Thesis presented in partial fulfilment of the requirements for the degree of Master of Technology (Food Technology) Department of Food Technology Faculty of Applied Sciences Cape Peninsula University of Technology, 2012 / The aim of this study was to evaluate bambara groundnut milk (BGNM) subjected to fermentation with lactic acid bacteria (LAB) as a probiotic beverage with a view to developing value-added product. Central Composite Rotatable Design (CCRD) was used to optimise the hydration time and temperature of BGN flour for optimum BGN milk (BGNM) production. The optimum time and temperature was 2 h at 25oC. The effect of variety was assessed on the quality and consumer acceptability of BGNM prepared from five varieties of BGN (black, red, brown, brown-eye, and black-eye) which were representatives of the BGN available in South Africa. BGNM from the five varieties differed significantly (p<0.05) in, lightness, chroma, redness, yellowness, hue and antioxidative activity, while the pH were not significantly different. The four BGNM samples were significantly different (p < 0.05) in appearance, colour, mouthfeel and overall acceptability but not in aroma and taste. A three factor design (4 x 3 x 3) consisting of probiotics (Lactobacillus acidophilus, L. bulgaricus, L. casei and L. plantarum), temperature and fermentation time, were used to estimate the optimal conditions for the production of BGN probiotic beverage (BGNPB). The optimal condition for the production of BGNPB was estimated to be 35oC for 24 h with a desirability of 0.854 for L. bulgaricus. The next promising probiotic was L. plantarum that could be fermented at 35oC for 24 h with 0.843 desirability. BGNM from the red variety were fermented with L. bulgaricus and L. plantarum and L bulgaricus (in combination), making plain and sweetened BGNPB which were evaluated for their quality and consumer acceptability. The four BGNPB samples were significantly different (p < 0.05) in aroma, taste, mouthfeel and overall acceptability but not in appearance and colour. The plain BGNPB were assessed for their proximate composition, antioxidant activity, in vitro probiotic tolerance to simulated gastric juices and bile and a 28 days shelf life study at 5, 15 and 25oC. The protein, total dietary fibre (TDF), ash and antioxidative activity of the BGNPB were significantly different while the fat and carbohydrates were not significantly different. Time and concentration of the gastric juice and bile had significant effects on the percentage bacterial survival of probiotics in the BGNPB. However, the probiotics did survive, in low numbers, in the simulated gastric juice and bile after 180 and 240 minutes of incubation. Titratable acidity, pH, microbial load and colour of the BGNPB were significantly affected by the storage time and temperature during the shelf life study. At the 5oC storage temperature the BGNPB had a right censored shelf life on day 28. At 15oC the shelf life was 18 and 10 days for L bulgaricus and L. plantarum and L. bulgaricus respectively. The outcome of this research showed that a novel BGNPB product can be made from fermenting BGNM with LAB.

A melancia [Citrullus lanatus (Thunb.) Matsum & Nakai] é originária da África Central, e bastante cultivada por todo o mundo. No ano de 2012, a produção mundial dessa fruta foi 105.372.341 milhões de toneladas, enquanto que o Brasil produziu 2.079.547 milhões toneladas. A cultura da melancia é de suma importância econômica para o estado do Tocantins, com rendimentos no ano de 2013 de 98,5 milhões de reais. A melancia é susceptível à ação de patógenos, sendo as doenças virais de maior relevância. Os vírus do gênero Tospovirus, transmitidos exclusivamente por tripes vetores em diversas culturas, têm causados prejuízos de bilhões de dólares em custos de controle. Os tripes são encontrados em todas as regiões do Brasil, com altas infestações em cultivos de melancia, podendo causar danos diretos e/ou indiretos. Neste trabalho foram coletados espécimes de tripes nos municípios produtores de melancia do estado do Tocantins: Gurupi, Formoso do Araguaia, Lagoa da Confusão e Porto Nacional. A identificação dos caracteres morfológicos dos tripes foi realizada utilizando chave taxonômica apropriada com alterações para identificação das espécies. Parte dos insetos coletados foi realizada a extração de RNA total. Posteriormente, foi sequenciado o extraído com tecnologia de sequenciamento de nova geração (NGS). Foram identificadas três espécies de tripes nos quatro municípios produtores, F. schultzei, F. tritici e F. insularis, com predominância de infestação da espécie F. schultzei. Sendo esta a única entre as identificadas, que é considerada vetor de Tospovirus. Foi identificado nas amostras de tripes, o vírus Groundnut ringspot virus (GRSV), o qual é eficientemente transmitido por F. schultzei. Entre as espécies de tripes identificadas, F. tritici e F. insularis, são registradas pela primeira vez em cultivos comerciais de melancia no Brasil. F. schultzei, é a única com relatos de danos diretos e indiretos causados em plantas de melancia, podendo ser considerada praga potencial à cultura, devido o alto nível populacional encontrado e por dificuldades no controle do tripes. / The Watermelon [Citrullus lanatus (Thunb.) Matsum & Nakai] is originally from Central Africa, and cultivated worldwide. In 2012, the world production of this fruit was 105.372.341 million tons, while Brazil produced 2.079.547 million tons. The watermelon cropping is of paramount economic importance to the state of Tocantins, with revenues of 98.5 million, in 2013. Watermelon is susceptible to the action of microorganisms, and viral diseases presents most relevance. The Tospovirus genus, transmitted exclusively by thrips vectors in diverse crops, have caused losses of billions of dollars in cost control. Thrips are found in all regions of Brazil, with high infestations in watermelon crops, and may cause direct and/or indirect damages. In this work, it was collected thrips specimens from watermelon producers of municipalities in the state of Tocantins: Gurupi, Formoso do Araguaia, Lagoa da Confusão and Porto Nacional. The thrips identification of morphological characters was performed using appropriate taxonomic key with changes to species identification. In part of the collected insects, the total RNA extraction was performed. The extracted was subsequently sequenced with next-generation sequencing technology (NGS). Three species of thrips were identified in the four producing municipalities, F. schultzei, F. tritici, and F. insularis, with predominance of F. schultzei. Among the identified species, only F. schultzei is considered a vector of Tospovirus. In thrips samples it was identified the virus Groundnut ringspot virus (GRSV), which is efficiently transmitted by F. schultzei. Among the species of thrips identified, F. tritici and F. insularis, are registered for the first time in watermelon commercial crops in Brazil. F. schultzei is the only one with reports of direct and indirect damage to watermelon plants and may be considered as a potential plague due the high population level found and difficulties in controlling it.

Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2008.

The term "lipid" covers an extremely diverse range of chemical or molecular species. Lipids, defined as molecules that are sparingly soluble in water but readily soluble in organic solvents, are broadly categorized into "neutral " or "apolar" lipids, and "amphiphilic” or "polar" lipids. Neutral lipids will include simple hydrocarbons, carotenes, triacylglycerols, wax esters, sterol eaters, as wel1 as other lipids such as fatty acids, polyprenols, and sterols In which the hydrophilic function has little Impact on the overall molecular characteristics. Polar lipids include phospholipids, glycolipids, sulfolipids, some sphingolipids, oxygenated carotenoids and chlorophylls.

Issues surrounding water scarcity will become topical in future as global fresh water resources become more limited thus threaten crop production. Predicted climate change and increasing population growth will place more pressure on agriculture to produce more food using less water. As such, efforts have now shifted to identifying previously neglected underutilised species (NUS) as possible crops that could be used to bridge the food gap in future. Taro (Colocasia esculenta L. Schott) and bambara groundnut (Vigna subterranea L. Verdc) currently occupy low levels of utilisation in South Africa. Both crops are cultivated using landraces with no improved varieties available. Information describing their agronomy and water–use is limited and remains a bottleneck to their promotion. The aim of this study was to determine the drought tolerance and water–use of selected landraces of taro and bambara groundnut from KwaZulu-Natal, South Africa. In order to meet the specific objectives for taro and bambara groundnut management, an approach involving conventional and modelling techniques was used. Three taro landraces [Dumbe Lomfula (DL), KwaNgwanase (KW) and Umbumbulu (UM)] were collected from the North Coast and midlands of KwaZulu-Natal, South Africa, in 2010. The UM landrace was classified as Eddoe type taro (C. esculenta var. antiquorum) characterised by a central corm and edible side cormels. The DL and KW landraces were classified as Dasheen (C. esculenta var. esculenta), characterised by a large edible main corm and smaller side cormels. A bambara groundnut landrace was collected from Jozini, KwaZulu- Natal, and characterised into three selections (‘Red’, ‘Light-brown’ and ‘Brown’) based on seed coat colour. Seed colour was hypothesised to have an effect on seed quality. Field and rainshelter experiments were conducted for both taro and bambara landraces at Roodeplaat in Pretoria and Ukulinga Research Farm in Pietermaritzburg, over two growing seasons (2010/11 and 2011/12). The objective of the field trials for taro and bambara groundnut was to determine mechanisms associated with drought tolerance in taro and bambara groundnut landraces. Experiments were laid out in a split-plot design where irrigation [fully irrigated (FI) and rainfed (RF)] was the main factor and landraces (3 landraces of either taro or bambara groundnut) were sub-factors. Treatments were arranged in a randomised complete block design (RCBD), replicated three times. Rainfed trials were established with irrigation to allow for maximum crop stand. Thereafter, irrigation was withdrawn. Whilst experimental designs and layouts for taro and bambara groundnut were similar, differences existed with regards to plot sizes and plant spacing. Trials were planted on a total land area of 500 m2 and 144 m2, for taro and bambara groundnut, respectively. Plant spacing was 1 m x 1 m for taro and 0.3 m x 0.3 m for bambara groundnut. Irrigation scheduling in the FI treatment was based on ETo and Kc and was applied using sprinkler irrigation system. Separate rainshelter experiments were conducted for taro and bambara groundnut landraces at Roodeplaat, to evaluate growth, yield and water-use of taro and bambara groundnut landraces under a range of water regimes. The experimental design was similar for both crops, a RCBD with two treatment factors: irrigation level [30, 60 and 100% crop water requirement (ETa)] and landrace (3 landraces), replicated three times. Irrigation water was applied using drip irrigation system based on ETo and Kc. Data collection in field and rainshelter trials included time to emergence, plant height, leaf number, leaf area index (LAI), stomatal conductance and chlorophyll content index (CCI). For taro field trials, vegetative growth index (VGI) was also determined. Yield and yield components (harvest index, biomass, corm number and mass) as well as water–use efficiency (WUE) were determined at harvest. Intercropping of taro and bambara groundnut was evaluated under dryland conditions using farmers’ fields at Umbumbulu, KwaZulu–Natal, South Africa. The experimental design was a RCBD replicated three times. Intercrop combinations included taro and bambara groundnut sole crops, a 1:1 (one row taro to one row bambara groundnut) and 1:2 intercrop combinations. The taro UM landrace and ‘Red’ bambara groundnut landrace selection were used in the intercropping study. Lastly, data collected from field and rainshelter experiments were used to develop crop parameters to calibrate and validate the FAO’s AquaCrop model for taro and bambara groundnut landraces. The UM landrace was used for taro while the ‘Red’ landrace selection was used for bambara groundnut. AquaCrop was calibrated using observed data from optimum (FI) experiments conducted during 2010/11. Model validation was done using observations from field and rainshelter experiments conducted during 2011/12 as well as independent data. Results showed that all taro landraces were slow to emerge (≈ 49 days after planting). Stomatal conductance declined under conditions of limited water availability (RF, 60% and 30% ETa). The UM landrace showed better stomatal regulation compared with KW and DL landraces under conditions of limited water availability. Plant growth (plant height, leaf number, LAI and CCI) of taro landraces was lower under conditions of limited water availability (RF, 60% and 30% ETa) relative to optimum conditions (FI and 100% ETa). The UM landrace showed moderate reductions in growth compared with the DL and KW landraces, suggesting greater adaptability to water limited conditions. The VGI showed a large reduction in growth under RF conditions and confirmed the UM landrace’s adaptability to limited water availability. Limited water availability (RF, 60% and 30% ETa) resulted in lower biomass, HI, and final yield in taro landraces relative to optimum conditions (FI and 100% ETa). For all trials, the DL landrace failed to produce any yield. WUE of taro landraces was consistent for the three irrigation levels (30, 60 and 100% ETa); however, on average, the UM landrace was shown to have a higher WUE than the KW landrace. Bambara groundnut landraces were slow to emerge (up to 35 days after planting). ‘Red’ and ‘Brown’ landrace selections emerged better than the ‘Light-brown’ landrace selection, confirming the effect of seed colour on early establishment performance. Plant growth (stomatal conductance, CCI, plant height, leaf number, LAI and biomass accumulation) was lower under conditions of limited water availability (RF, 60% and 30% ETa) relative to optimum conditions (FI and 100% ETa). The ‘Red’ landrace selection showed better adaptation to stress. Limited water availability resulted in early flowering and reduced flowering duration as well as early senescence and maturity of bambara groundnut landrace selections. The ‘Red’ landrace selection showed delayed leaf senescence under conditions of limited water availability. Yield reductions of up to 50% were observed under water limited conditions (RF, 60% and 30% ETa) relative to optimum conditions (FI and 100% ETa). Water use efficiency increased at 60% and 30% ETa, respectively, relative to 100% ETa, implying adaptability to limited water availability. The ‘Red’ landrace selection showed better yield stability and WUE compared with the ‘Brown’ and ‘Light-brown’ landrace selections suggesting that seed colour may be used as a selection criterion for drought tolerance in bambara groundnut landraces. The intercropping study showed that intercropping, as an alternative cropping system, had more potential than monocropping. Evaluation of growth parameters showed that taro plant height was generally unaffected by intercropping but lower leaf number was observed as compared with the sole crop. Bambara groundnut plants were taller and had more leaves under intercropping relative to the sole crop. Although not statistically significant, yield was generally lower in the intercrops compared with the sole crops. Evaluation of intercrop productivity using the land equivalent ratio (LER) showed that intercropping taro and bambara groundnut at a ratio of 1:1 was more productive (LER = 1.53) than intercropping at a ratio of 1:2 (LER = 1.23). The FAO’s AquaCrop model was then calibrated for the taro UM landrace and ‘Red’ bambara groundnut landrace selection. This was based on observations from previous experiments that suggested them to be drought tolerant and stable. Calibration results for taro and bambara groundnut landraces showed an excellent fit between predicted and observed parameters for canopy cover (CC), biomass and yield. Model validation for bambara groundnut showed good model performance under field (FI and RF) conditions. Model performance was satisfactory for rainshelters. Validation results for taro showed good model performance under all conditions (field and rainshelters), although the model over-estimated CC for the declining stage of canopy growth under RF conditions. Model verification using independent data for taro showed equally good model performance. In conclusion, the taro UM landrace and ‘Red’ bambara groundnut landrace selection were shown to be drought tolerant and adapted to low levels of water–use. The mechanisms responsible for drought tolerance in the taro UM landrace and ‘Red’ bambara groundnut landrace selection were described as drought avoidance and escape. The taro UM landrace and ‘Red’ bambara groundnut landraces avoided stress through stomatal regulation, energy dissipation (loss of chlorophyll) as well as reducing canopy size (plant height, leaf number and LAI), which translates to minimised transpirational water losses. This indicated landrace adaptability to low levels of water–use. The ‘Red’ bambara groundnut landrace selection showed phenological plasticity and escaped drought by flowering early, delaying leaf senescence, and maturing early under conditions of limited water availability. Performance of the ‘Red’ landrace selection lends credence to the use of seed coat colour as a possible selection criterion for drought tolerance in bambara groundnut, and possibly for other landraces with variegated seed. The taro UM landrace escaped drought by maturing early under conditions of limited water availability. The FAO’s AquaCrop model was successfully calibrated and validated for taro UM and ‘Red’ bambara groundnut landraces. The calibration and validation of AquaCrop for taro is the first such attempt and represents progress in the modelling of neglected underutilised crops. The calibration and validation of AquaCrop for taro requires further fine-tuning while that for bambara groundnut still needs to be tested for more diverse landraces. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Issues surrounding water scarcity will become topical in future as global fresh water resources become more limited thus threaten crop production. Predicted climate change and increasing population growth will place more pressure on agriculture to produce more food using less water. As such, efforts have now shifted to identifying previously neglected underutilised species (NUS) as possible crops that could be used to bridge the food gap in future. Taro (Colocasia esculenta L. Schott) and bambara groundnut (Vigna subterranea L. Verdc) currently occupy low levels of utilisation in South Africa. Both crops are cultivated using landraces with no improved varieties available. Information describing their agronomy and water–use is limited and remains a bottleneck to their promotion. The aim of this study was to determine the drought tolerance and water–use of selected landraces of taro and bambara groundnut from KwaZulu-Natal, South Africa. In order to meet the specific objectives for taro and bambara groundnut management, an approach involving conventional and modelling techniques was used. Three taro landraces [Dumbe Lomfula (DL), KwaNgwanase (KW) and Umbumbulu (UM)] were collected from the North Coast and midlands of KwaZulu-Natal, South Africa, in 2010. The UM landrace was classified as Eddoe type taro (C. esculenta var. antiquorum) characterised by a central corm and edible side cormels. The DL and KW landraces were classified as Dasheen (C. esculenta var. esculenta), characterised by a large edible main corm and smaller side cormels. A bambara groundnut landrace was collected from Jozini, KwaZulu- Natal, and characterised into three selections (‘Red’, ‘Light-brown’ and ‘Brown’) based on seed coat colour. Seed colour was hypothesised to have an effect on seed quality. Field and rainshelter experiments were conducted for both taro and bambara landraces at Roodeplaat in Pretoria and Ukulinga Research Farm in Pietermaritzburg, over two growing seasons (2010/11 and 2011/12). The objective of the field trials for taro and bambara groundnut was to determine mechanisms associated with drought tolerance in taro and bambara groundnut landraces. Experiments were laid out in a split-plot design where irrigation [fully irrigated (FI) and rainfed (RF)] was the main factor and landraces (3 landraces of either taro or bambara groundnut) were sub-factors. Treatments were arranged in a randomised complete block design (RCBD), replicated three times. Rainfed trials were established with irrigation to allow for maximum crop stand. Thereafter, irrigation was withdrawn. Whilst experimental designs and layouts for taro and bambara groundnut were similar, differences existed with regards to plot sizes and plant spacing. Trials were planted on a total land area of 500 m2 and 144 m2, for taro and bambara groundnut, respectively. Plant spacing was 1 m x 1 m for taro and 0.3 m x 0.3 m for bambara groundnut. Irrigation scheduling in the FI treatment was based on ETo and Kc and was applied using sprinkler irrigation system. Separate rainshelter experiments were conducted for taro and bambara groundnut landraces at Roodeplaat, to evaluate growth, yield and water-use of taro and bambara groundnut landraces under a range of water regimes. The experimental design was similar for both crops, a RCBD with two treatment factors: irrigation level [30, 60 and 100% crop water requirement (ETa)] and landrace (3 landraces), replicated three times. Irrigation water was applied using drip irrigation system based on ETo and Kc. Data collection in field and rainshelter trials included time to emergence, plant height, leaf number, leaf area index (LAI), stomatal conductance and chlorophyll content index (CCI). For taro field trials, vegetative growth index (VGI) was also determined. Yield and yield components (harvest index, biomass, corm number and mass) as well as water–use efficiency (WUE) were determined at harvest.Intercropping of taro and bambara groundnut was evaluated under dryland conditions using farmers’ fields at Umbumbulu, KwaZulu–Natal, South Africa. The experimental design was a RCBD replicated three times. Intercrop combinations included taro and bambara groundnut sole crops, a 1:1 (one row taro to one row bambara groundnut) and 1:2 intercrop combinations. The taro UM landrace and ‘Red’ bambara groundnut landrace selection were used in the intercropping study. Lastly, data collected from field and rainshelter experiments were used to develop crop parameters to calibrate and validate the FAO’s AquaCrop model for taro and bambara groundnut landraces. The UM landrace was used for taro while the ‘Red’ landrace selection was used for bambara groundnut. AquaCrop was calibrated using observed data from optimum (FI) experiments conducted during 2010/11. Model validation was done using observations from field and rainshelter experiments conducted during 2011/12 as well as independent data. Results showed that all taro landraces were slow to emerge (≈ 49 days after planting). Stomatal conductance declined under conditions of limited water availability (RF, 60% and 30% ETa). The UM landrace showed better stomatal regulation compared with KW and DL landraces under conditions of limited water availability. Plant growth (plant height, leaf number, LAI and CCI) of taro landraces was lower under conditions of limited water availability (RF, 60% and 30% ETa) relative to optimum conditions (FI and 100% ETa). The UM landrace showed moderate reductions in growth compared with the DL and KW landraces, suggesting greater adaptability to water limited conditions. The VGI showed a large reduction in growth under RF conditions and confirmed the UM landrace’s adaptability to limited water availability. Limited water availability (RF, 60% and 30% ETa) resulted in lower biomass, HI, and final yield in taro landraces relative to optimum conditions (FI and 100% ETa). For all trials, the DL landrace failed to produce any yield. WUE of taro landraces was consistent for the three irrigation levels (30, 60 and 100% ETa); however, on average, the UM landrace was shown to have a higher WUE than the KW landrace. Bambara groundnut landraces were slow to emerge (up to 35 days after planting). ‘Red’ and ‘Brown’ landrace selections emerged better than the ‘Light-brown’ landrace selection, confirming the effect of seed colour on early establishment performance. Plant growth (stomatal conductance, CCI, plant height, leaf number, LAI and biomass accumulation) was lower under conditions of limited water availability (RF, 60% and 30% ETa) relative to optimum conditions (FI and 100% ETa). The ‘Red’ landrace selection showed better adaptation to stress. Limited water availability resulted in early flowering and reduced flowering duration as well as early senescence and maturity of bambara groundnut landrace selections. The ‘Red’ landrace selection showed delayed leaf senescence under conditions of limited water availability. Yield reductions of up to 50% were observed under water limited conditions (RF, 60% and 30% ETa) relative to optimum conditions (FI and 100% ETa). Water use efficiency increased at 60% and 30% ETa, respectively, relative to 100% ETa, implying adaptabilityto limited water availability. The ‘Red’ landrace selection showed better yield stability and WUE compared with the ‘Brown’ and ‘Light-brown’ landrace selections suggesting that seed colour may be used as a selection criterion for drought tolerance in bambara groundnut landraces. The intercropping study showed that intercropping, as an alternative cropping system, had more potential than monocropping. Evaluation of growth parameters showed that taro plant height was generally unaffected by intercropping but lower leaf number was observed as compared with the sole crop. Bambara groundnut plants were taller and had more leaves under intercropping relative to the sole crop. Although not statistically significant, yield was generally lower in the intercrops compared with the sole crops. Evaluation of intercrop productivity using the land equivalent ratio (LER) showed that intercropping taro and bambara groundnut at a ratio of 1:1 was more productive (LER = 1.53) than intercropping at a ratio of 1:2 (LER = 1.23). The FAO’s AquaCrop model was then calibrated for the taro UM landrace and ‘Red’ bambara groundnut landrace selection. This was based on observations from previous experiments that suggested them to be drought tolerant and stable. Calibration results for taro and bambara groundnut landraces showed an excellent fit between predicted and observed parameters for canopy cover (CC), biomass and yield. Model validation for bambara groundnut showed good model performance under field (FI and RF) conditions. Model performance was satisfactory for rainshelters. Validation results for taro showed good model performance under all conditions (field and rainshelters), although the model over-estimated CC for the declining stage of canopy growth under RF conditions. Model verification using independent data for taro showed equally good model performance. In conclusion, the taro UM landrace and ‘Red’ bambara groundnut landrace selection were shown to be drought tolerant and adapted to low levels of water–use. The mechanisms responsible for drought tolerance in the taro UM landrace and ‘Red’ bambara groundnut landrace selection were described as drought avoidance and escape. The taro UM landrace and ‘Red’ bambara groundnut landraces avoided stress through stomatal regulation, energy dissipation (loss of chlorophyll) as well as reducing canopy size (plant height, leaf number and LAI), which translates to minimised transpirational water losses. This indicated landrace viii adaptability to low levels of water–use. The ‘Red’ bambara groundnut landrace selection showed phenological plasticity and escaped drought by flowering early, delaying leaf senescence, and maturing early under conditions of limited water availability. Performance of the ‘Red’ landrace selection lends credence to the use of seed coat colour as a possible selection criterion for drought tolerance in bambara groundnut, and possibly for other landraces with variegated seed. The taro UM landrace escaped drought by maturing early under conditions of limited water availability. The FAO’s AquaCrop model was successfully calibrated and validated for taro UM and ‘Red’ bambara groundnut landraces. The calibration and validation of AquaCrop for taro is the first such attempt and represents progress in the modelling of neglected underutilised crops. The calibration and validation of AquaCrop for taro requires further fine-tuning while that for bambara groundnut still needs to be tested for more diverse landraces. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

An introduction to nematode systematics is provided which deals broadly with the history of the classification of nematodes, the controversial usage of the Phylum names Nemata Cobb, 1919 and Nematoda (Rudolphi, 1808) Lankester, 1877 and the reason why the name Nematoda was used in the present study. The classification, diagnosis and bionomics of the genus Aphelenchoides Fischer, 1894, the genus to which A. arachidis Bos, 1977 belongs is discussed. The section on bionomics is included to capture the astounding ability of this group of organisms to adapt to different trophic levels, a concept that is used to attempt an explanation for the ability of a supposedly African nematode, A. arachidis, to infest an alien crop species (groundnut). The ability of Aphelenchoides spp. to adapt to different host plant species is discussed, as well as the ability of the groundnut plant to mature its pods underground, a characteristic that predisposes these plants to a host of pathogens. The damage caused by two of the most important endoparasitic nematode species on groundnut, A. arachidis and Ditylenchus africanus Wendt, Swart, Vrain & Webster, 1995 were compared with each other. The South African population of A. arachidis was found predominantly in the shells of groundnut, whereas they were found in the shells, roots, hypocotyls and testas of the groundnut plants in Nigeria. The present study showed that A. arachidis and D. africanus occur together in groundnut in South Africa with D. africanus usually being the dominant species. In only one instance, at Bullhill (Vaalharts Irrigation Scheme, Northern Cape), the groundnut shells and testas were infested by A. arachidis alone. The importance of plant quarantine in South Africa is dealt with and the aims and principles of quarantine, as well as the different guidelines that have to be adhered to when deciding on the quarantine status of an organism are explained. Descriptions are provided of the methods used to prepare specimens for viewing with the light microscope (LM) and the scanning electron microscope (SEM), as well as the procedures of the molecular study. A morphological and morphometrical description of A. arachidis specimens from South Africa, as well as a comparison with specimens from Nigeria was done. Differences between the South African and Nigerian populations included, respectively, a lower b-value (7 - 11 vs 10 - 18), more lateral lines (2 - 4 vs 2), a slightly shorter stylet (8-10 m vs 10 - 12 m) and a longer length of the post-uterine sac as a percentage of the distance from vulva to anus (41 - 96 % vs ± 50 %). Scanning electron micrographs of this species are presented for the first time and shows the morphology of the lip region and lateral lines. Since both A. arachidis and A. blastophthorus were detected in the pods, a study was done to evaluate a PCR-based diagnostic method for the identification of these species and to compare the results with those reported in literature. Restriction fragment length polymorphisms (RFLPs) in the rDNA fragment were used to compare and differentiate between nematode species. The differences encountered within the South African population (morphological, morphometrical and molecular) warrant a study of more specimens from more localities. Through this it could be ascertained whether the South African population is a subspecies of A. arachidis or if this species just differs widely between localities. Future research should focus on a survey of the groundnut producing areas in South Africa to determine the distribution and economic impact of A. arachidis. The incidence of A. arachidis on other agricultural crops, especially those used in rotation with groundnut, also needs to be determined. The next issue to address is what enables a supposedly endemic species to Africa, A. arachidis, to parasitize an alien plant species (groundnut) from South America. Screening of the endemic bean family (Fabaceae) in Africa for the presence of A. arachidis, could hold the answer to this question. / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2008.

Resumo: O experimento foi conduzido na área da Fazenda de Ensino, Pesquisa e Produção e nos Laboratórios de Análises de Sementes e Fitopatologia da Faculdade de Ciências Agrárias e Veterinárias - FCAV/UNESP, Câmpus de Jaboticabal. O objetivo foi verificar as perdas quantitativas e qualitativas da cultura do amendoim, cultivar Runner IAC 886, em função das velocidades de arranquio e recolhimento. Foram utilizadas duas velocidades de arranquio (4,3 e 5,0 km h-1) e três de recolhimento (3,5; 4,6 e 6,2 km h-1). Foram avaliadas as perdas visíveis (PVA), perdas invisíveis (PIA), as perdas totais (PTA), perdas visíveis totais (PVT) e perdas totais na colheita (PTC), dimensionamento das leiras, distribuição de palhas e produtividade real. Para a qualidade fisiológica foram utilizados os seguintes testes: germinação em areia e em papel, primeira contagem de germinação, índice de velocidade de emergência, massa seca de plântulas, envelhecimento acelerado, condutividade elétrica, emergência de plântulas em campo e tetrazólio. Para análise estatística no arranquio, foi utilizado o delineamento inteiramente casualizado com duas velocidades de arranquio. No recolhimento foi utilizado o delineamento em blocos casualizados em parcelas sub-divididas. As parcelas foram compostas por duas velocidades de arranquio, e as sub-parcelas por três velocidades de recolhimento Para a qualidade fisiológica os dados foram submetidos ao teste de Shapiro-Wilk e após foram submetidos à análise de variância pelo teste F juntamente com os dados de sanidade, e as médias comparadas por meio do teste de Tukey com nível 5% de significância. As velocidades máximas e mínimas não influenciaram nas perdas quali-quantitativas das sementes de amendoim. / Abstract: The experiment was carried out in the experimental FARM and in the Analysis and in the Phytopatology Laboratories belonging to College of Agriculture, São Paulo State University, Campus of Jaboticabal. The objective of this research was to quantify the losses of peanut crop harvesting and, also the qualitative, it was used a cultivar Runner IAC 886, and was analyzed the effect of the groundnut digger and the harvesting combine speeds. Were used two groundnut digger speeds (4.3 and 5.0 km h-1) and three harvesting combine speeds (3.5; 4.6 and 6.2 km h-1). In the digging process were evaluated the visible losses (PVA), the invisible losses (PIA), total losses groundnut digger (PTA), total visible losses (PVT), total losses in the crop (PTC), design of piles, distribution of straws and the real production. To the physiological quality were used the germination percentage (in sand and paper), first germination count, speed of emergence, seedling dry matter, accelerated aging, electrical conductivity, seedling emergence at field conditions and tetrazolium. For the statistical analysis it was used a completely randomized design consisting of two groundnut digger speed. In the combined harvesting was used a randomized block design in a split plot scheme. The plots were composed by two digger speeds and the split split plot by three combine harvesting speeds. The physiological seed quality were analized by the Shapiro-Will test and after were submitted to the analys of variance together with the sanity data, and the means compared by the Tukey test at 5% level of probability. The maximum and minimum speeds didn't influence in the qualitatife and quantitative losses of the peanut seeds. / Orientador: Rubens Sader / Coorientador: Roberval Daiton Vieira / Banca: Carlos Eduardo Angeli Furlani / Banca: Denise Mahl / Mestre

Made available in DSpace on 2014-06-11T19:28:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-03Bitstream added on 2014-06-13T18:34:39Z : No. of bitstreams: 1 barrozo_lm_me_jabo.pdf: 1167406 bytes, checksum: a14ce668242f0784c3da533068d2476d (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O experimento foi conduzido na área da Fazenda de Ensino, Pesquisa e Produção e nos Laboratórios de Análises de Sementes e Fitopatologia da Faculdade de Ciências Agrárias e Veterinárias – FCAV/UNESP, Câmpus de Jaboticabal. O objetivo foi verificar as perdas quantitativas e qualitativas da cultura do amendoim, cultivar Runner IAC 886, em função das velocidades de arranquio e recolhimento. Foram utilizadas duas velocidades de arranquio (4,3 e 5,0 km h-1) e três de recolhimento (3,5; 4,6 e 6,2 km h-1). Foram avaliadas as perdas visíveis (PVA), perdas invisíveis (PIA), as perdas totais (PTA), perdas visíveis totais (PVT) e perdas totais na colheita (PTC), dimensionamento das leiras, distribuição de palhas e produtividade real. Para a qualidade fisiológica foram utilizados os seguintes testes: germinação em areia e em papel, primeira contagem de germinação, índice de velocidade de emergência, massa seca de plântulas, envelhecimento acelerado, condutividade elétrica, emergência de plântulas em campo e tetrazólio. Para análise estatística no arranquio, foi utilizado o delineamento inteiramente casualizado com duas velocidades de arranquio. No recolhimento foi utilizado o delineamento em blocos casualizados em parcelas sub-divididas. As parcelas foram compostas por duas velocidades de arranquio, e as sub-parcelas por três velocidades de recolhimento Para a qualidade fisiológica os dados foram submetidos ao teste de Shapiro-Wilk e após foram submetidos à análise de variância pelo teste F juntamente com os dados de sanidade, e as médias comparadas por meio do teste de Tukey com nível 5% de significância. As velocidades máximas e mínimas não influenciaram nas perdas quali-quantitativas das sementes de amendoim. / The experiment was carried out in the experimental FARM and in the Analysis and in the Phytopatology Laboratories belonging to College of Agriculture, São Paulo State University, Campus of Jaboticabal. The objective of this research was to quantify the losses of peanut crop harvesting and, also the qualitative, it was used a cultivar Runner IAC 886, and was analyzed the effect of the groundnut digger and the harvesting combine speeds. Were used two groundnut digger speeds (4.3 and 5.0 km h-1) and three harvesting combine speeds (3.5; 4.6 and 6.2 km h-1). In the digging process were evaluated the visible losses (PVA), the invisible losses (PIA), total losses groundnut digger (PTA), total visible losses (PVT), total losses in the crop (PTC), design of piles, distribution of straws and the real production. To the physiological quality were used the germination percentage (in sand and paper), first germination count, speed of emergence, seedling dry matter, accelerated aging, electrical conductivity, seedling emergence at field conditions and tetrazolium. For the statistical analysis it was used a completely randomized design consisting of two groundnut digger speed. In the combined harvesting was used a randomized block design in a split plot scheme. The plots were composed by two digger speeds and the split split plot by three combine harvesting speeds. The physiological seed quality were analized by the Shapiro-Will test and after were submitted to the analys of variance together with the sanity data, and the means compared by the Tukey test at 5% level of probability. The maximum and minimum speeds didn't influence in the qualitatife and quantitative losses of the peanut seeds.

1 Viruses are submicroscopic obligate parasites that depend on the host cell for their growth and reproduction. Plants are infected by diverse group of viruses that mostly possess RNA as their genome. In the recent times, many new RNA viruses have evolved that possess the potential threat to plants and animals. One among them is Tospovirus (Family Bunyaviridae) which has severely affected the agricultural productivity in India. One of the Tospoviruses GBNV is a major challenge of crop production in south India. Tospoviruses shares several features such as morphology, genome structure and organization with members of other genera in the family Bunyaviridae. Virus particles are 80–120 nm in diameter. The genome includes three RNAs referred to as large (L), medium (M) and small (S). The L RNA is in negative-sense while the M and S RNAs are ambisense. The L RNA codes for the RNA-dependent RNA polymerase (RdRp), and the M RNA for the precursor of two glycoproteins (GN and GC) and a non-structural protein (NSm). The S RNA codes for the N protein and another non-structural protein (NSs). Tospovirus infection is an emerging threat for agricultural productivity in India. Therefore, biochemical and molecular characterization of these viruses is essential for developing various strategies for control of these diseases. 2 Present thesis deals with biochemical characterization of nonstructural protein, NSs of GBNV. 3 A review of literature on Tospovirus genome organization, replication, transcription, translation and assembly is presented in Chapter I. This chapter also includes the recent work on all the proteins encoded by the tospoviruses. 4 The objectives of the present study are as follows; a. Cloning, expression, purification and biophysical characterizations of rNSs. b. Analysis of its NTPase/dATPase activity c. Demonstration of nucleic acid 5’ phosphatase activity d. Characterization of nucleic acid unwinding activity of rNSs 5 The materials used in this study and the experimental protocols followed such as construction of recombinant clones, their overexpression in bacteria, protein purification techniques, site directed mutagenesis and all other biochemical, molecular biology are described in chapter II 6 NSs of TSWV was shown to be suppressor of gene silencing (PTGS) in 2002. Since then there has been no further work on this protein. Till date neither in vitro nor in vivo study of NSs of any tospovirus has been carried out in detail. To gain insight into the biochemical function of rNSs, the NSS gene was cloned, overexpressed in E.coli and purified. The NSS gene, was cloned into pRSET-C vector. 7. Chapter 3 deals with cloning, overexpression, purification and biophysical characterization of GBNV NSs in terms of secondary structure analysis as well as its interaction with siRNA and ssRNA. The results provide the evidence that rNSs was successfully expressed in E.coli and purified (Fig. 3.1). Molecular mass of purified rNSs was confirmed by MALDI TOF, which gave the molecular mass of expected size 51.5 kDa (Fig. 3.2) Circular dichroism study revealed that rNSs has negative ellipticity peak at 215 and 223 nm typical of a globular protein. The protein had an emission maximum at 340 nm (Fig 3.3 B) when exited at 280 nm, which reflects that rNSs is well folded. Thermal melting study (Fig 3.3 C) showed rNSs had a reasonably high Tm (65°C). So overall, spectral study suggested that purified rNSs was soluble, well folded and thermally stable and could be used for further biochemical assay. The oligomeric status of the protein was determined by size exclusion chromatography to be trimeric (156 kDa, Fig 3.5). Purified rNSs was used to raise the polyclonal antibodies in rabbit. The antiserum could detect rNSs specific band only in IPTG induced sample not in uninduced sample (Fig 3.6). 50% binding was observed at 100 ng/ml of antigen showing that these antibodies were of high affinity (Fig 3.7 B). Further, the 50% binding was observed at 1:34000 dilution of the antiserum, which suggests that high titer antibodies against rNSs were obtained (Fig 3.7 A). 8 Further, the RNA binding property of rNSs was examined. Synthetic 21 bp siRNA and in vitro transcribed 100 nt ssRNA was used to analyze the RNA binding property of rNSs. Indeed rNSs was able to bind with 100 nt ssRNA (Fig 3.8 A) or 21 nt siRNA in a protein concentration dependent manner (Fig 3.8 B). The binding however did not require presence of divalent cation such as Mg 2+ (Fig 3.8 C). In order to understand the biological function of rNSs, its interaction with the structural protein, NP by ELISA was investigated. rNSs could interact with the NP protein (Fig 3.9) . Further 15 amino deletions from C terminus of NP did not affect its interaction with rNSs protein (Fig 3.9), which suggest that the C terminal 15 amino acid residues of NP are not essential for interaction with rNSs in vitro. 9. Sequence analysis of GBNV NSs revealed the presence of Walker motifs A (GxxxxGKT) and B (DExx) in its primary structure (Fig 4.2). The proteins that possess the Walker motifs A and B exhibit ATPase activity. Therefore, the purified rNSs was tested for its ability to hydrolyze ATP in the absence and presence of poly(A) (chapter IV). rNSs could hydrolyze [γ-32P] ATP in a concentration-dependent manner (Fig. 4.3 A). Further, ATPase activity was stimulated in presence of poly(A) (Fig. 4.3 B). Quantitative analysis of reaction product suggested that the reaction was linear in the presence of poly(A) upto 1.6 µg of rNSs (Fig. 4.3 C). 10. The product of ATP hydrolysis by rNSs had the same mobility as the phosphate released by RecoP51 ATPase, a positive control used in the assay. In contrast, another viral protein from the Cotton leaf curl virus, His tagged-AV2, purified in same way as rNSs, did not show the release of phosphate, suggesting that the activity was not due to the histidine tag present at the N-terminus of rNSs. Further, no release of phosphate could be seen when immunodepleted rNSs was used suggesting that the activity was inherent to the protein and was not due to bacterial contamination (Fig 4.3 lane 7). Time course analysis of ATPase activity revealed that the reaction is linear up to 25 mins (Fig 4.4). Further, pH profile was a typical bell shaped curve with a distinct pH optimum at pH 7.0 (Fig 4.5 A) and the temperature optimum was at 25 °C(Fig 4.5 B). Most of the known viral ATPases require the divalent cation for their activity. The rNSs exhibited the optimum ATPase activity between 2-2.5 mM of MgCl2. The reaction was inhibited by increasing concentration of EDTA demonstrating the requirement of Mg2+ for ATP hydrolysis (Fig. 4.7). Further, the ATPase activity of rNSs was inhibited by increasing concentrations of non-hydrolyzable analog of ATP (Fig. 4.8) and was not inhibited by AMP (Fig 4.9) suggesting that rNSs is not a nucleotidyl phosphatase and is a true ATPase. Limited proteolysis of rNSs suggested that core domain was 23 kDa in size and could catalyze ATP hydrolysis (Fig. 21 and 4.22). 11. Interestingly rNSs not only cleaved ATP rather it could hydrolyze all rNTPs as well as dATP (Fig 4.10). Kinetic parameters were determined for its enzymatic activity. Comparison of the kinetic constants of rNSs NTPase activity revealed little variation, suggesting that the rNSs has a broad substrate specificity (Fig 4.10- 4.15 and table 4.1). 12. To assess the role of amino acids in Walker motif A and B (Fig. 4.16) site specific mutants K189A and D159A were generated ( Fig 4.17) confirmed by sequencing, overexpressed in E.coli and purified (Fig. 4.18). Point mutation in Walker motif B (D159A) reduced the ATPase activity (Fig 4.19) where as point mutation in Walker motif A (K189A abolishes the activity (Fig 4.19). 13. Chapter V deals with the nucleic acid 5’ phosphatase activity of rNSs. Experimental evidence presented in this chapter clearly shows that rNSs can cleave the single phosphate from the ssDNA, ssRNA, dsRNA and dsDNA. Nucleic acid 5’ phosphatase activity of rNSs was inhibited by AMP and ATP (Fig 5.2 and Fig 5.3). Interestingly the K189A mutant rNSs was as active as wild type rNSs where as D159A mutant showed slightly reduced activity (Fig 5.7 C). 14. As mentioned earlier, rNSs was shown to possesses the RNA stimulated NTPase/dATPase activity, a hallmark of all known helicases. Therefore, its nucleic acid unwinding activity was examined using dsDNA and dsRNA as a substrate. rNSs was able to unwind the dsDNA as well as dsRNA in a ATP dependent manner (chapter VI, Fig. 6.1 and 6.5 respectively). ATP and Mg2+ are essential cofactors for the unwinding activity (Fig. 6.1). While the unwinding activity could be observed with ATP and to some extent with dATP, all other NTPs and dNTPs failed to support the helicase function of rNSs (Fig 6.2) Further experimental evidence suggested that rNSs is a bidirectional helicase (Fig. 6.3). D159A mutation in Walker motif B resulted in reduced helicase activity where as K189A mutation in walker Motif A completely abolished the DNA as well as RNA helicase activity of rNSs (Fig. 6.6 and Fig 6.7 respectively). Therefore, mutational analysis clearly suggests that helicase activity is an intrinsic property of rNSs. 15. In conclusion rNSs of GBNV is multifunctional enzyme. This is the first report on the demonstration that rNSs is an non canonical ATP dependent helicase in the Bunyaviridae family. In addition to being a suppressor of PTGS, NSs may also regulate the viral replication and transcription by modulating the secondary structure of the viral genome. This new research finding on NSs might pave way for further studies on its role in viral replication and transcription.