Glyphosat ist eines der weltweit am häufigsten eingesetzten Herbizide. Sein Einsatz wird u.a. auf Grund einer möglichen karzinogenen Wirkung und eines möglichen negativen Einflusses auf die Biodiversität kritisch diskutiert. Um Aussagen über die Verbreitung von Glyphosat in der Umwelt treffen zu können, werden verlässliche Nachweissysteme benötigt. Das Ziel der vorliegenden Arbeit bestand darin, ein einfaches optisches System zum schnellen Nachweis von Glyphosat in wässrigen Proben, basierend auf einer Hydrophobin-funktionalisierten Oberfläche, die das Glyphosat Zielprotein präsentiert, zu entwickeln. Hierfür wurden verschiedene Fusionsproteine aus dem Glyphosat Zielprotein, der 5-Enolpyruvylshikimat-3-phosphatsynthase (EPSPS, hier aus dem Bakterium Escherichia coli (EcEPSPS)) und dem zur Selbstassemblierung an hydrophilen/hydrophoben Grenzflächen befähigten Hydrophobin Ccg2 aus Neurospora crassa erzeugt, welche für die Oberflächenfunktionalisierung eingesetzt wurden. Die Expression und Reinigung der Fusionsproteine und von Ccg2 in E. coli verlief erfolgreich. Nach initialen Kontaktwinkelmessungen zur Untersuchung der Funktionalität des Hydrophobins und Enzymaktivitätsmessungen für die Fusionsproteine, konnte deren Aktivität auch nach der Reinigung nachgewiesen werden. Dabei erwies sich das Fusionsprotein Ccg2_GS_EcEPSPS, aufgrund einer hohen enzymatischen Aktivität nach Immobilisierung, als am besten geeignet. Es wurden verschiedene Belegverhältnisse zwischen Hydrophobin und Fusionsprotein untersucht, um etwaige sterische Behinderungen zu minimieren. Hierbei erwies sich ein Belegverhältnis von 1 µM Ccg2_GS_EcEPSPS und 5 µM Ccg2 für die künftigen Messungen als gut geeignet. Auf Basis der so funktionalisierten Oberfläche wurden zwei Verfahren zum optischen Nachweis von Glyphosat entwickelt. Eines der Verfahren, der Malachitgrün-Assay, weist die enzymatische Aktivität der EPSPS auf der Oberfläche nach, genauer das entstehende anorganische Phosphat (Pi). Durch Glyphosathemmung entsteht weniger Pi, dies kann mittels Malachitgrün-Assay nachgewiesen werden. Unter Laborbedingungen konnte ein Detektionslimit von 50 nM erreicht werden. Des Weiteren zeigte der Assay keine nennenswerte Querempfindlichkeit und erwies sich damit als sehr spezifisch. Zusätzlich wurde der Einfluss unterschiedlicher Temperaturen und pH-Werte untersucht. Es zeigte sich, dass Schwankungen dieser Parameter den Assay beeinflussen. Auch ein Einfluss der Ionenstärke konnte festgestellt werden. Deshalb sind entsprechende Kontrollen unerlässlich. Der Einfluss nicht-reaktionsbedingten Phosphates konnte durch Vorinkubation der Oberfläche mit der Glyphosat-haltigen Analyselösung mit anschließender Entfernung der Selbigen und Durchführung des Malachitgrün-Assays minimiert werden. Das zweite Verfahren, der Hydrogelsonden (HGS)-Assay, weist direkt die Interaktion von Glyphosat und der immobilisierten EcEPSPS nach. Hierfür wurden verformbare, Glyphosat-dekorierte HGS aus Polyethylenglykol benötigt. Bei Abwesenheit von freiem Glyphosat liegen die Bindestellen der immobilisierten EPSPS frei vor, sodass sie für die Bindung des immobilisierten Glyphosats an den HGS zur Verfügung stehen. Zwischen den HGS und der Oberfläche entsteht auf diese Weise eine große Kontaktfläche, welche mittels Reflektionsinterferenzkontrastmikroksopie messbar ist. Freies Glyphosat in der Analyselösung reduziert die verfügbaren Bindestellen an der Oberfläche. Dies resultiert in einer kleineren Kontaktfläche. Auf diese Weise kann durch Ermittlung der Größe der Kontaktfläche zwischen HGS und funktionalisierter Oberfläche und der daraus berechneten Adhäsionsenergie, auf das Vorhandensein von Glyphosat in der Analyselösung geschlossen werden. Im Rahmen dieser Arbeit konnte nach Optimierung der Oberflächenbeschichtung, ein positiver Machbarkeitsbeweis für dieses Verfahren erbracht werden.

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Une étude ayant pour objectif d'évaluer différentes séquences d'herbicides en condition de doses réduites, a été mise en place à la station agronomique de l'Université Laval pendant trois ans. Huit séquences culturales de maïs (Zea mays) et de soya (Glycine max) tolérant au glyphosate et au glufosinate ont été testées avec quatre doses d'herbicides. Les résultats montrent que l'emploi du glufosinate trois ou deux années de suite en remplacement d'une utilisation continue du glyphosate paraît difficilement envisageable. Des problèmes de répression et des baisses de rendement sont visibles en présence d'une flore mixte (présence de graminées annuelles en particulier). L'utilisation de doses réduites de glufosinate n'est pas conseillée pour les mêmes raisons. Concernant le glyphosate, une réduction d'un quart de la dose homologuée semble réalisable du point de vue des rendements des cultures, mais présente toutefois un risque d'augmentation de la banque de graines au bout de deux années d'utilisation répétée.

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Zoysiagrass (Zoysia spp.) is utilized as a warm-season turfgrass because of its density, visual quality, stress tolerance, and reduced input requirements. Turf managers often exploit winter dormancy in warm-season turfgrass to apply nonselective herbicides such as glyphosate and glufosinate to control winter annual weeds. Although this weed control strategy is common in bermudagrass (Cynodon spp.), it has been less adopted in zoysiagrass due to unexplainable turf injury. Many university extension publications recommend against applying nonselective herbicides to dormant zoysiagrass despite promotional language found in a few peer-reviewed publications and product labels. Previous researchers have used vague terminology such as "applied to dormant zoysiagrass" or "applied prior to zoysiagrass green-up" to describe herbicide application timings. These ambiguous terms have led to confusion since zoysiagrass typically has subcanopy green leaves and stems throughout the winter dormancy period. No research has sought to explain why some turfgrass managers are observing zoysiagrass injury when the literature only offers evidence that these herbicides do not injure dormant zoysiagrass. We sought to explore various herbicides, prevailing temperatures surrounding application, heat unit based application timings, and spray penetration into zoysiagrass canopies as possible contributors to zoysiagrass injury. The results indicated that a wide range of herbicides may be safely used in dormant zoysiagrass. However, as zoysiagrass begins to produce more green leaves, herbicides such as metsulfuron, glyphosate, glufosinate, flumioxazin, and diquat become too injurious. Glufosinate was consistently more injurious regardless of application timing than glyphosate and other herbicides. When temperatures were 10 °C for 7 d following treatment, a delayed effect of glyphosate and glufosinate effect on digitally-assessed green cover loss was noted on zoysiagrass sprigs. In subsequent studies on turf plugs, a 14-d incubation period at 10 °C reduced glyphosate but not glufosinate effects on turf green color reduction. Glyphosate applied at 125, and 200 GDD5C can safely be applied to zoysiagrass while glufosinate applied at the same timings caused inconsistent and often unacceptable zoysiagrass injury in field studies conducted at Blacksburg, VA, Starkville, MS, and Virginia Beach, VA. Zoysiagrass green leaf density was described as a function of accumulated heat units consistently across years and locations but variably by turf mowing height. Turf normalized difference vegetative index was primarily governed by green turf cover but reduced by herbicide treatments, especially when applied at greater than 200 GDD5C. Substantial spray deposition occurred to subcanopy tissue regardless of nozzle type, pressure and height above the zoysiagrass canopy based on spectrophotometric assessment of a colorant admixture. However, increasing nozzle height above the turf canopy and avoiding air induction type nozzles significantly reduced the percentage of green tissue exposed at lower canopy levels. Absorption of radio-labeled glyphosate and glufosinate was up to four times greater when exposed to zoysiagrass stems compared to leaves. Glyphosate translocated more than glufosinate and both herbicides moved more readily from stem to leaf than from leaf to stem / Doctor of Philosophy / Zoysiagrass (Zoysia spp.) is utilized as a warm-season turfgrass because of its density, visual quality, stress tolerance, and reduced input requirements. Being that zoysiagrass is a warm-season turfgrass, it enters a dormancy period during the winter months. During this period, zoysiagrasses' active growth is halted, and leaves lose their green color and turn a golden-brown color. The winter dormancy period presents turfgrass managers with a unique opportunity to apply nonselective herbicides such as glyphosate and glufosinate to control a broad spectrum of winter annual weeds. Although this weed control strategy is common in bermudagrass (Cynodon spp.), it has been less adopted in zoysiagrass due to turfgrass managers observing unexplainable turfgrass injury. Many university extension publications recommend against applying nonselective herbicides to dormant zoysiagrass despite language found in peer-reviewed publications and product labels suggesting they could be safely applied. Previous researchers have used vague terminology such as "applied to dormant zoysiagrass" or "applied prior to zoysiagrass green-up" to describe herbicide application timings. These terms have led to confusion about when to make these applications since zoysiagrass typically has subcanopy green leaves and stems throughout the winter dormancy period. No research has sought to explain why some turfgrass managers observe zoysiagrass injury when the literature only offers evidence that these herbicides do not injure dormant zoysiagrass. Research projects were designed to explore various herbicides, temperatures surrounding herbicide applications, application timings, and spray penetration into zoysiagrass canopies as possible contributors to zoysiagrass injury. The results indicated that a wide range of herbicides may be safely used in dormant and semidormant zoysiagrass. However, as zoysiagrass begins to produce more green leaves and stems, herbicides such as metsulfuron, glyphosate, glufosinate, flumioxazin, and diquat become too injurious and should be avoided. Across multiple research studies, glufosinate was consistently more injurious regardless of application timing than glyphosate and other herbicides. When temperatures were 10 °C for 7-d following treatment, it delayed zoysaigrass response to glyphosate and glufosinate. In a subsequent study, when temperatures were at 10 °C for a 14-d period, glyphosate and the nontreated reached 50% green cover at the same time, which suggests cold temperatures could mitigate glyphosate injury on zoysiagrass over a 14-d period. The 10 ° temperature only delayed glufosinate injury on zoysiagrass, and no safening was observed. The results also indicated that as temperatures increased, glyphosate and glufosinate rate in which injury was observed increased on the zoysiagrass. Glyphosate applied at 125, and 200 GDD5C can safely be applied to zoysiagrass while glufosinate applied at the same timings caused inconsistent and often unacceptable zoysiagrass injury in field studies conducted at Blacksburg, VA, Starkville, MS, and Virginia Beach, VA. Zoysiagrass injury increased when glyphosate and glufosinate were applied later into the spring when more green leaves were present regardless of location. Accumulated heat units and zoysiagrass green leaf density were closely related, indicating that accumulated heat units could be a useful tool for turfgrass managers to track zoysiagrass spring green-up. Substantial spray deposition was found on subcanopy zoysiagrass leaves and stems regardless of nozzle type, pressure, and height above the zoysiagrass canopy based on recovered colorant at the upper, middle and lower levels of the zoysiagrass canopy. However, avoiding air induction-type nozzles and raising spray height may slightly decrease penetration of spray droplets into a zoysiagrass subcanopy, but a large percentage of droplets still reached the middle and lower canopy layers in this research. Absorption of radio-labeled glyphosate and glufosinate was up to four times greater when applied directly to zoysiagrass stolen compared to leaves. Glyphosate translocated more than glufosinate, and both herbicides moved more readily from stem to leaf than from leaf to stem. These data suggest limiting the number of green zoysiagrass leaves at application would be an effective method to avoid injury zoysiagrass when applying nonselective herbicides

Soil is an important ecosystem that supports a wide variety of organisms such as bacteria, fungi, arthropods and nematodes. This sensitive ecosystem may be influenced by various factors, including agricultural management practices. With the introduction of genetically modified (GM) glyphosate-tolerant (RoundUp ® Ready: RR) crops, herbicides such as glyphosate have been increasingly used. However, little is known about the effect of glyphosate on the biological communities in these herbicide-sprayed soils. With the intimate proximity that microorganisms and nematodes have with the roots of plants, these organisms can be used to assess changes that may occur in the soil surrounding roots of RR crops. The aim of this study was to determine microbial community structure and nematode diversity, with emphasis on that of non-parasitic nematodes, in soil samples from conventional soybean (CS) - and RR- soybean fields compared to that in adjacent natural veld (NV) areas. Samples were collected from twenty three sites at six localities that are situated within the soybean-production areas of South Africa. These sites represented fields where RR and CS soybean grew, as well as surrounding NV. All RR fields have been treated with glyphosate for no less than five years. Microbial community structures of the twenty three sites in the RR, CS and NV ecosystems were determined by phospholipid fatty acid (PLFA) analyses. Nematode diversity was determined by extracting the nematodes from soil samples and conducting a faunal analysis. Soil physical and chemical properties were determined by an independent laboratory, Eco-Analytica (North West University, Potchefstroom) according to standard procedures. Results from this study indicated differences in microbial community structure between the various localities. However, there were no significant (p ≤ 0.05) differences in microbial community structures between RR- and CS ecosystems. Soils of both RR- and CS crops were primarily dominated by bacteria. Nematode identification and faunal analysis also indicated no significant (p ≤ 0.05) differences between the different non-parasitic/beneficial nematodes that were present in soils of these two ecosystems during the time of sampling. Non-parasitic nematode communities were primarily dominated by bacterivores. A faunal analysis indicated that most of the sites contained enriched, but unstructured soil food-webs. However, four of the sites showed enriched and structured food webs due to the presence of non-parasitic nematodes with high coloniser-persister (cp) values. Relationships between non-parasitic nematode – and microbial communities showed that there was a positive relationship between nematode functional groups and their corresponding microbial prey. From the results obtained in this study, it can be concluded that the community structures of both non-parasitic nematodes and microorganisms shared similarities. These community structures showed no long-term detrimental effects of glyphosate application in the soils surrounding roots of RR soybean crops. Relationships existed between non-parasitic nematode and microbial communities in the rhizosphere of soybean crops and natural veld. For example, bacterivore nematodes had a strong positive relationship with gram-negative bacteria. Similar but weaker relationships also existed between carnivores, omnivores, plantparasitic nematodes and gram-negative bacteria. A positive relationship also existed between fungivores and fungal fatty acids. This emphasises the value of these organisms as indicators of soil health and also the impact that agricultural practices can have on soils. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

Soil is an important ecosystem that supports a wide variety of organisms such as bacteria, fungi, arthropods and nematodes. This sensitive ecosystem may be influenced by various factors, including agricultural management practices. With the introduction of genetically modified (GM) glyphosate-tolerant (RoundUp ® Ready: RR) crops, herbicides such as glyphosate have been increasingly used. However, little is known about the effect of glyphosate on the biological communities in these herbicide-sprayed soils. With the intimate proximity that microorganisms and nematodes have with the roots of plants, these organisms can be used to assess changes that may occur in the soil surrounding roots of RR crops. The aim of this study was to determine microbial community structure and nematode diversity, with emphasis on that of non-parasitic nematodes, in soil samples from conventional soybean (CS) - and RR- soybean fields compared to that in adjacent natural veld (NV) areas. Samples were collected from twenty three sites at six localities that are situated within the soybean-production areas of South Africa. These sites represented fields where RR and CS soybean grew, as well as surrounding NV. All RR fields have been treated with glyphosate for no less than five years. Microbial community structures of the twenty three sites in the RR, CS and NV ecosystems were determined by phospholipid fatty acid (PLFA) analyses. Nematode diversity was determined by extracting the nematodes from soil samples and conducting a faunal analysis. Soil physical and chemical properties were determined by an independent laboratory, Eco-Analytica (North West University, Potchefstroom) according to standard procedures. Results from this study indicated differences in microbial community structure between the various localities. However, there were no significant (p ≤ 0.05) differences in microbial community structures between RR- and CS ecosystems. Soils of both RR- and CS crops were primarily dominated by bacteria. Nematode identification and faunal analysis also indicated no significant (p ≤ 0.05) differences between the different non-parasitic/beneficial nematodes that were present in soils of these two ecosystems during the time of sampling. Non-parasitic nematode communities were primarily dominated by bacterivores. A faunal analysis indicated that most of the sites contained enriched, but unstructured soil food-webs. However, four of the sites showed enriched and structured food webs due to the presence of non-parasitic nematodes with high coloniser-persister (cp) values. Relationships between non-parasitic nematode – and microbial communities showed that there was a positive relationship between nematode functional groups and their corresponding microbial prey. From the results obtained in this study, it can be concluded that the community structures of both non-parasitic nematodes and microorganisms shared similarities. These community structures showed no long-term detrimental effects of glyphosate application in the soils surrounding roots of RR soybean crops. Relationships existed between non-parasitic nematode and microbial communities in the rhizosphere of soybean crops and natural veld. For example, bacterivore nematodes had a strong positive relationship with gram-negative bacteria. Similar but weaker relationships also existed between carnivores, omnivores, plantparasitic nematodes and gram-negative bacteria. A positive relationship also existed between fungivores and fungal fatty acids. This emphasises the value of these organisms as indicators of soil health and also the impact that agricultural practices can have on soils. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014

Thesis (MScAgric)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Conyza bonariensis (flaxleaf fleabane) was reported for the first time, as a weed in California in 1893-1896. The first report of the occurrence of this weed in South Africa was made in May 1895 in Franschoek, South Africa. Paraquat resistance in C. bonariensis was first reported in the 1970s and early 1980s when resistance was observed from vineyards and citrus plantations in Egypt. More recently a report of herbicide resistance in South Africa was made in January 2003 when resistance occurred in C. bonariensis in the Breede Valley, South Africa. The resistance was to glyphosate, but recently reports of resistance to glyphosate and paraquat were received. C. bonariensis seeds were found to be positively photoblastic and germinated only under unfiltered white light and red light whilst no germination occurred under far-red light and in the dark. The optimum temperature range for C. bonariensis seed was found to be between 15 and 30°C, with no germination occurring at 0-5°C and at 35- 40°C. Optimum germination occurred at the soil surface. No germination occurred at depths of 2 cm and deeper. Although the optimum temperature range was found to be the same for the different biotypes tested. However, germination was highest in the susceptible biotype. Since farmers first reported paraquat and glyphosate resistance in C. bonariensis in the Breede Valley, South Africa, reports of resistance increase every year. Seed collected from populations suspected of being resistant to paraquat and glyphosate were obtained from the Breede Valley and screened for resistance. To determine the easiest, quickest, and most effective method to screen for paraquat and glyphosate resistance, two tests were evaluated viz. the petri dish assay method and the whoIeplant dose-response method. Both screening methods identified paraquat and glyphosate resistant biotypes. The petri dish assay method was found to be a more rapid method of screening for resistance in C. bonariensis. During this study it was found that both paraquat and glyphosate resistance does occur in the Breede Valley. The effect of growth stage on the level of herbicide resistance in C. bonariensis was tested. Herbicides other than paraquat and glyphosate were tested to determine if they could be used to control resistant C. bonariensis seedlings. The alternative herbicides tested included MCPA and Sorgomil Gold 600. The four herbicides were sprayed at different leaf stages. During the study it was found that growth stage does play an important role in the level of herbicide resistance. It was found that the control of different herbicides decreased with an increase in growth stage. The different herbicides showed varying levels of control depending on growth stage and resistant profile. Overall MCPA gave the best control at all leaf stages tested. What is gratifying was the finding that every biotype tested could be controlled by at least one of the treatments applied. This means that the producer using the most appropriate herbicide applied at the optimum application stage will be able to control most if not all the resistant populations of C. bonariensis that occur in the Western Cape. / AFRIKAANSE OPSOMMING: Conyza bonariensis (Kleinskraalhans) is vir die eerste keer as 'n onkruid gerapporteer in Kalifornia in 1893-1896. Die eerste waarneming van hierdie onkruid in Suid-Afrika is gemaak in Mei 189S in Franschoek. Parakwat weerstandbiedendheid in C. bonariensis is die eerste maal in die 1970s en vroeë 1980s waargeneem, toe weerstandbiedendheid opgemerk is in wingerde en sitrus plantasies in Egipte. Meer onlangs is 'n geval van onkruiddoder weerstandbiedendheid in Suid-Afrika aangemeld in Januarie 2003, toe 'n biotipe van C. bonariensis in die Breede Vallei weerstand-biedendheid getoon het teen 'n onkruiddoder. Die weerstand was teen glifosaat, maar onlangse berigte van weerstandbiedendheid teen glifosaat sowel as parakwat is ontvang. Daar is gevind dat die saadjies van C. bonariensis positief fotoblasties is en slegs ontkiem onder ongefiltreerde wit- en rooi lig, terwyl geen ontkieming voorkom onder ver-rooi lig en in die donker nie. Die optimum temperatuurreeks vir C. bonariensis saad is tussen IS en 30°C, met geen ontkieming wat by O-SoCen by 3S-40°C voorkom nie. Optimum ontkieming kom voor op die grondoppervlak. Geen ontkieming kom by dieptes van 2 cm of dieper voor nie. Alhoewel die optimum temperatuurreeks dieselfde is vir die verskillende biotipes wat getoets is, is daar tog 'n verskil in die persentasie ontkieming tussen die biotipes met die beste ontieming by die sensitiewe biotipe. Sedert boere die eerste geval van parakwat en glifosaat weerstandbiedendheid in C. bonariensis in die Breede Vallei, Suid-Afrika gerapporteer het, word meer gevalle van weerstandbiedendheid jaarliks aangemeld. Saad van populasies wat vermoedelik parakwat en glifosaat weerstandbiedend is, is in die Breede Vallei versamel en getoets vir weerstandbiedendheid. Om die maklikste, vinnigste en mees effektiewe metode van weerstandbiedendheidstoetsing te vind, is twee verskillende metodes van toetsing, naamlik die petribakkietoets en die heel plant dosis respons metode gebruik. Beide metodes van toetsing het parakwat en glifosaat weerstandbiedende biotipes geïdentifiseer. Daar is gevind dat die petri bakkie metode 'n vinniger manier van toetsing vir weerstandbiedendheid is. Die studie het ook bewys dat parakwat en glifosaat weerstandbiedendheid wel in die Breede Vallei, Suid-Afrika voorkom. Die effek van groeistadium op die vlak van onkruiddoder weerstandbiedendheid in C. bonariensis is ook tydens die studie getoets. Ander onkruiddoders buiten parakwat en glifosaat is getoets om te bepaal of hulle gebruik kan word vir die effektiewe beheer van weerstandbiedende C. bonariensis saailinge. Die alternatiewe onkruiddoders wat getoets is, was MCPA en Sorgomil Gold 600. Die vier onkruiddoders is gespuit by verskillende blaarstadiums. Gedurende die studie is daar gevind dat groeistadium wel 'n belangrike rol speel in die vlak van onkruiddoder weerstandbiedendheid. Die persentasie beheer van verskillende onkruiddoders neem af met 'n toename in die groeistadium. Die verskillende onkruiddoders se beheer het gewissel afhangend van weerstandbiedendheid en groeistadium. MCPA het die beste beheer by alle blaarstadiums wat getoets is getoon. Daar is ook gevind dat een of die ander van die onkruiddoders wat getoets is, gebruik kan word vir die suksesvolle beheer van onkruiddoder weerstandbiedendheid in elke biotipe wat getoets is. Dit beteken dat 'n produsent wat die korrekte onkruiddoder op die korrekte groeistadium toedien, in staat sal wees om die meeste, indien nie alle weestandbiedende C. bonariensis populasies wat in die Wes Kaap voorkom, te beheer.