysiological traits was significant and positive. Cluster analysis showed that grouping of genotypes on the basis of AFLP data and agro-morphological data fairly agreed. A number of AFLP fragments were significantly correlated with the various traits evaluated under stress conditions. AFLP fragments were observed to affect a number of traits that were strongly correlated with drought susceptibility index.
Open Access
Analysis of genetic variability of grain mould resistance in grain sorghum
(University of the Free State, 2009-09) Mpofu, Leo Thokoza; McLaren, N. W.; Van Deventer, C. S.
English: In an effort to characterize the relationship between fungal pathogenicity and host genetic resistance, sorghum panicles of 11 genotypes were inoculated with five fungi frequently isolated from sorghum grain. Panicles were inoculated at anthesis with Fusarium graminearum, Fusarium thapsinum, Curvularia lunata, Phoma sorghina and Alternaria alternata spores. There were highly significant differences in the levels of fungal pathogenicity on different sorghum genotypes. These differences accounted for 58.4% of observed variation in ergosterol concentration. Genotype by pathogen (G x P) interactions accounted for 33.5% of the observed ergosterol concentration variation. The implication is that different genotypes reacted differently to different fungi. The genotypic reactions of the hosts accounted for 8.1% of the observed ergosterol concentration variation. Overall, fungal pathogenicity is the most important factor to consider in the evaluation of germplasm for grain mould resistance. Possible sources of resistance could be identified by use of biplot analysis of G x P interactions. Visual scoring for grain mould has limited value without identifying causal fungi. Fusarium thapsinum and Phoma sorghina were the most abundant fungi across all genotypes. Gene action and heritability for grain mould resistance in sorghum were investigated using a selection of 9 random pollen parents with varying levels of grain mould resistance to a different set of three random seed parents. Differences in ergosterol concentration were used as a measure of level of grain mould resistance among all genotypes. Use of ergosterol concentration as a measure of grain mould severity did not correlate with visual field scoring. Other traits measured include plant yield, plant height, kernel hardness, field grade score, days to flowering, glume color and seed color. The combined analysis of variance showed no genotypic variance for grain mould resistance. The expression of grain mould resistance was also not stable with significant genotype x location interaction. The analysis from Potchefstroom showed significant differences among genotypes whereas Cedara-1 and 2 showed no differences. Additive genetic variance was greater than dominance variance for all traits except grain mould resistance. A significant heterosis of -20.15% was observed for grain mould resistance indicating the importance of use of hybrid seed. LM124 (white seeded female) and LM130 (brown seeded female) produced resistant hybrids. Due to very high environmental variance, grain mould heritability could not be detected. Yield indicated the highest heritability of 0.41 at Cedara-1, plant height 0.39 at Cedara-2 and kernel hardness 0.38 at Potchefstroom. The significance of genotype by environment interaction was assessed over three environments. Differences in ergosterol concentration were used as a measure of level of grain mould resistance among all genotypes. Significant G x E interaction was detected after analysis of variance across all three locations. Single site analysis was then done to better explain the nature of the G x E interaction. Potchefstroom is the only location that showed significant genotypic responses to grain mould infection. Low grain mould pressure at Cedara-1 and Cedara-2 caused very low genotypic responses. A biplot was then used to indicate all genotypic performances across the three locations in a graphical design. Fungal species infecting sorghum kernels were isolated, counted and identified. Low genotypic responses at the Cedara locations seems to have been caused by prevalence of a less aggressive fungal species, Mucor spp. Weather variables did not correlate significantly with ergosterol concentration. Mycotoxin levels of aflatoxin, deoxyvalenol (DON), and zearalenone together with ergosterol concentration levels across 39 sorghum genotypes were measured. Varying levels of ergosterol and mycotoxins were observed across the three locations. There was no correlation between ergosterol concentration and any of the mycotoxins which indicates that mycotoxins concentration is not related to total fungal biomass. Mycotoxin concentration must therefore be related only to the biomass of the fungal species that is producing the mycotoxin among all fungal species infesting the grain. Farmers in Potchefstroom should be wary of aflatoxins and zearalenone while farmers in Cedara should choose varieties that tend to be low in DON and aflatoxins while not ignoring zearalenone.
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. ___________________________________________________________________
Open Access
The application of fossil grass-phytolith analysis in the reconstruction of cainozoic environments in the South African interior
(University of the Free State, 2009-05) Rossouw, Lloyd; Scott, L.
Grass-dominated ecosystems occupy a primary position with regard to current debates concerning key events in faunal turnover and human evolution in Africa. Our knowledge of how grass-dominated ecosystems in southern Africa have reacted to periods of global warming and cooling in the past is provided by a broad range of proxy data sources, most notably pollen and stable isotope records. Phytolith analyses provide an alternative fossil record of environmental change and are now progressively becoming a conventional analytical approach in palaeoenvironmental research.The grass family (Poaceae) produces abundant silica bodies, especially within specific and specialized silica cells located in costal zones of the leaf epidermis. In addition to being highly resistant to decomposition, grass phytoliths show markedly varied and distinct morphologies. In this study a central hypothesis, namely that the morphology of grass short-cell phytoliths consistently follows meaningful environmental traits that are rooted in the relationship between phytolith shape and the ecological niche of grasses, was investigated by interpreting morphologically diagnostic grass phytolith assemblages according to their association with the ecological requirements of the grass species that produces them, irrespective of taxonomic affiliation.An effort was therefore made to assign ecological meaning to short-cell phytoliths by comparing a range of ecological preferences in modern grasses with the phytoliths that they produce, rather than just using phytoliths to discriminate between grass subfamilies, tribes or genera. This entailed a systematic investigation of grass leaf epidermis from a collection of 309 species, followed by an assessment of the ecological significance of grass-short-cell phytoliths within a quantitative model. The model allowed for comparison of geographically diverse grass phytolith assemblages by converting them into one homogenous group represented by ecological categories. Several meaningful ecological trends were demonstrated by results in this study, and it is suggested that short cell phytolith association in grasses is primarily driven by a temperature gradient, marked by cool versus warm growing temperatures and reflected by grasses utilizing the C3 and C4 photosynthetic pathway. The study gives emphasis to the importance of investigating phytolith systematics with the aid of adequate comparative reference collections.
Open Access
Assessment of cup quality, morphological, biochemical and molecular diversity of Coffea arabica L. genotypes of Ethiopia
(University of the Free State, 2005-09) Bekele, Yigzaw Dessalegn; Labuschagne, M. T.; Herselman, L.; Osthoff, G.
The genetic diversity of C. arabica genotypes collected from the northwestern and southwestern parts of Ethiopia was evaluated using morphological, biochemical and molecular (AFLP and SSR) markers. The objectives of the study were to (1) estimate the level of genetic variation among currently grown C. arabica genotypes, (2) estimate the level of associations among agro-morphological characters, coffee quality traits and green bean biochemical compounds, (3) compare the level of genetic variation between coffee genotypes collected from the northwestern and southwestern parts of Ethiopia and (4) compare the efficiency of AFLP and SSR markers in detecting genetic variation in C. arabica. Results of diversity analysis using nine qualitative and 18 quantitative morphological characters indicated the presence of substantial variability among evaluated coffee genotypes. Genotypes were significantly different for all 18 quantitative morphological characters. Higher Shannon-Weaver diversity indices ranging from 0.401 to 0.989 were recorded for nine qualitative morphological characters. Cluster analysis using qualitative, quantitative and a combination of qualitative and quantitative characters classified genotypes into two groups. Qualitative characters failed to independently distinguish seven of the genotypes. Principal component analysis grouped 18 quantitative characters into 15 principal components and the first four explained 82.3% of the entire variability among genotypes. Average green bean yield per tree and bean length were important for the variation explained in three of the first four principal components. Correlation analysis performed among 18 agro-morphological characters indicated positive associations between average green bean yield per tree and all other characters. Average green bean yield per tree had statistically significant correlations with percentage of bearing primary branches per tree, bean weight, canopy and trunk diameters, tree height, bean length and thickness, internode lengths of orthotropic and primary branches and fruit and petiole lengths. Broad sense heritability varied from 38% for bean thickness to 94% for bean weight and number of secondary branches per tree. Average green bean yield per tree, canopy diameter, tree height and bean weight had higher genetic coefficients of variation and broad sense heritabilities. These characters were positively and significantly correlated, allowing simultaneous improvement of these traits. Genotypes were evaluated for variability in four cup quality traits (acidity, body, flavour and overall standard) and four green bean physical characters (bean shape, size, uniformity and weight). Considerable variation was observed among genotypes both for cup quality as well as green bean physical characters. Of all the genotypes, AD0691 and AD1691 had both desirable cup quality and green bean physical characters. Cluster analysis performed using cup quality and green bean physical characters, classified genotypes into two groups. Genotypes were not clustered according to collection regions. All cup quality traits were positively and significantly correlated and correlation coefficients ranged from 0.69 to 0.93. Positive correlations were observed among desirable cup quality and green bean physical characters indicating that cup quality and green bean physical characters can be improved simultaneously. Genotypes were evaluated for green bean caffeine, chlorogenic acids, sucrose and trigonelline content variability. Analysis of variance indicated significant differences among genotypes for all biochemical compounds. Green bean caffeine, chlorogenic acids, sucrose and trigonelline contents ranged between 0.91-1.32%, 2.34-4.67%, 5.30-8.98% and 1.04-1.71%, respectively on dry matter basis. Cluster analysis classified coffee genotypes into eight groups and genotypes were not clustered according to collection regions. Coffee genotypes collected from northwestern Ethiopia were more diverse in green bean caffeine, chlorogenic acids and sucrose contents compared to southwestern Ethiopia genotypes. On average, southwestern Ethiopia coffee genotypes had higher green bean caffeine, chlorogenic acids, sucrose and trigonelline contents compared to northwestern Ethiopia genotypes. Green bean caffeine content showed negative and statistically significant associations with all desirable cup quality attributes and correlation coefficients ranged from -0.305 to -0.407. Desirable cup quality traits and low green bean caffeine content can be selected simultaneously. Green bean sucrose content showed positive associations with all desirable cup quality and green bean physical characters. Higher green bean sucrose content, desirable cup quality as well as green bean physical characters can be improved together. The genetic diversity of coffee genotypes was investigated using 10 AFLP primer combinations. Results revealed differences among AFLP primer combinations in detecting genetic variation among genotypes. Of the 10 primer combinations EcoRI-ACA/MseI-CAA, EcoRI-ACC/MseI-CAG, EcoRI-ACT/MseI-CAG and EcoRI-AAC/ MseI-CAA were superior in detecting genetic variation. Almost all primer combinations were positively correlated in estimating pair-wise genetic similarity coefficients, indicating the similarity of primer combinations in detecting genetic variation among genotypes. All genotypes were independently distinguished and pair-wise genetic similarity coefficients ranged from 0.851 to 0.982 with an average of 0.915. Ten genotypes had 18 specific AFLP markers which could be utilised for genotype fingerprinting. AD1291 and AD1491 were the most dissimilar while AD3591 and AD3991 were the most similar genotypes. Genotypes were classified into two groups using UPGMA method of cluster analysis. Genotypes did not cluster according to collection regions. Results unveiled the presence of genetic variation among genotypes collected in each region and the presence of close genetic similarity among some coffee genotypes collected from different regions. The genetic diversity among coffee genotypes from southwestern Ethiopia was higher compared to those from northwestern Ethiopia. Genetic diversity analysis performed using six SSR primer pairs indicated the presence of considerable genetic variation among some of the genotypes collected from northwestern and southwestern Ethiopia. Twenty polymorphic SSR markers were amplified of which two were specific to genotypes AD1491 and AD2991. Using 20 polymorphic SSR markers, 64.3% of the genotypes were independently distinguished. Pair-wise genetic distances ranged from 0.286 to 1.000. The lowest pair-wise genetic similarity coefficient was recorded between AD0591 and AD1491 as well as AD1491 and AD4591. Genotypes from southwestern Ethiopia were more diverse compared to those from northwestern Ethiopia. Molecular markers differ in the amount of information generated per PCR reaction. The efficiency of 10 AFLP primer combinations and six SSR primer pairs in detecting genetic variation was compared using 28 C. arabica genotypes. AFLP primer combinations amplified 220 and SSR primer pairs 20 polymorphic alleles among evaluated genotypes. AFLP markers independently distinguished all evaluated coffee genotypes whereas SSR markers distinguished 64.3% of the genotypes. Moreover, 18 genotype specific AFLP markers compared to two SSR genotype specific markers were identified. The assay efficiency index of AFLP markers (22) was far superior to that of SSR markers (3.3). AFLP markers surpassed SSR markers in detecting genetic variation among evaluated arabica coffee genotypes. Results of this study indicated the presence of good congruence between the two genetic markers in estimating pair-wise genetic similarity coefficients among genotypes.
Open Access
Assessment of genetic diversity, combining ability, stability and farmer preference of cassava germplasm in Ghana
(University of the Free State, 2012-11-11) Parkes, Elizabeth Yaa; Labuschagne, Maryke T.; Fregene, Martin A.
English: The aim of this study was to assess genetic diversity, combining ability, heterosis, heritability of traits, stability and farmer preference of cassava germplasm in Ghana in order to structure an effective breeding programme. The assessment of genetic diversity in a collection of local cassava genotypes using 33 SSR markers revealed a high overall heterozygosity. The SSR markers identified 63 duplicates in the collection. The heterotic pools were distinct for the savannah and the forest ecological zones and this was the basis for the selection of progenitors for the rest of the study. Seven and five cassava progenitors were crossed in a diallel design without reciprocals in the forest and savannah ecologies respectively. The progenitors and progenies were evaluated for CMD, CBB, yield and its components in two seasons in the forest and one season in the savannah zone. The GCA:SCA ratio indicated that the GCA was larger than SCA for average root number, indicating additive gene effects and a possibility for improvement by selection. SCA was larger than GCA for harvest index and fresh root yield, indicating environmental effects on the trait and confirms the non-additive effects mainly determining expression of root yield. The parental genotypes were selected from a particular region where CMD is the main economic constraint. Debor had the best GCA effect for cassava mosaic severity reaction; while Tuaka had the best GCA effect for fresh root weight. Debor x Kwasea was the best combination for CMD resistance. Debor and Kwasea were the best parents for resistance breeding to CMD. The predictability ratio varied between 0.58 and 0.84. CMD and root number had a ratio close to one for the savannah and forest zones, showing that these traits were highly heritable and influenced by additive gene action. Marker assisted selection was used to search for new sources of CMD resistance in the progenies and progenitors. Crosses among CIAT elite genotypes and landraces revealed progenitor Dabodabo as a putative source of new genes to CMD resistance.
Open Access
Biochemical events associated with rust resistance in sunflower
(University of the Free State, 2003-11) Mohase, Lintle; Van der Westhuizen, A. J.; Pretorius, Z. A.
English: The biochemical resistance response of sunflower (Helianthus annuus L.) to rust (Puccinia helianthi Schw., pathotype UVPhe 2) was investigated in a comparative study using susceptible (S37-388) and resistant (PhRR3) sunflower cultivars and related to rust development. In addition, the potential of a plant activator, benzothiadiazole (BTH), to induce rust resistance in susceptible sunflower was investigated. Rust infection induced higher enzyme activities of the apoplastic pathogenesis-related (PR) proteins, β-1,3-glucanase, chitinase and peroxidase in the resistant than susceptible plants. The accumulation of β-1,3-glucanase isoenzymes was confirmed by Western blots. According to the labelling data of the immunogold localisation studies, β-1,3-glucanases were concentrated in the mesophyll cell walls during the rust resistance response. As the enzyme activities of the PR proteins were induced, the development of infection structures in the resistant plants was retarded, i.e. the number of vesicles, infection hyphae and haustorium mother cells. An early transient accumulation of H2O2 in the infected resistant plants is proof of the involvement of active oxygen species (AOS) in the resistance response. Active oxygen species’ burst correlated with an early induction (after 3 h of infection) of the activities of AOS-generating enzymes, NADPH oxidase, superoxide dismutase (SOD) and the NADH-dependent peroxidase. The phenolic, salicylic acid (SA), has been implicated in the resistance response as a potential signal molecule. The increase in SA content occurred with a concomitant increase in the activity of phenylalanine ammonia-lyase (PAL), a key enzyme in the synthesis of phenolic compounds. Induction of the activity of lipoxygenase, a key enzyme in lipid biosynthesis, in infected resistant plants, suggests that lipid-derived signals may play a role in the rust resistance response. As rust infection progressed, the number of aborted substomatal vesicles increased in resistant than susceptible plants. The number of infection hyphae, haustorium mother cells and rust colonies, which all favoured disease expression, were lower in the resistant than susceptible plants. Furthermore, in the resistant cultivar, haustorium mother cells were more often associated with host cell necrosis. The hypersensitivity index exceeded one, 96 hours after infection, indicating that the resistant plants were exhibiting the hypersensitive reaction (HR). Since it was found that SA is a possible signal for rust resistance reactions, the effectiveness of BTH, a functional analogue of SA, to induce the resistance response was investigated. This was measured in terms of activities of defence-related enzymes, β-1,3-glucanase and peroxidase. Benzothiadiazole induced an increase in the activities of these enzymes and further reduced disease symptoms by about 30%. In this study some of the biochemical components of the rust resistance response in sunflower have been identified and related to the development of rust infection structures. Furthermore, the potential of BTH as a plant activator in the control of rust diseases in sunflower has been demonstrated. The information gathered in this study contributes to a better understanding of the mechanisms in sunflower involved in rust resistance, which eventually could help to manipulate sunflower for improved resistance.
Open Access
Biochemistry of Russian wheat aphid resistance in wheat: involvement of lipid-like products
(University of the Free State, 2007-11-03) Berner, Jacques; Van der Westhuizen, A. J.
Physical damage and disease are known to cause changes in the lipid composition of plants. The biochemical pathways that lead to changes in the lipid composition were investigated in a comparative study using Russian wheat aphid (RWA) (Diuraphis noxia) infested and uninfested resistant (‘Tugela DN’, ‘Gariep’ and ‘Betta’ DN) and near isogenic susceptible (‘Tugela’, ‘Molopo’ and ‘Betta ’) wheat cultivars. Lipoxygenase (LOX) activity was determined spectrophotometrically while LOX proteins were analysed by means of Western blots. Lipid peroxidation was determined by measuring malondialdehyde levels spectrophotometrically. To investigate the involvement of prostanoids in the RWA resistance response, prostanoid biosynthesis was inhibited with indomethacin where after the activities of defence related enzymes peroxidase (POD) and LOX were determined spectrophotometrically. Lipid-like products were analysed by means of gas liquid chromatography (GC/MS) and mass spectrometry. Western blots were used to analyse the cyclooxygenase (COX) proteins. LOX, which catalyzes the first step of the lipoxygenase pathway, was selectively induced in the infested resistant wheat. This increase in LOX activity coincided with lipid peroxidation. Several lipid-like compounds were found to be newly induced after infestation and the synthesis of others were enhanced after infestation in the resistant wheat. Many of these lipid products were identified as hydroxyl and keto fatty acids. Some of these fatty acids could be detected as early as 1 h.p.i. (hours post infestation). The levels of some fatty acids increased as infestation proceeded while the levels of others reached a peak 48 h.p.i. and declined towards 96 h.p.i. Hydroxyl and keto fatty acids are important signalling compounds during defence responses. A second pathway, COX pathway, which was believed to exist only in mammals, was also found during the wheat-RWA interaction. The COX protein was selectively induced in resistant wheat after infestation . It has high homology to mammalian COX, which is responsible for prostanoid synthesis. The involvement of prostanoids in the RWA resistance response was confirmed by inhibition studies of prostanoid biosynthesis. Downstream defence reactions e.g. LOX and peroxidase (POD) activities were inhibited upon inhibition of the prostanoid biosynthetic pathway. These results emphasize the importance of two lipid biosynthesis pathways (LOX and COX) that may be essential for the establishment of a successful defence response in wheat to the RWA.
Open Access
Biodiversity in plant, grain and nutritional characteristics of sorghum [Sorghum bicolor (L.) Moench] accessions from Ethiopia and South Africa
(University of the Free State, 2010-11) Gerrano, Abe Shegro; Labuschagne, M. T.; Geleta, N.; Van Biljon, A.
This study was conducted with the objectives of assessing the genetic diversity of sorghum accessions that were obtained from different geographical locations in Ethiopia, as well as South Africa, using phenotypic traits, AFLP markers, minerals, starch and protein. Twenty phenotypic traits were subjected to ANOVA and highly significant differences were observed for all traits among accessions. Some important characteristics were found to be significantly correlated, which could allow for selection of related secondary characteristics to improve primary characteristics. Cluster analysis grouped accessions into five clusters based on similarity. These results would be useful in a breeding programme for selecting sorghum accessions to improve production. Nine qualitative morphological traits were also analysed using the Shannon Weaver diversity index (H’). The results showed that the average H’ was 0.59. The highest polymorphism was recorded for the glume colour (0.84), while the lowest was recorded in panicle compactness and shape (0.31). This indicated that there was wide variability among accessions studied. The cluster analysis for the qualitative traits also identified accessions based on their similarity and differences based on their genetic distances. Hence, visual selection and measurements of the sorghum attributes in field trials could be used to select the desirable traits and improving yield and stability of the accessions in breeding programmes. AFLP analysis showed high genetic similarity of Ethiopian landraces, as well as breeding material from the Ethiopian and South African sorghum breeding programmes, even though the phenotypical characterisation showed high variability. This indicated the possibility that South African and Ethiopian breeding material may have a common ancestry. The morphological, AFLP and combined morphological and AFLP cluster analysis clearly distinguished between all accessions, even though they were genetically similar. Sorghum kernels were used to determine the mineral, starch and protein composition of the sorghum accessions. The ANOVA of the traits revealed highly significant variation among the accessions studied. Furthermore, PCA grouped accessions according to their similarity/differences in the four quadrants which confirmed that there was variation among them for these nutritional traits. Therefore, there would be potential for selecting accessions for specific mineral and protein content for human consumption, and specific starch and amylose content for industrial use. The sugar content of the stalks also indicated the potential to develop dual sorghum cultivars where both the seed and sugar can be produced from the same plants.
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.
Open Access
Biology and control of bean anthracnose in Ethiopia
(University of the Free State, 2003-06) Beshir Mohamed, Tesfaye; Pretorius, Z. A.; Assefa, H.
English: Common bean (Phaseolus vulgaris L.) is an important food legume for millions of Ethiopian people. However, diseases are major constraints in bean production with anthracnose, caused by Colletotrichum lindemuthianum (Sacc. & Magn.) Briosi and Cav. (teleomorph = Glomerella cingulata (Stonem.), being of particular concern. Anthracnose is the most important disease of bean in Ethiopia and can cause complete yield losses on susceptible cultivars when contaminated seed are planted. The impact of epidemics is further enhanced when favourable environmental conditions prevail. The main objective of this study was to provide information that can be used as part of an integrated control program for anthracnose in Ethiopia. Field surveys were conducted in the foremost bean growing areas of Ethiopia to acquire information on the geographical distribution of anthracnose. Similar to bean rust, anthracnose is favoured by high moisture and relative humidity. In surveys of the different agro-ecological zones (AEZ) anthracnose was found severe at Ambo (08°58'N, 03r26'E), Meki (08°05'N, 038°51 'E), Arsi-Negele (OrOO'N, 038°35'E), Areka 0 and Bako (09°06'N, 37°09'E), intermediate at Awassa (06°52'N, 038°27'E), Jimma (07°28'N, 035°37'E), and Metu (07°02'N, 35°63'E), and slight to absent at Melkassa (08°23'N', 039°19'E). Other diseases observed included bacterial blight, angular and floury leaf spot and rust. The success rate in breeding for resistance depends to a large extent on the degree of variability in a pathogen. Using the standard differential cultivar set and binomial system proposed by CIAT (Centro Internacional de Agricultutra Tropical), eight races of C. Iindemuthianum, viz. 65, 73 128, 296, 511, 589, 961, and 1027, were identified from Ethiopia and compared to races 3, 6, 81,323,390 and 593 from Southern Africa. Races 128 and 511 constituted more than 50% of all isolates characterised. Considering the differential cultivars, G 2333 was resistant to all races in Ethiopia, and AB 136, G 2333, Kaboon, Cornell 49-242 to all races in Southern Africa. Field studies were conducted at Ambo and Bako (Ethiopia) to evaluate resistant and susceptible cultivar mixtures as a means of anthracnose control. Results showed that disease incidence and severity were consistently lower in mixtures containing 50%, 67% and 80% of the resistant component at both locations. Disease progress curves and disease gradients indicated that disease development and spread were slower or reduced in all treatments that contained an increased proportion of the resistant cultivar. It was thus found that anthracnose could be controlled by growing cultivar mixtures containing at least 60% of a resistant cultivar. In an assessment of the efficacy of chemical control of anthracnose, a benomyl seed dressing followed by a foliar difenoconazole spray, or difenoconazole application alone, showed the most promising results. Finally, greenhouse and multi location field experiments were carried out to identify bean genotypes as possible sources of resistance to Ethiopian isolates of C. lindemuthianum. The study established that cultivars Widusa, GLP X 1132, A 482, A 193, G-7, HAL 5 and G 2333 were valuable sources of anthracnose resistance.
Open Access
Biology and control of Sclerotium rolfsii on groundnut in South Africa
(University of the Free State, 2001-05) Cilliers, André John; Pretorius, Z. A.; Van Wyk, P. S.
English: Information gleaned from this study has stimulated new questions regarding the genetic structure, population dynamics and control of Sclerotium rolfsii. This work can hopefully serve as a foundation for the further study of this fungus in South Africa and elsewhere in the world. The population of the fungus in South Africa was studied and the 121 isolates collected were found to belong to 13 different mycelial compatibility groups (MCG), occurring on various crop and other plants in different geographic localities. Vague associations between MCG, host plant and geographical area could be observed. The genetic fingerprinting technique of amplified fragment length polymorph isms was found to be an effective tool to distinguish between MCGs of S. rolfsii. This technique has never before been used for this purpose, even though other genetic techniques have been used in other studies. No previous study, however, was able to distinguish isolates within MCGs, as this study was able to do. The hypothesis was also made that isolates within a MCG are not clonally derived, based on the differences observed between isolates in the same MCG, the different host plants and the different geographic areas from which the isolates originated. The pathogenicity of MCGs of S. rolfsii to groundnut appeared to be significantly different from group to group, but the results of field trials suggested that isolates were not fixed into specific MCGs, even though a controlled glasshouse experiment did not support this. The phenomenon of MCG switching has never been reported for this fungus, even though it has been recorded for other fungi. Certain isolates also appeared to be more pathogenic to groundnut than others. Mycelial compatibility group E consistently emerged as a virulent pathogen of groundnut. No groups were found to e specific to any of three other crop plants tested. The control of the fungus in groundnut fields was found to be possible with the use of an inversion plough in trials conducted over two consecutive seasons. A fungicide was identified that suppressed S. folfsii but not Trichoderma harzianum, a naturally occurring biological antagonist of the pathogen in the soil. The application of the fungicide difenoconazole at 12.5% of the registered concentration for groundnuts did not reduce disease in the field. The application of T. harzianum and difenoconazole in the early stages of groundnut plant development had no effect on yield or disease incidence. levels of disease in the field were found to increase with a decreased plant density. This observation was attributed to increased soil erosion in the field as a result of the sparser plant density. Sclerotium roltsii is a virulent pathogen of numerous crop plants in South Africa and this study has provided some answers to questions regarding the biology and control of this fungus. However, much still has to be discovered and may future scientists find some inspiration from what was done here. Key words: Sclerotium faItsii, southern stem rot, population, mycelial compatibility groups, AFlP analysis, pathogenicity, control, inversion plough, integrated pest management.
Open Access
Biometrical approaches for investigating genetic improvement in wheat breeding in South Africa
(University of the Free State, 2015-10-28) Boyse, Mardé; Labuschagne, M. T.; Pakendorf, K. W.
English: Wheat is the biggest winter cereal crop in South Africa and the second largest cereal to feed the population of South Africa. The population of South Africa grows with approximately one million people a year. Consistent wheat production is necessary for food security and is therefore of extreme agricultural and economic significance. Future production increases depend on the ability to improve, or at least maintain, the rate of increase to feed the population. The study was undertaken to investigate genetic improvement (genetic advance) in wheat by various statistical methods of analysis. This was done to determine the most suitable procedure to evaluate genetic improvement in the three wheat production areas of South Africa, namely the Western Cape Province, the Free State province and the irrigation areas. The second objective of this study was to demonstrate the trend of yield and the two quality traits [HLM (hectolitre mass) and protein content] over 16 years (1995-2010) by various statistical techniques. The third objective was to compare the AMMI (additive main effects and multiplicative interaction) and the GGE (genotype plus genotype-by-environment interaction) analyses in assessing genotype-by-environment interaction (GEI) for yield and the two quality traits. The fourth objective was to study the relationship among wheat grain yield and the two quality traits by various statistical techniques. Linear regression (TRET) and various variance component methods were investigated to determine genetic advance. The recommended method of determining genetic advance in this study is TRET. In the Western Cape elite trials TRET predicted a genetic advance of 1% per year and genetic advance estimated at 1% genetic improvement for protein content in the cultivar trials. No significant trend was observed in the elite trials of the Free State with TRET. Yield showed 0.5% and 0.6% per year improvement for the two planting dates of the eastern cultivar trials of Free State. A yield improvement of 0.3% per year improvement for the two planting dates of the central cultivar trials of Free State was determined. A genetic advance for yield of 0.7% per year was found in the warm region of the elite irrigation trials and 9% yield improvement per year for the first planting date of the eastern region of the cultivar irrigation trials. A negative trend was observed for the second planting date of eastern region of both elite and cultivar irrigation trials. The effects of GEI on yield and quality traits were studied by comparing the AMMI and GGE analyses. These methods portrayed similar results. An advantage of these techniques is their complementary nature. Although both models portray GEI in various biplots, the AMMI provides statistical evidence to the visual presentation of the GGE biplots. Pearson product moment correlation matrix provided a linear relationship among the variables studied. Principal component analysis (PCA), cluster analysis (CA) and discriminant analysis (DA) offered auxiliary information on the relationship among the factors (e.g. genotypes, years, localities and/or environments) and the variables. DA was not able to indicate direction of genetic improvement in either of the three production areas in this study.
Open Access
Breeding potential of cassava (Manihot esculenta Crantz) in Mozambique
(University of the Free State, 2008-05) Da Silva, Anabela Matangue Zacarias; Koen, Elizma; Kanju, Edward Eneah
English: This study represents the first comprehensive genetic study based on local cultivars of Mozambique. Five cassava (Manihot esculenta Crantz) parents were crossed in a full diallel. The parents and F1 progeny were evaluated for eight agronomical and 17 morphological traits in two consecutive seasons. General combining ability (GCA) was higher than specific combining ability (SCA) for number of roots, root taste, fresh root yield, root pulp hardness and harvest index. Positive heterosis was seen for number of roots, fresh root weight and harvest index. Negative heterosis was present for cassava brown streak virus disease. Harvest index showed high broad sense heritability, which indicated that phenotypic variance was additive. The predictability ratio was close to unity, which confirmed that this trait is highly heritable, and determined by additive gene action. Cassava brown streak root necrosis had SCA effects significantly higher than GCA effects indicating the presence of non-additive genetic effects. A selection index comprised of the most relevant characters was compiled. Amplified fragment length polymorphism (AFLP) analysis showed high genetic similarity among 17 accessions. A total of 425 fragments were produced by eight primer combinations. Mocuba and Nikwaha were the most distinct among the accession and two heterotic groups were suggested. Morphological analysis showed genetic distance ranging from 0.105 to 0.833. The correlation coefficient between AFLP and agronomic clustering was not significant. The AFLP and morphological analysis did not group the accessions according to geographical origin. The principal component analysis provided diversity structure, suggesting the effectiveness of this analysis in genetic diversity studies. The narrow genetic similarity observed, indicates the need to introduce new cassava germplasm.
Open Access
Characterisation and diallel analysis of commercially planted cotton (Gossypium hirsutum L.) germplasm in Tanzania
(University of the Free State, 2005-11) Lukonge, Everina Paul; Labuschagne, M. T.; Herselman, L.
English: 1. Thirty cotton varieties were studied. Seven parents generated 21 F1 diallel progenies. The main objective was to evaluate genotypes through oil content, fatty acids, morphological and molecular characteristics. Statistical procedures were performed with Agrobase, NCSS and NTYSS computer programmes. Correlation, heterosis, heritability, G x E interaction and stability values were obtained. Stability across four environments was analysed by five stability measures, that of Shukla, Wricke, Lin and Binns, Eberhart and Russell and the AMMI analysis. 2. Significant differences were observed for oil and fatty acids content. Oil content results revealed an average of 20.23%. The ratio for polyunsaturated:saturated fatty acid was 2:1 and for unsaturated:saturated 3:1. The predominant fatty acids were linoleic, palmitic, oleic, stearic, myristic, palmitoleic, arachidic and behenic. Palmitic fatty acid was high while oleic and stearic fatty acids were low for almost all varieties studied. Oil and fatty acid content were significantly influenced by the environment. CIM 70 and Cyto 12/74 can be selected as parents for increased oleic and stearic fatty acid and Stoneville 506 as a parent for reduced palmitic fatty acid, increased oleic, stearic and linolenic fatty acids in hybridisation. 3. ANOVA results indicated significant differences between varieties for all agronomical characteristics studied, where NTA 93-21 and NTA 93-15 were the best performers almost for all characteristics. Genetic distances for morphological (qualitative characteristics) analysis ranged from 0.18 to 0.80. Two major groups, one including many varieties from Africa and the other including varieties from the USA, were revealed. 4. AFLP analysis showed high genetic similarities (0.85 to 0.98). Varieties were distinguished according to pedigree and origin. The increased genetic similarity observed, indicates the need for the introduction of more diverse cotton material. Comparison of agronomical and AFLP markers for determining genetic diversity indicated that genetic similarities between varieties were low for agronomical compared to AFLP markers. The correlation coefficient between AFLP analysis and agronomical genetic similarities was not significant, 5. Highly significant differences between genotypes over environments were observed and NTA 93-21 crosses outperformed the seven parents for almost all characteristics. The parent Okra leaf performed poorly for many characteristics. Trial sites Ukiriguru followed by Kanziga were high yielding environments. 6. Combining ability analysis results indicated significant variation in GCA estimates among parents for different characteristics. Genotypes having high positive GCA estimates for yield components exhibited negative GCA estimates for fibre quality, excluding NTA 93-21. Some combinations indicated positive and significant SCA effects for seedcotton, boll weight, GOT and lint yield. The GCA/SCA ratios for most of the studied characteristics were larger than one. 7. Correlation among characteristics indicated highly significant positive and negative correlations. The observed high and significant GCA and SCA effects for those characteristics are promising for cotton population improvement. Positive heterosis for boll weight was observed for more than 90% of the combinations. Hybrids with high SCA values presented high values of heterosis. Heritability values for the studied characteristics were moderate to high. 8. The combined ANOVA over four environments indicated significant differences between genotypes and locations for all characteristics. The percentage contribution of location to total variation was high compared to genotype variation except for a few characteristics. Highly significant differences of G x E for lint yield and fibre strength were observed. 9. Three stability procedures (Wricke, Shukla and ASV) identified similar stable genotypes. Eberhart and Russell’s procedure, was in harmony with the above three methods but with some divergence. Cultivar superiority measure deviated from the other four measures. Therefore the procedures of Wricke, Shukla and AMMI can be applied for cotton stability studies.
Open Access
Characterisation of both Hoodia gordonii and the associating wilt causing pathogen Fusarium oxysporum
(University of the Free State, 2014-01) Philippou, Onoufrios Agathoclis; Van Biljon, A.; Minnaar-Ontong, A.; Swart, W. J.
English: Hoodia gordonii has been used by the San people for centuries as an appetite suppressant while they were on long hunting trips. These succulents are globally known as an important component in diet supplements and products which assist in weight-loss. Together with other plants these contribute towards a multimillion US dollar market, as many pharmaceutical and nutritional companies have made significant financial investments in the research and development for people who suffer from weight problems and obesity. In South Africa, H. gordonii is classified as being endangered, because so many pharmaceutical companies cashed-in on the dietary characteristics of these succulents. The plant is found in the South Western parts of Southern Africa mainly in the Karoo, Kalahari and Namib deserts. Plants were also grown in cultivated nurseries under favourable conditions for commercial use. However, diseases have dramatically hampered production in nurseries with almost total loss of crop. Although not many diseases have been documented on this plant, Fusarium wilt, a devastating fungal disease of H. gordonii, caused by F. oxysporum had been identified. This disease is not only responsible for economic losses, but also contaminates the soils with spores, which remain dormant until the next season as inoculum. This study concentrated on the morphological characterisation and molecular identification of the Fusarium wilt causing pathogen present in four areas (Kakamas, Klein Pella, Pofadder and Prieska) as well as the morphological evaluation of the host. Fusarium oxysporum was identified as the causal agent of Fusarium wilt on H.gordonii plants. The AFLP analysis and DNA sequences resulted in two distinct groups. Those that clustered in the AFLP cluster B were also grouped in a TEF cluster B, however some isolates from AFLP cluster A also grouped together with the TEF cluster B. The low genetic variation revealed by the AFLP analysis indicated that differences amongst the pathogen isolates occur, but the DNA sequences confirmed that these isolates share a common ancestor. DNA sequencing analysis was used to place 44 South African F. oxysporum isolates into the phylogenetic groups as described by O’Donnell and associates. South African F. oxysporum isolates clustered into two groups. The observed genetic variation amongst individual isolates was lower than the genetic variation between out-group isolates. Two clusters were identified; within each cluster isolates had a relatively high frequency of clones. These clones confirm that these pathogen isolates share similar allele frequencies. Results from sequencing data showed that the isolates fall within the FOSC, however there was no isolates identified that show 100% similarities when compared with all three genes sequences with recorded genetic sequences of F. oxysporum isolates in other parts of the world. Therefore, based on the current taxonomic system, of host specificity the fungus only infects H. gordonii, so the study has proved that a possible new formae specialis has been identified. New preventative measures must be applied to the host when planting in nurseries. Knowledge gained from analyses of the genetic fingerprinting, DNA sequencing of these isolates and the mporphological evaluation of the host might assist with the development of effective control strategies, i.e. resistance breeding against Fusarium wilt. This will provide an incentive to potential farmers to plant H. gordonii, thus improving production of this succulent for pharmaceutical companies and nurseries in South Africa.
Open Access
Characterisation of Malawian cassava germplasm for diversity, starch extraction and its native and modified properties
(University of the Free State, 2005-11-30) Benesi, Ibrahim Robeni Matete; Saka, J. K.; Labuschagne, Maryke T.; Herselman, Liezel
English: Cassava has been neglected and considered as a primitive crop, food for the poor and a crop with poor nutritional value for a long time. Cassava provides more dietary energy per unit area than any staple crop. Population increase, multiplicity of cassava uses, unusual climatic variations and prospects of global warming highlight advantages of possible uses of this hardy drought tolerant crop. Policy makers realised the importance of cassava and are encouraging its promotion through research, production and utilisation. Since the crop has lagged behind for a long time, there exist knowledge gaps in many areas. Commercialisation of cassava, and biotic and abiotic factors encourage genetic erosion. Management and use of germplasm in production and breeding programmes need knowledge of genetic diversity. Hence, there existed a need to collect, characterise, evaluate, conserve and use Malawian cassava germplasm. Indigenous knowledge revealed wide genetic diversity because farmers reported a wide range of quantitative and quality attributes, which they preferred or disliked in existing Malawian cassava germplasm. Morphological markers failed to uniquely distinguish all analysed accessions. Manyokola5 and Mbundumali1, Gomani1 and 2, and Matuvi and Depwete were morphologically identical. However, morphological markers revealed high levels of genetic diversity with Dice genetic similarity (GS) ranging between 0.083-1.000. The most distant accessions were Beatrice (local cultivar) and Yizaso (CH92/112; locally screened). AFLP characterisation using cluster analysis indicated narrow genetic diversity with a GS range between 0.778-0.946 and uniquely differentiated all analysed accessions. The closest accessions were Mabundumali1 and Manyokola5 (GS=0.946), and Gomani1 and 2 (GS=0.938). PCA analysis for AFLP markers revealed that PCA axis 1 displayed narrow genetic diversity but PCA axes 2 and 3 displayed wide genetic diversity. AFLP analysis of cassava germplasm grouped accessions according to pedigree and geographical origins. Morphological and AFLP markers were not similar but significantly correlated, indicating the need to use both markers in characterisation of cassava germplasm. Screening of AFLP primers revealed that M-CAA/E-ACA, M-CAA/E-AAC, M-CAT/E-ACA, M-CAT/E-AAC, M-CTT/E-ACT and M-CTT/E-ACC were convenient and efficient in analysing cassava. Further analysis indicated that a combination of four primer pairs was more cost and time efficient and gave similar resolution compared to a combination of six primer pairs, the best being M-CAA/E-ACA+M-CAA/E-AAC+M-CTT/E-ACT+M-CTT/E-ACC. Evaluation of genotypes for starch extraction was done just before or at onset of the rainy season (October-November), within the rainy season (January) and towards the end of the rainy season (March-April). Results indicated that the best genotypes were Mkondezi, Silira, Mbundumali and CH92/082. Starch extraction done just before or at onset of the rainy season gave the highest starch extraction rates compared to the middle of the rainy season. Apart from high recovery of starch in October-November, it is convenient for drying starch using open-air method and releases land for other crops in the following rainy season. Makoka was the best site for growing cassava for starch extraction. Starch extraction rate on fresh root weight basis had a direct relationship with starch yield as opposed to starch extraction on dry root weight basis. Cassava starch from different genotypes were characterised for physical and functional properties. Results indicated that Silira, 81/00015, Mbundumali and Sauti were easily dextrinised and 80% solubility was achieved within 60min of dextrinisation at 100 o C after acidification with 0.1M HCl. Pyrodextrin from Mkondezi variety after dextrinisation for 40min turned out to be similar in functional properties to amylmaize starch. Cassava starches had lower gelatinisation temperatures desired for hot-setting adhesives since less heating is required to gelatinise cassava starch, which leads to energy saving. Functional properties of 83350 native starch was different from the rest of the genotypes but was very close to amylmaize starch as revealed by DSC. Afrikaans: Cassava is in die verlede verwaarloos en beskou as ‘n primitiewe gewas, kos vir arm mense en ‘n gewas met swak voedingswaarde. Cassava verskaf meer voedingsenergie per eenheid area as enige ander stapelgewas. Populasie toename, veelvoudige gebruike, ongewone klimaatswisseling, en die moontlikheid van globale verwarming beklemtoon die voordele van hierdie geharde, droogte tolerante gewas. Beleidmakers het die belangrikheid van cassava besef en moedig nou navorsing, produksie en gebruik van cassava aan. Omdat navorsing op die gewas vir lank agterweë gebly het, is daar kennisgapings wat gevul moet word om dit effektief te produseer, prosesseer en gebruik. Kommersialisering van cassava, en biotiese en abiotiese faktore bevorder genetiese erosie. Bestuur en gebruik rakende kiemplasma in produksie en teelprogramme vereis kennis van genetiese diversiteit. Daarom bestaan daar ‘n behoefte aan versameling, karakterisering, evaluering, bewaring en gebruik van Malawiese kiemplasma. Inheemse kennis het wye genetiese diversiteit aangetoon, omdat boere ‘n wye spektrum in bestaande kwantitatiewe en kwalitatiewe eienskappe aangetoon het wat hulle in bestaande Malawiese cassava kiemplasma verkies of afkeur. Morfologiese merkers kon nie tussen al die inskrywings onderskei nie. Manyokola5 en Mbundumali1, Gomani1 en 2, en Matuvi en Depwete was morfologies identies. Tog het morfologiese merkers wye genetiese diversiteit aangetoon met die Dice genetiese similariteits (GS) spektrum van 0.083-1.000. Die genotipes met die grootste genetiese afstand was Beatrice (plaaslike cultivar) en Yizaso (CH92/112; plaaslik geëvalueer). AFLP karakterisering met groeperings analise het baie nou genetiese diversiteit aangedui met ‘n GS spektrum van 0.778-0.946, maar alle inskrywings kon gedifferensieer word. Die mees verwante inskrywings was Mabundumali1 en Manyokola5 (GS=0.946), en Gomani1 en 2 (GS=0.938), wat met die morfologiese karakterisering as duplikate geïdentifiseer is. PCA analise vir AFLP merkers het getoon dat die PCA as 1 min genetiese diversiteit wys, maar PCA asse 2 en 3 het wye genetiese diversiteit getoon. AFLP analise van cassava kiemplasma het die inskrywings volgens geografiese oorsprong gegroepeer, wat verwant was aan die stamboom van die inskrywings. Morfologiese en AFLP merkers was nie dieselfde nie, maar was tog betekenisvol gekorreleer. Dit is dus goed om beide merkersisteme karakterisering van cassava kiemplasma te gebruik in. Evaluering van AFLP voorvoerders het getoon dat M-CAA/E-ACA, M-CAA/E-AAC, MCAT/E-ACA, M-CAT/E-AAC, M-CTT/E-ACT en M-CTT/E-ACC cassava effektief analiseer. Verdere analise het getoon dat ‘n kombinasie van vier voorvoerders net so goed was as ‘n kombinasie van ses voorvoerders. Die beste voorvoerders was M-CAA/E-ACA+M-CAA/EAAC+M-CTT/E-ACT+M-CTT/E-ACC. Evaluasie van genotipes vir stysel ekstraksie is net voor die begin van die reënseisoen (Oktober-November), in die reënseisoen (Januarie) en naby die einde van die reënseisoen (Maart-April) gedoen. Resultate het getoon dat die beste genotipes Mkondezi, Silira, Mbundumali en CH92/082 was. Styselektraksies wat net voor die begin van die reënseisoen gedoen is het hoër ekstraksie persentasies gegee as in die middel van die reënseisoen. Behalwe dat die ekstraksies so hoog was tydens Oktober-November, is dit ook ‘n baie geskikte tyd om die stysel te droog met die opelug metode, en dit maak die lande ook beskikbaar vir ander gewasse in die opvolgende reënseisoen. Makoka was die beste area om cassava te verbou vir stysel ekstraksie. Cassava stysel van verskillende genotipes is vir fisiese en funksionele eienskappe gekarakteriseer. Resultate het gewys dat Silira, 81/00015, Mbundumali en Sauti maklik gedektriniseer is en 80% oplosbaarheid is binne 60 min van dekstrinisering gekry by 100oC na asidifisering met 0.1M HCl. Pirodekstrinering van Mkondezi vir 40min het baie dieselfde funksionele eienskappe gewys as die van amylmaize stysel. Gelvorming vind by laer temperature plaas in cassava stysel as ander stysel, wat voordelig is vir hitte geaktiveerde plakmiddels, omdat minder energie nodig is om gelvorming te induseer in die cassava stysel, wat tot energiebesparing lei. Funksionele eienskappe van 83350 natuurlike stysel was verskillend van die van die ander genotipes, maar dit was baie dieselfde as amylmaize stysel soos gesien met die DSC.