<p align="justify">Dans les pays en voie de développement et plus particulièrement en Afrique, la médecine traditionnelle est parfois la seule source de soins abordable et accessible,surtout pour les patients les plus pauvres.</p><p><p align="justify">Le présent travail a été réalisé dans le but de rechercher les preuves scientifiques de l’activité anthelminthique des extraits d’amandes de Balanites aegyptiaca utilisés en médecine traditionnelle africaine et d’évaluer une activité potentiellement anti-tumorale du ou des principe(s) actif(s) responsable(s) de l’activité anthelminthique.</p><p><p align="justify">Pour caractériser l’activité nématocide des extraits des amandes de Balanites aegyptiaca,nous avons tout d’abord mis au point un test d’évaluation de l’activité toxique en tenant compte des limitations des tests existants. La validation pharmacologique (mesurant la sélectivité, la linéarité, l’exactitude et la précision) a consisté en la détermination de l’activité nématocide d’anthelminthiques couramment utilisés. Pour la caractérisation de l’activité nématocide des amandes de Balanites aegyptiaca, puis le fractionnement, l’isolement et la purification de(s) agent(s) nématocide(s) nous avons adopté la stratégie du fractionnement bio-guidé. Les résultats obtenus montrent que le produit isolé (déterminé comme étant la balanitine-7 ou Bal-7) induit une activité toxique plus élevée sur les vers adultes que sur les stades larvaires.</p><p><p align="justify">Bal-7 s’est avéré moins toxique que le levamisole, le mébendazole et le thiabendazole, mais plus toxique que le pyrantel, le niclosamide et la pipérazine. La présente étude a donc permis de montrer que les amandes de Balanites aegyptiaca, utilisée en médecine traditionnelle au Burkina Faso, pourraient être efficaces dans le traitement des parasitoses intestinales.</p><p><p align="justify">Certains anthelminthiques comme les benzimidazoles, du fait de leur activité d’inhibition de la polymérisation des tubulines, présentent une activité anti-tumorale. Aussi, faisant suite à la mise en évidence de l’activité nématocide de Bal-7 nous avons entrepris de caractériser l’activité anti-tumorale de balanitines. La méthode d’extraction que nous avons utilisé pour évaluer l’effet anti-tumoral de la Bal-7 est distincte de celle que nous avions utilisée pour évaluer l’effet anthelminthique de cette balanitine. Ainsi, alors que la méthode d’extraction que nous avons utilisée pour obtenir de la Bal-7 pour nos tests liés à l’activité anthelminthique semble avoir conduit à l’isolement de la balanitine-7 pure, la méthode d’extraction que nous avons utilisée pour observer les effets anti-tumoraux potentiels de cette balanitine-7 nous ont conduit à isoler un mélange de balanitine-6 et de balanitine-7 dans des proportions de 28/72%. Nous avons dénommé ce mélange Bal-6/7. L’activité anti-tumorale a été évaluée sur deux lignées cancéreuses humaines (A549, cancer du poumon non-à-petites cellules et U373, glioblastome). Dans ce travail, nous avons montré que Bal-6/7 induit la mort des cellules tumorales par une déplétion marquée de l’[ATP]i et une désorganisation majeure du cytosquelette d’actine. In vivo, Bal-6/7 a montré une activité anti-tumorale modeste, mais néanmoins statistiquement significative. A ce jour, il n’existe pas sur le marché, d’anti-cancéreux dirigé contre les filaments d’actine. Etant donné le rôle de ces filaments d’actine dans la prolifération et la migration des cellules tumorales, le développement de médicaments ayant cette protéine pour cible constituerait une avancée majeure dans la recherche de nouvelles thérapies anti-tumorales. Le mélange Bal-6/7, isolé pour la caractérisation de l’activité anti-tumorale des balanitines, du fait de son potentiel anti-tumoral, présente donc un intérêt certain en thérapeutique anti-cancéreuse. Il serait donc envisageable de développer par synthèse ou hémisynthèse des dérivés de balanitines présentant un meilleur index thérapeutique que le mélange Bal-6/7.</p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Thesis (M. A. Agriculture (Plant Protection)) -- University of Limpopo, 2019 / Potato (Solanum tuberosum L.) is highly susceptible to root-knot (Meloidogyne species) nematodes, with no known nematode resistant genotypes. In Limpopo Province, two cucurbitacin-containing phytonematicides had been researched and developed. The active ingredients of the cucurbitacin-containing phytonematicides are cucurbitacins, which are allelochemicals that could induce phytotoxicity on crops being protected against nematode damage. The objectives of this study were to determine: (1) mean concentration stimulation point (MCSP) of Nemarioc-AL phytonematicide on potato cultivar ꞌMondial G3ꞌ for managing M. javanica and (2) application interval of Nemarioc AL phytonematicide on potato cultivar ꞌMondial G3ꞌ. Sprouted tubers were planted in 10 cm deep/pot with each pot filled with steam-pasteurised soil and Hygromix at 3:1 (v/v) ratio in the field under microplot conditions. After 100% emergence (2 weeks), each plant was inoculated with 5 000 M. javanica eggs and second-stage juveniles (J2). Seven treatments, namely, 0, 2, 4, 8, 16, 32 and 64% Nemarioc-AL phytonematicide were arranged in a randomised complete block design, with 11 replications. In Objective 2, four treatments, namely, 1, 2, 3 and 4 weeks were arranged in randomised complete block design, with 15 replications. Plant variables and nutrient elements were subjected to the Curve-fitting Allelochemical Response Data (CARD) model to generate biological indices used to compute MCSP using the relation MCSP = Dm + Rh/2 and the overall sensitivity value (∑k). The MCSP for plant variables and nutrient elements, were empirically derived as 4.31% and 1.33%, with the ∑k of 18 and 4 units, respectively. Nematode variables and increasing concentrations of Nemarioc-AL phytonematicide exhibited negative quadratic relations where eggs, J2 in soil and roots and total population (Pf) were optimised at xv 14.43, 28.23, 23.30 and 13.55%. To conduct Objective 2 which is application interval, empirically derived MCSP value of 4.31% from Objective 1 was used. Application interval was optimised using the concept of 1, 2, 3, and 4 weeks in weeks-per-month-of-30-days. The application interval of 4.31% was established at 2.43 weeks which translated to 18 days [(2.43 weeks/4 weeks) × 30 days]. All nematode variables in Objective 2 were not significantly different at all intervals. In, conclusion Nemarioc-AL phytonematicide can be used at 4.31% concentration to control nematodes population densities without being phytotoxic to crops at 18 days application interval. / National Research Foundation (NRF) , Agricultural Research Council (ARC) and the Flemish Interuniversity Council of Belgium

Thesis (M.Sc. (Plant Production)) -- University of Limpopo, 2013 / Two phytonematicides were researched and developed from fermented crude extracts of wild watermelon (Cucumis africanus) and wild cucumber (Cucumis myriocarpus) fruits for use as alternatives to methyl bromide in managing root-knot (Meloidogyne species) nematodes in tomato (Solanum lycopersicum) production. Fruits of C. africanus contain cucurbitacin B (C32H48O8), while those of C. myriocarpus contain cucurbitacin A, which comprises cucumin (C27H40O9) and leptodermin (C27H38O8). Phytonematicides from C. africanus and C. myriocarpus fruits are referred to as nemafric-B and nemarioc-A, respectively. The two phytonematicides, due to their origin from plant species with allelochemicals, have high potential of being phytotoxic to crops. The use of the Curve-fitting Allelochemical Response Dosage (CARD) computer-based model assisted in the establishment of concentrations which were stimulatory to growth of tomato (Solanum lycopersicum) plants, while exhibiting nematoxic properties to Meloidogyne species. The two phytonematicides were developed from crude extracts of fruits dried at 52˚C in air-forced ovens and ground in a Wiley mill through 1-mm-opening sieves. However, equipment for drying and grinding fruits would not be accessible to smallholder farmers who wished to prepare their own products on-farm. The objective of this study therefore, was to determine whether nemafric-BL and nemarioc-AL produced from fresh fruit of the two Cucumis species would be suitable for use (i.e. non phytotoxic) in tomato production for managing population densities of M. incognita race 2. In order to distinguish the products of fresh (F) fruits from those of dried (D) fruits, they were code-named nemafricF-BL or nemariocF-BL and nemafricD-BL or nemariocD AL, respectively, where G and L denoted granular and liquid formulations, respectively. Tomato cv. ‘Floradade’ seedlings were infested with 3 000 eggs and second-stage xv juveniles of M. incognita race 2. An equivalent of 40 g and 80 g dried fruit mass of nemafric-B and nemarioc-A, namely, 284 g and 411 g fresh fruit mass for nemafric-B and nemarioc-A, respectively, were separately fermented using EMROSA effective micro-organisms mixed with 16 L chlorine-free tapwater in 20 L container for 14 days at ± 25˚C, allowing pH to gradually decline to ± 3.7. Separate experiments for each product run concurrently. Treatments, namely, 0, 2, 4, 8, 16, 32 and 64% concentrations, where for instance, 2% = 20 ml/1000 ml x 100, were arranged in a randomised complete block design, with 10 replications. Blocking in the greenhouse was done for wind direction which was regularly erected by fans for cooling down the greenhouse. At 56 days after weekly application of each treatment, flower number, fruit number, dry shoot mass, dry root mass, dry fruit mass, plant height, stem diameter and nematode numbers were each subjected to analysis of variance. Nematode data were, prior to analysis, transformed using log10(x + 1), but untransformed data were reported. Using the sum of squares, nemafric-BL and nemarioc-AL treatments affected dry root mass, dry shoot mass, flowers number, fruit number, plant height and stem diameter. Nemafric-BL contributed 67%, 78%, 58%, 43%, 60% and 26%, while nemarioc-AL contributed 71%, 61%, 19%, 35%, 34% and 24% to total treatment variation of the six respective variables. Plant variables with significant (P ≤ 0.05) treatment effects were further subjected to the CARD model to generate seven biological indices, with three distinct phases, namely, stimulation, neutral and inhibition phases. Using the quantified stimulation phase, the mean concentration stimulation range (MCSR) was computed for each variable using two biological indices, namely, threshold stimulation point (Dm) and saturation point (Rh). The CARD model explained 98%, 99%, 98% and 98% of the quadratic models of dry root mass, dry shoot mass, plant height and stem diameter, xvi respectively, against increasing concentrations of nemarioc-AL. Similarly, the CARD model explained 99%, 96%, 84% and 93% of total treatment variation in the respective plant variables. The integrated MCSR [MSCR = Dm + (Rh/2)] for nemafric-BL on tomato plants was 7%, while that for nemarioc-AL was 4%. In the CARD model, the overall sensitivities (∑k) of tomato plants exposed to nemafric-BL and nemarioc-AL were 3 units and 5 units, respectively. Tomato plants were therefore, less sensitive to nemarioc-AL since it had higher ∑k value than nemafric-BL. At 4% nemarioc-AL and at 7% nemafric-BL, the two phytonematicides were each highly suppressive to population densities of M. incognita race 2. In conclusion, on the basis of non-phytotoxicity of the computed MCSR values and their suppressive effects on population densities of M. incognita race 2, the smallholder farmers could produce nemafric-BL and nemarioc-AL phytonematicides on-farm. However, the production of the two products from fresh fruits would not be sustainable since fruits of the two Cucumis species are highly seasonal due to the high incidence of post-harvest decays. / The Land Bank Chair of Agriculture – University of Limpopo, Limpopo Agro-processing Technology Station,and the Flemish Interuniversity Council of Belgium

Thesis (MSc. (Horticulture)) -- University of Limpopo, 2016. / Pelargonium sidoides has numerous medicinal applications, with economic potential to serve as a future cultigen in smallholder farming systems. However, it is highly susceptible to the root-knot (Meloidogyne species) nematodes, without any identifiable nematode resistant genotypes. Nemarioc-AL and Nemafric-BL phytonematicides, with cucurbitacin A and cucurbitacin B active ingredients, respectively, are being researched and developed as an alternative to synthetic nematicides at the University of Limpopo. However, since active ingredients in phytonematicides are allelochemicals, the two phytonematicides have the potential of inducing phytotoxicity on crops protected against nematode damage. The objectives of the study, therefore, were (1) to determine the non-phytotoxic concentration of Nemarioc-AL phytonematicide on plant growth of P. sidoides, and (2) to determine the non-phytotoxic concentration of Nemafric-BL phytonematicide in plant growth of P. sidoides. Cuttings were raised in 30-cm-diameter plastic pots containing 10 000 ml steam-pasteurised river sand and Hygromix-T at 3:1 (v/v) under microplot conditions in autumn (March-May) and repeated in spring (August October) 2015. After establishment each plant was inoculated with 5 000 eggs and second-stage juveniles (J2s) of M. javanica. Six treatments, namely, 0, 2, 4, 6, 8 and 10% concentrations of each phytonematicide on separate trials were arranged in a randomised complete block design, with seven replicates. At 56 days after inoculation, in Experiment 1, Nemarioc-AL phytonematicide, treatment significantly (P ≤ 0.05) affected plant height, dry root mass and root galls, contributing 62, 69 and 70% to total treatment variation of the three variables, respectively. Relative to untreated control Nemarioc-AL phytonematicide increased plant height and dry root mass by 34 to 61% xxi and 20 to 76%, respectively, with a slight decrease by 5% in plant height at the highest concentration. However, the material decreased root galls by 5 to 50%. Significant (P ≤ 0.05) plant variables were subjected to Curve fitting-allelochemical respond dosage model, to generate biological indices which were used to compute the mean concentration stimulation point (MCSP) using the relation: MCSP = Dm + Rh/2 and the overall sensitivity value (∑k). In Experiment 1, MCSP = 6.18% and ∑k = 3. Plant variables and increasing concentration of phytonematicide exhibited quadratic relations. Treatments reduced nematode variables, at all levels including at the lowest, but the effect were not different. In Experiment 2, Nemarioc-AL phytonematicide treatment effects were not significant on plant variables except for root galls, but were significant for root nematodes except for eggs. Data for plant variables in Experiment 2 were not subjected to Curve fitting-allelochemical respond dosage model because they were not significant (P ≤ 0.05). In Experiment 1, Nemafric-BL phytonematicide treatment significantly (P ≤ 0.05) affected plant height and root galls, contributing 63 and 67% to total treatment variation of the two variables, respectively. Relatively to untreated control, plant height was increased by 10 to 36%, while root galls was reduced by 2.43 to 60%. In Experiment 1, MCSP = 2.87% and ∑k = 3. Concentrations of Nemafric-BL phytonematicide significantly (P ≤ 0.05) reduced eggs, juveniles and Pf at all levels including at the lowest, but the effect were not significant different, with treatments contributing 78, 72 and 90% to the total treatment variation. In Experiment 2, Nemafric BL phytonematicide treatment effects were not significant on plant variables except for root galls, but were significant for root. In conclusion, Nemarioc-AL and Nemafric-BL xxii phytonematicides could be applied at the lowest concentration of 2% where it was shown to be effective in suppressing population densities of M. javanica. / Agricultural Research Council (ARC), National Research Fund (NRF) , Flemish Inter university Council of Belgium and Land Bank Chair of Agriculture ─ University of Limpopo

Thesis (M.Sc. (Agriculture, Plant Protection)) -- University of Limpopo, 2020 / The unavailability of environment-friendly nematicides for managing root-knot (Meloidogyne species) nematodes in crop husbandry have led to various alternative methods being sort which includes the development of cucurbitacin-containing phytonematicides. The cited phytonematicides consistently suppressed nematode numbers on different crops under greenhouse, microplot and field conditions, although there is lack of information on how the products would affect susceptible Swiss chard infected by root-knot nematodes. Swiss chard is one of most nutritious vegetables, grown throughout the year and is well adapted to different soil types. However, these products have the potential to induce phytotoxicity on various crops, if applied improperly. Phytotoxicity of phytonematicides on different crops, has been resolved by deriving Mean Concentration Stimulation Point (MCSP). The MCSP, developed using the Curve-fitting Allelochemical Response Data (CARD) computer-based model, is crop-specific, hence it should be developed for every crop. The objectives of this study were to investigate (1) whether population densities of Meloidogyne species, growth and accumulation of selected nutrient elements in Swiss chard would respond to increasing concentration of Nemarioc-AL and Nemafric-BL phytonematicides under greenhouse and microplot conditions and (2) whether the nemarioc-group and nemafric-group phytonematicides in liquid and granular formulations would affect population densities of Meloidogyne species and the productivity of Swiss chard with related accumulation of nutrient elements in leaf tissues under field conditions. Parallel experiments for Nemarioc-AL and Nemafric-BL phytonematicides were conducted concurrently under greenhouse and microplot conditions. Greenhouse experiment was prepared by arranging 25-cm-diameter plasticpods on greenhouse benches, whereas microplot experiment was prepared by digging holes and inserting 30-cm-diameter plastic pots in the field. The four-week-old Swiss chard seedlings were transplanted into the pots, filled with steam-pasteurised loam, sand and Hygromix-T at 3:1:1 (v/v) ratio. Treatments comprised 0, 2, 4, 8, 16, 32 and 64% phytonematicides arranged in randomised complete block design (RCBD), with six replications. Treatments were applied seven days after inoculation, with 3000 eggs and J2 of M. incognita race 4 under greenhouse conditions, whereas under microplot conditions were inoculated with 6000 eggs and J2 of M. javanica. Under field conditions, treatments comprised untreated control (0), 2 g Nemarioc-AG and 3% Nemarioc-AL phytonematicides (nemarioc-group) or 0, 2 g Nemafric-BG and 3% Nemafric-BL phytonematicides (nemafric-group), arranged in RCBD, each experiment with 8 replications. At 56 days after initiation of treatments, eggs in roots, J2 in roots and Pf exhibited negative quadratic relations under both greenhouse and microplot conditions. Under greenhouse conditions, dry shoot mass (R2 = 0.81), dry root mass (R2 = 0.87) and leaf number (R2 = 0.91) over Nemarioc-AL phytonematicide exhibited positive quadratic relations. In contrast, dry shoot mass (R2 = 0.78), dry root mass (R2 = 0.93) and leaf number (R2 = 0.70) over Nemafric-BL phytonematicide exhibited positive quadratic relations. Under microplot conditions, dry shoot mass (R2 = 0.95) and gall rating (R2 = 0.96) over Nemarioc-AL phytonematicide, exhibited positive quadratic relations. Dry shoot mass (R2 = 0.84) and gall rating (R2 = 0.97) versus Nemafric-BL phytonematicide exhibited positive quadratic relations. Selected nutrient elements under greenhouse conditions K (R2 = 0.96), Ca (R2 = 0.79), Mg (R2 = 0.64), Fe (R2 = 0.78) and Zn (R2 = 0.77) over Nemarioc-AL phytonematicide exhibited positive quadratic relations. In contrast, only Ca (R2 = 0.90), Mg (R2 = 0.68) and Zn (R2 = 0.84) over Nemafric-BL phytonematicide exhibited positive quadratic relations, whereas K (R2 = 0.72) and Fe (R2 = 0.63) over the product exhibited negative quadratic relations. Under microplot conditions, K (R2 = 0.82), Ca (R2 = 0.90) and Mg (R2 = 0.98) over Nemarioc-AL phytonematicide exhibited positive quadratic relations, whereas Fe (R2 = 0.91) and Zn (R2 = 0.79) over the product exhibited negative quadratic relations. In contrast, K (R2 = 0.60), Ca (R2 = 0.68) and Zn (R2 = 0.95) over Nemafric-BL phytonematicide exhibited positive quadratic relation, whereas Mg and Fe over the product did not have significant relationships. Under greenhouse conditions, MCSP values for Nemarioc-AL and Nemafric-BL phytonematicides on Swiss chard were 3.03 and 2.36%, whereas overall sensitivity (∑k) values of the crop to the product were 3 and 0 units, respectively. In contrast, MCSP values of Nemarioc-AL and Nemafric-BL phytonematicides on Swiss chard under microplot conditions was successfully established at 3.71 and 3.33%, whereas the ∑k values were 2 and 1 units, respectively. Under field conditions, at 64 days after initiating the treatments, the nemarioc-group phytonematicides had highly significant effects on eggs in roots and reproductive potential (RP), contributing 79 and 77% in total treatment variation (TTV) of the respective variables. In contrast, the nemafric-group phytonematicides had highly significant effects on eggs in roots and RP, contributing 67 and 76% in TTV of the respective variables. Under field conditions, all plant growth variables were not significantly affected by the treatments. The nemarioc-group phytonematicides had significant effects on K and Mg in leaf tissues of Swiss chard, contributing nemafric-group phytonematicides had significant effects on Mg, contributing 62% in TTV of the variable. In conclusion, the products could be used on Swiss chard for managing population densities of Meloidogyne species. However, due to the sensitivity of Swiss chard to the products, it would be necessary to use the derived MCSP values to determine the application intervals of the products on the test cultigen / National Research Foundation (NRF) Agricultural Research Council (ARC)

No description available.

Thesis (M.Sc. Agriculture (Plant Protection)) -- University of Limpopo, 2021 / In use of phytonematicides as an alternative to synthetic chemical nematicides, the major challenge had been the development of appropriate application technologies, which are currently limited to the ground leaching technology (GLT) and botinemagation (BNT) systems. The former is labour-intensive, whereas the latter requires infrastructure that could be costly for smallholder farmers. The priming of seeds with hypogenous germination properties in phytonematicide solutions could serve as an alternative method of the application of phytonematicides, where the cotyledons would serve as carriers of the active ingredients that are leached into the rhizosphere for suppression of nematode numbers. However, since germination is a chemical process, it is not known whether the active ingredients in cucurbitacin containing phytonematicides would interfere with germination and the subsequent emergence of the seedlings through the incidence of phytotoxicity as observed in the use of the products in crop production. The objectives of the study, therefore, were (1) to investigate the sensitivity and overall sensitivity of pea (Pisum sativum L.) plants to Nemarioc-AL and Nemafric-BL phytonematicides, and (2) to determine the mean concentration point (MCSP) for pea-inoculated with Meloidogyne incognita under greenhouse and microplot conditions, where seeds were previously primed in phytonematicide solutions. Two separate trials were conducted with seven treatments, namely, 0, 2, 4, 8, 16, 32 and 64% Nemarioc-AL or Nemafric-BL phytonematicide, arranged in completely randomised design (CRD), with 8 replications each. Pea seeds were primed in Nemarioc-AL and Nemafric-BL phytonematicide solutions for two hours and shade dried prior to sowing. In vitro trial, 10 seeds were spread uniformly on a moistened filter paper in sterilised petri-dishes with lids and placed in an incubator at 25oC. In vivo trials were under greenhouse and micro-plot conditions, pea seeds were sown in 25-cm and 30-cm diameter plastic pots, respectively. Pots were filled with pasteurised loam soil. Seedlings were inoculated with 5 000 eggs + second-stage juveniles (J2) of M. incognita. Treatments in each case included priming seeds as explained earlier, arranged in a randomised complete block design (RCBD), with 6 replications under greenhouse conditions and 8 replications under micro-plot conditions. In all cases, plant growth variables were assessed using the Curve-fitting Allelochemical Response Dose (CARD) model to generate biological indices which were used to calculate MCSP and the overall sensitivity (Σk). Nematode variables in inoculated trials were assessed using the regression model. In vitro trials, germination variables had positive quadratic relation versus Nemafric-BL phytonematicide, with MCSP= 0.62 % and ∑k = 34 units. In contrast, tested germination variables exhibited negative quadratic relations versus Nemarioc-AL phytonematicide. In greenhouse trials, MCSP values for Nemarioc-AL and Nemafric-BL phytonematicides were 0.62 and 2.18 %, respectively, with ∑k = 0. Plant height (R2 = 0.86), stem diameter (R2 = 0.93) and chlorophyll content (R2 = 0.85), exhibited positive quadratic relationship against Nemarioc-AL phytonematicide, whereas, plant height (R2 = 0.95), stem diameter (R2 = 0.92), chlorophyll content (R2 = 0.89), number of flowers (R2 = 0.93) and dry shoot mass (R2 = 0.94), exhibited positive quadratic relationship against Nemafric-BL phytonematicide. In micro-plot trials, MCSP values for Nemarioc-AL and Nemafric-BL phytonematicides were 0.71 and 2.45 %, respectively, with ∑k = 0. Plant height (R2 = 0.95), stem diameter (R2 = 0.98), chlorophyll content (R2 = 0.98), and gall ratings (R2 = 0.98), exhibited positive quadratic relationships against Nemarioc-AL phytonematicide, while chlorophyll content (R2 = 0.97) and gall ratings (R2 = 0.96) exhibited positive quadratic relationships against Nemafric-BL phytonematicide. All degrees of Nemarioc-AL and Nemafric-BL phytonematicides profoundly reduced nematode numbers under greenhouse and micro-plot trials. In conclusion, both Nemarioc-AL and Nemafric-BL phytonematicides could be applied through the priming technology on pea seeds which have hypogenous germination properties in suppression of nematode population densities. / National Research Foundation (NRF)

Thesis (Ph. D. Agriculture (Plant Protection)) -- University of Limpopo, 2013 / Global withdrawal of synthetic fumigant and non-fumigant nematicides due to their ecounfriendly impacts and high toxicity to non-target organisms, respectively, increased the research and development of alternatives for managing population densities of plantparasitic nematodes, particularly the root-knot (Meloidogyne species) nematodes. Although Meloidogyne species had been managed using genotypes that are resistant to plant-parasitic nematodes in various crops, various challenges negate the available or introgressed nematode resistance. In tomato (Solanum lycopersicum) production, nematode races and instability of nematode resistant genotypes under certain conditions necessitated the continued research and development of alternatives since most of the existing commercial tomato cultivars are highly susceptible to various biological races of Meloidogyne species. The aim of the study was to research and develop appropriate dosages of two phyto- nematicides which could be applied through drip irrigation system in open field tomato production systems, while the specific objectives were to: (1) determine whether a computer-based model could provide nonphytotoxic concentrations to tomato plants using fresh fruits of wild watermelon (Cucumis africanus) and wild cucumber (C. myriocarpus) under greenhouse conditions, (2) determine whether computer-based concentrations from the two plant species when using dried fruits would be less phytotoxic and more suppressive to nematodes, (3) investigate application time intervals for the two products, (4) determine responses of plant growth in tomato and nematode suppression in respect to the derived dosages, and and (5) validate dosages of fermented crude extracts from the two plant species with respect to plant growth of tomato and suppression of nematode numbers. xxxiii Greenhouse, microplot and field studies were set to test the hypotheses intended to achieve the stated objectives, with reliability of measured variables being ensured by using statistical levels of significance (P ≤ 0.05) and coefficients of determination (R2), while validity was ensured by conducting experiments at the same location over two seasons and/or by setting up factorial treatments. Firstly, fermented plant extracts of fresh fruits from C. africanus and C. myriocarpus consistently reduced population densities of Meloidogyne species by 80-92% and 50-90%, respectively. Tomato plants were highly sensitive to the two products as shown by the total degree of sensitivities (Σk) and biological index of 0 and 3, respectively. Also, the mean concentration stimulation range (MCSR) of 11% and 7% concentrations, respectively, attested to this phytotoxicity. Secondly, fermented crude extracts of dried fruits from C. africanus and C. myriocarpus also reduced population densities of Meloidogyne species by 78-97% and 87-97%, respectively. Tomato plants were highly tolerant to the two products in dried form as shown by the total degree of sensitivities (Σk) and biological index of 4 and 3, respectively. The MCSR values for C. africanus and C. myriocarpus dried fruits on tomato were 2.64% and 2.99%, respectively, which for the purpose of this study were individually adjusted to 3%, which translated to 36 L undiluted material/ha of 4 000 tomato plants. In subsequent studies, 3% concentration was used as the standard, along with double strength concentration, namely, 6% concentration. Thirdly, the MCSR values derived in Objective 4, namely 3% and 6% concentration for both Cucumis species using the CARD model were used in the optimisation of application time interval using the innovative concept of weeks (0, 1, 2, 3 and 4) in a 30-day month period. Application time interval for 3% and 6% concentrations of C. africanus fruits was xxxiv optimised at 2.40 and 2.61 weeks in a 30-day month period, respectively, which translated to 18 days [(2.4 weeks/4 weeks) × 30 days] and 20 days [(2.6 weeks/4 weeks) × 30 days], respectively. In contrast, for both concentrations from fermented crude extracts of C. myriocarpus fruits, application time interval was optimised at 16 days for 2.2 and 2.1 weeks, respectively. During optimisation of application frequencies, fermented crude extracts from C. africanus and C. myriocarpus reduced final population densities of M. incognita race 2 by 70-97% and 76-96%, respectively. Fourthly, optimum application intervals (time), allowed computation of dosage, which is a product of concentration and application frequency (dosage = concentration × application frequency). Fifthly, validation of the dosages under open field conditions suggested that 6% × 16-day dosage under crude extracts from C. myriocarpus fruit significantly (P ≤ 0.05) improved growth of tomato plants when compared with those of either 0% (untreated control) or 3% at 16 days. In contrast, dosages of C. africanus fruit at two application frequency had no effect on growth of tomato plants – suggesting that either of the dosages was suitable for use in tomato production since both reduced nematode numbers. During validation, the materials reduced nematode numbers by margins similar to those observed previously under other environments. In conclusion, crude extracts of the two Cucumis species have stimulatory concentrations which have potential similar reductive effects on population densities of Meloidogyne species and could serve as botanical nematicides. However, since plant responses to the two products differed in terms of their respective dosages and active ingredients, it implied that for further improvement of the two, the overriding focus should be on their interaction with the protected plants and nematode numbers. Ideally, future research xxxv should include environmental impact studies, especially on the influence of the products fruit quality of tomato, earthworms, fish and bees.

Thesis (Ph. D. Agriculture (Plant Production)) -- University of Limpopo, 2016. / Nemarioc-AL and Nemafric-BL phytonematicides, had been researched and developed from indigenous plants at the University of Limpopo, Green Technologies Research Centre, under the auspices of the Indigenous Cucurbitaceae Technologies (ICT) Research Programme. After the international 2005 cut-off withdrawal date of the highly effective methyl bromide nematicide from the agrochemical markets, management options on nematode population densities shifted to more environment-friendly alternatives. Nemarioc-AL and Nemafric-BL phytonematicides as environment-friendly alternatives to synthetic chemical nematicides had been consistent in nematode suppression under diverse conditions. In order to avoid challenges similar to those experienced with the use of synthetic chemical nematicides, the South African Fertiliser, Farm Feeds, Agricultural Remedies and Stock Remedies Act No. 36 of 1947 (amended) require that the product to be used in agriculture must first be registered with the National Department of Agriculture, Forestry and Fisheries, after extensive efficacy and bioactivity tests. The information on bioactivity of the phytonematicides is also critical in the effective application of the product for efficient management of nematodes. Information on bioactivities of Nemarioc-AL and Nemafric-BL phytonematicides on nematodes, plant and soil was not available. This study comprised eight objectives: (1) to examine whether (i) increasing concentration of cucurbitacin A and B would have impact on second-stage juvenile (J2) hatch of M. incognita, (ii) the Curve-fitting Allelochemical Response Dosage (CARD) model would quantify the three phases of density-dependent growth (DDG) patterns on J2 hatch when exposed to increasing cucurbitacin concentrations, (iii) computed J2 hatch inhibition concentration (EHIC) and xli CARD-generated D-values would be statistically similar, (iv) the CARD model would provide information on minimum inhibition concentration (MIC) and (v) J2 hatch inhibition would be reversible when cucurbitacins were diluted, (2) to determine whether (i) increasing concentration of Nemarioc-AL and Nemafric-BL phytonematicides would have impact on J2 hatch of M. incognita, (ii) the CARD model would quantify the three phases of DDG pattern on J2 hatch when compared to increasing phytonematicide concentrations, (iii) comparison of computed EHIC and CARD-generated D-values would be statistically comparable in magnitudes, (iv) the CARD model would provide information on MIC and (v) J2 hatch inhibition would be reversible when phytonematicides were diluted, (3) to establish whether (i) increasing concentration of cucurbitacin A and B would have impact on M. incognita J2 immobility, (ii) the CARD model would quantify the three phases of DDG pattern on J2 immobility when compared to increasing cucurbitacin concentration, (iii) comparison of computed J2 immobility concentration and CARD-generated D-values would be statistically comparable in magnitudes, (iv) the CARD model would provide information on MIC and (v) juvenile immobility would be reversible when cucurbitacins were diluted, (4) to test whether (i) increasing concentration of Nemarioc-AL and Nemafric-BL phytonematicides would have impact on M. incognita J2 immobility, (ii) the CARD model would quantify the three phases of DDG pattern on J2 immobility when compared to increasing phytonematicide concentrations, (iii) comparison of computed J2 immobility concentration and CARD generated D-values would be statistically comparable in magnitudes, (iv) the CARD model would provide information on MIC and (v) juvenile immobility would be reversible when phytonematicides were diluted, (5) to determine whether (i) increasing xlii concentration of cucurbitacin A and B would have impact on M. incognita J2 mortality, (ii) the CARD model would quantify the three phases of DDG patterns on J2 mortality when compared to increasing cucurbitacin concentration, (iii) comparison of computed lethal concentration (LC) and CARD-generated D-values would be statistically comparable in magnitudes and (iv) the CARD model would provide information on minimum lethal concentration (MLC), (6) to investigate whether (i) increasing concentration of Nemarioc-AL and Nemafric-BL phytonematicides would have impact on M. incognita J2 mortality, (ii) the CARD model would quantify the three phases of DDG pattern on J2 mortality when compared to increasing phytonematicide concentrations, (iii) comparison of computed LC and CARD-generated D-values would be statistically comparable in magnitudes and (iv) the CARD model would provide information on MLC, (7) to test whether (i) increasing concentrations of Nemarioc-AL and Nemafric-BL phytonematicides would impact on M. incognita J2 infectivity of susceptible tomato plant, (ii) the CARD model would quantify the three phases of DDG pattern (iii) generated inhibition concentration (IC) and CARD-generated D-values would be statistically comparable in magnitudes and (iv) the CARD model would provide information on MIC and (8) to determine whether nematodes can serve as bioindicators of Nemarioc-AL and Nemafric-BL phytonematicides in tomato plant roots/fruits, soil types and organic matter at different depths. To achieve these objectives, reliability of measured variables was ensured by using statistical levels of significance (P ≤ 0.05) and coefficient of determination (R2), with validity ensured by conducting three independent experiments over time. In Objective 1, pure cucurbitacin A and B concentration effects on J2 hatch were significant, with both exhibiting DDG patterns. xliii The DDG patterns demonstrated that J2 hatch was inhibited at low pure cucurbitacin concentrations and slightly stimulated at higher cucurbitacin concentrations. At 24-, 48- and 72-h exposure periods, cucurbitacin A reduced J2 hatch by 40‒67, 34‒66 and 34‒45%, respectively, whereas cucurbitacin B reduced J2 hatch by 12‒57, 3‒36 and 9‒54%, respectively. CARD model quantified the concentration ranges of the two pure cucurbitacins associated with the phases of DDG patterns. The J2 hatch was highly sensitive to cucurbitacin B and highly tolerant to cucurbitacin A, as shown by sensitivities values of 0‒2 and 5‒20 units, respectively. The CARD-generated MIC values for cucurbitacin A and B were 1.75‒2.88 and 1.31‒1.88 µg.mL-1, respectively. The conventionally generated J2 hatch inhibition concentrations were higher than CARD-generated D-values at all exposure periods for both pure cucurbitacins. The J2 hatch inhibition effect was not reversible for both pure cucurbitacins. In Objective 2, Nemarioc-AL and Nemafric-BL phytonematicide concentration effects on J2 hatch were highly significant (P ≤ 0.01), with both exhibiting DDG patterns. The DDG patterns demonstrated that J2 hatch inhibition increased with increase in phytonematicide concentrations. Relative to water control, Nemarioc-AL phytonematicide significantly reduced J2 hatch at 48-, 72-h and 7-d by 22‒92, 3‒79 and 1‒42%, respectively, whereas Nemafric-BL phytonematicide reduced it by 41‒93, 1‒80 and 12‒84%, respectively. The J2 hatch inhibition was highly sensitive to Nemarioc-AL and Nemafric BL phytonematicides, with sensitivity of 0‒1 and 0‒4 units, respectively. The conventionally generated J2 hatch inhibition concentrations at 50 and 100% were higher than CARD-generated D-values for both phytonematicides. The J2 hatch inhibition effect was not reversible for both phytonematicides. In Objective 3, pure cucurbitacin A xliv and B concentration effects on J2 immobility were significant, with both exhibiting DDG patterns. The J2 immobility over increasing concentrations of pure cucurbitacins had DDG patterns which were similar for conventional method and those from CARD model. The DDG patterns were characterised by stimulation of J2 immobility at low concentrations, followed by saturation at higher concentrations. The CARD model could not generate the D-values for comparison with JMC-values, but generated MIC-values for cucurbitacin A and B which were 0.5‒0.6 and 0.5‒0.7 µg.mL-1, respectively. The J2 immobility was moderately sensitive to both cucurbitacins with sensitivity of 4 units and the inhibition effect of the two pure cucubitacins was not reversible. In Objective 4, Nemarioc-AL and Nemafric-BL phytonematicide concentration effects on J2 immobility were highly significant (P ≤ 0.01), with both phytonematicides exhibiting DDG patterns. The DDG pattern had stimulation, saturation and inhibition effects for Nemarioc-AL phytonematicide, whereas for Nemafric-BL phytonematicide they had stimulation and saturation effects on J2 immobility as concentrations increased. The MIC-values for Nemarioc-AL and Nemafric-BL phytonematicides were 3.6‒115.2 and 0.1‒6.5%, respectively. The CARD generated D-values were comparable with computed JMC values for Nemafric-BL phytonematicide unlike for Nemarioc-AL phytonematicide. The J2 immobility was highly sensitive to the two phytonematicides with sensitivity values of 0‒4 and 0‒2 units, respectively. The effects on J2 immobility of the two phytonematicides were not reversible. In Objective 5, pure cucurbitacin A and B concentration effects on J2 mortality were highly significant (P ≤ 0.01), with both cucurbitacins exhibiting DDG patterns. The DDG pattern had stimulation, saturation and slight inhibition effects for both cucurbitacin A and B as concentrations increased. The xlv MIC-values for cucurbitacin A and B were 0.63 and 0.61 µg.mL-1, respectively. The CARD-generated D-values were higher than the computed LC-values for both cucurbitacin A and B, with J2 mortality being highly sensitive to cucurbitacin A and B, with sensitivity of 4 units for both cucurbitacins. In Objective 6, Nemarioc-AL and Nemafric-BL phytonematicide effects on J2 mortality were highly significant (P ≤ 0.01), with both phytonematicides exhibiting DDG patterns. The DDG pattern had stimulation effect at low phytonematicide concentrations and saturation effects at higher concentrations for both relative impact and CARD-generated graphs of J2 exposed to both phytonematicides. The MIC-values for Nemarioc-AL and Nemafric-BL phytonematicides were 1.12 and 0.67%, respectively. The CARD-generated D-values were higher than the computed LC-values for both phytonematicides and J2 mortalities were highly sensitive to Nemarioc-AL and Nemafric-BL phytonematicides with sensitivity value of 2 and 1 units, respectively. In Objective 7, Nemarioc-AL and Nemafric-BL phytonematicide concentrations had a highly significant effect on infectivity of M. incognita post-exposure on susceptible tomato seedlings. The relationship between infectivity and increasing concentrations of the two phytonematicides exhibited DDG patterns. The DDG patterns were characterised by stimulation effect at low Nemarioc AL phytonematicide concentrations and saturation effects at higher phytonematicide concentrations, whereas for Nemafric-BL phytonematicide slight inhibition, saturation and stimulation effects were observed. The CARD-generated inhibition concentrations for Nemarioc-AL phytonematicide were comparable with computed inhibition concentrations, whereas for Nemafric-BL phytonematicides, the values were not comparable. The MIC-values for Nemarioc-AL and Nemafric-BL phytonematicides were xlvi 0.2 and 0.7%, respectively and J2 infectivity were highly sensitive to the two phytonematicides, with sensitivity value of 2 and 0 units, respectively. In Objective 8, M. incognita was an excellent bioindicator in response to the application of two phytonematicides. The two phytonematicides significantly affected distribution of population densities of M. incognita across the tested soil types, with Nemafric-BL phytonematicide reducing population densities of M. incognita relative to Nemarioc-AL phytonematicide. The active ingredient of Nemafric-BL phytonematicide, cucurbitacin B tended to remain in the top layers of soil, where more roots accumulated, thereby reducing a relatively higher population densities of M. incognita than did active ingredient of Nemarioc-AL phytonematicide, cucurbitacin A which moved with water beyond the effective root zone. Soil type alone and phytonematicide alone had no effect on nematode numbers, whereas the interaction of soil type, phytonematicides and depth, the nematode population densities were inversely proportional to soil depth. The interaction of clay with any of the two phytonematicides, reduced M. incognita population densities compared to sand and loam interactions. More than 62% tomato root systems occurred in the top 0–25 cm depth. The interactions between organic matter levels, phytonematicides and depth had no effect on the population densities of M. incognita. The two phytonematicides were able to reduce nematode population densities throughout the soil column in all four soil types and organic matter levels. Cucurbitacin residues were not detected in all tomato fruit samples. In conclusion, Nemarioc-AL and Nemafric-BL phytonematicides have bioactivities on J2 hatch, J2 immobility, J2 mortality and J2 infectivity. The CARD model quantified the three phases of DDG patterns for most of the variables. Even though CARD-generated inhibition xlvii concentrations at 50 and 100% were not comparable with computed values for pure cucurbitacins they were for most phytonematicide variables, the model was able to generate excellent MIC-values for all variables. The inhibition effects of the two phytonematicides were irreversible. The major findings of this study were that the two phytonematicides exhibited DDG patterns for all variables tested and that the CARD model could be adopted for the in vitro evaluation of phytonematicides. Meloidogyne incognita was an excellent bioindicator on movement of two phytonematicides across soil types and organic matter levels at different depths. Nemarioc-AL and Nemafric-BL phytonematicides did not leave any cucurbitacin residues in tomato fruit. The information on bioactivities of the two phytonematicides generated in this study provides a much needed data for the registration of the products as required by the law. Proposed future research area includes, microscopy study of molecular effects of the phytonematicides on nematodes post-exposure. / National Research Foundation (NRF), Flemish Interuniversity Council (VLIR) and Land Bank Chair-University of Limpopo.