Les oomycètes représentent un groupe de microorganismes eucaryotes filamenteux distincts phylogénétiquement des champignons incluant de nombreuses espèces phytopathogènes. CBEL est une glycoprotéine pariétale de Phytophthora parasitica constituée d'une répétition de deux régions séparées par un linker. Chaque région protéique est constituée d'un domaine protéique de liaison à la cellulose (CBM1) et un motif PAN /Apple impliqué dans des interactions protéines-protéines ou protéines-polysaccharides. Cette étude doctorale porte sur la caractérisation de la protéine CBEL et plus particulièrement de ses CBM1s ainsi que sur l'évaluation et optimisation du potentiel de cette protéine à : (i) stimuler les défenses naturelles des plantes (ii) augmenter l'activité de glycosides hydrolases. Dans la première partie de ce travail doctoral différents tests visant à reproduire un traitement éliciteur externe sur plante entière ont pour cela été développés. Ces tests ont permis de mettre en évidence que formulée en présence de surfactants CBEL est capable d'induire diverses réponses de défense chez A. thaliana. Une production en masse de cette protéine a été réalisée dans la levure Pichia pastoris et la bactérie Escherichia coli dans l'optique d'une future application agronomique. Les protéines recombinantes CBELcol et CBELpic produite dans ces différents systèmes d'expression présentent des profils de glycosylation différents de celui de la protéine native CBELnat. Alors que ces protéines semblent se lier de manière identique à la cellulose les différents tests d'élicitation développés au cours de ce travail mettent en évidence des variations dans leur activité élicitrice suggérant que la nature des résidus glucidiques présents sur cette glycoprotéine peut avoir un impact sur sa capacité induire des réponses de défenses en application externe. Lors de la deuxième partie de ce travail de thèse la capacité de CBEL à interagir avec différents substrats cellulosiques a été caractérisée. Les résultats obtenus ont permis de montrer que CBEL se lie avec une haute affinité à la cellulose cristalline avicel et que la présence de CBM1 fonctionnels est nécessaire à cette interaction. De manière intéressante, le CBM1-1 et CBM1-2 ne semblent pas contribuer de manière égale à cette interaction. Par ailleurs la laison de CBEL à la cellulose induit des perturbations structurales sur le substrat et permet d'améliorer l'activité de la xylanase XynB de Talaromyces versatilis sur paille de blé. En outre une xylanase chimère possédant dans sa séquence le CBM1-1 de CBEL possède également une activité augmentée sur paille blé. L'ensemble de ces résultats met en évidence le potentiel de CBEL et de son CBM1-1 pour l'amélioration de l'activité de glycoside hydrolases utilisables par exemple en bioraffinerie. En dernier lieu un travail de caractérisation structurale de la protéine CBEL a également été entamé au cours de cette étude. L'enveloppe de la protéine CBEL en solution à notamment été déterminée par SAXS (Small Angle X-ray Scattering) et un modèle 3D de cette protéine a été obtenu. / Oomycetes are fungal like microorganisms evolutionary distinct from true fungi that include pathogens of plants. CBEL is a cell wall glycoprotein isolated from the oomycete Phytophthora parasitica that is composed of two distinct regions linked by a threonine/proline rich linker. Each region owns a cellulose binding module (CBM1) and a PAN-Apple domain involved in protein-protein or proteins-polysaccharides interactions. Since CBEL is able to induce defense responses in numerous plant species, its use for the development of products able to protect crops has been envisaged. For this purpose we analysed the effect of an external CBEL treatment on plants. We found that in the presence of surfactants CBEL is able to induce cytosolic calcium changes, defense gene expression, and cell death on A. thaliana. CBEL application for crop protection requires the development of economically reliable production processes. In the case of proteinaceous elicitors, an attractive strategy to obtain large amount of elicitors is to express them in heterologous hosts such as bacteria or yeasts. CBELcol and CBELpic were produced respectively in E. coli and in P. pastoris. CBELcol is unglycosylated whereas CBELpic displays a glycosylation profile distinct from the native protein (CBELnat). We found that all these proteins are able to bind crystalline cellulose. On the other side we found that the elicitor activity of CBELpic is distinct from CBELnat and CBELcol suggesting that the glycosylation on CBEL can have an impact on its ability to induce plant defense responses after external treatment on A. thaliana. In the second part of this work the two CBMs (1-1 and 1-2) that form part of CBEL have been submitted to detailed characterization, first to better quantify their interaction with cellulose and second to determine whether these CBMs can be useful for biotechnological applications, such as biomass hydrolysis. A variety of biophysical techniques were used to study the interaction of the CBMs with various substrates and the data obtained clearly indicate that CBEL's CBM1-1 exhibits much greater cellulose binding ability than CBM1-2. Engineering of the family 11 xylanase from Talaromyces versatilis (TvXynB), an enzyme that naturally bears a family 1 CBM, has produced two variants. The first one lacks a CBM, whereas the second contains the CBEL CBM1-1 in the place of the natural CBM1. The study of these enzymes has revealed that wild type TvXynB binds to cellulose, probably via its CBM1, and that the substitution of its CBM by oomycetal CBM1-1 does not affect its activity on this substrate. Moreover, the presence of CBEL during the hydrolysis of wheat straw actually potentiates the action of TvXynB, a result that is consistent with the hypothesis that CBM1-1 can alter cellulose surface fibres rather like some other members of CBM family 1.

Cryphonectria parasitica is the causal agent of chestnut blight, which devastated the American Chestnut tree population in the early 20th century. The discovery of hypoviruses that reduce the severity of the chestnut blight infection offers the potential for biological control. However, the spread of the hypoviruses is hampered by a diverse genetically controlled nonself-recognition system, vegetative incompatibility (vic). CPVIB-1 was identified as a transcription regulator playing an important role in the programmed cell death response to this stimulus. In this study, we have found that CPVIB-1 is ubiquitin-decorated which might lead to its degradation in the proteasome pathway. RNA-Seq and ChIP-Seq were used to further explore the downstream targets of CPVIB-1 that mediate the various metabolic changes that lead to the altered phenotype of the Δcpvib-1 mutant. Due to inaccuracies in the prior annotation, we performed a genome re-annotation to improve the accuracy using a MAKER2-two-pass pipeline. To validate the improvement a second pipeline, PEPA, was developed to compare quality metrics between the old and new annotations. Approximately 1/3 of the original annotations from 2009 were found to be inaccurate. Experimental confirmation by testing 27 predicted genes using a diagnostic PCR protocol to differentiate between prior and new transcript structures showed that over 80 % of tested genome locations supported for the new annotation. Using rapamycin treatment to mimic stimulation of the vic response and applying the RNA-seq and ChIP-seq data to this new information, we found that CPVIB-1 is related to TOR signaling pathways, promoting autophagy and the proteasome pathway, but repressing carbon metabolism, protein and lipid biosynthesis. In depth analysis of CPVIB-1-bound DNA targets showed that this protein is a member of the GAGA regulator family, a group of multifaceted transcription factors with diverse roles in gene activation and repression, maintenance of mitosis, and cell development. Following treatment with rapamycin the recognition sequence bound by CPBVIB-1 was altered leading to the regulation of different suite of genes with diverse metabolic functions. Ultimately, we have developed a revised model of TOR signaling pathway where TORC1 and TORC2 signaling pathways are connected by the action of CPVIB1.

No description available.

<u>Endothia parasitica</u> (Murr.) P. J. & H. E. Anderson (syn: <u>Cryphonectria parasitica</u>(Murr.) Barr), the causal agent of chestnut blight, was able to grow in total aqueous and tannin extracts from blight-susceptible American chestnut as well as in blight-resistant Chinese chestnut bark extracts, from winter and summer bark. Differences in the amount of conidial germination and growth in extracts of the two species were small. The, <u>E. parasitica</u> tannase was more abundant intracellularly than extracellularly. Total tannase activities from cultures in American chestnut aqueous and tannin extracts were greater than in the Chinese chestnut extracts, for both winter and summer bark. The tannase was isolated from the mycelium of E. parasitica and purified 142-fold with a 10% yield by anion exchange chromatography and gel filtration. The estimated molecular weight was 240 kD and the molecule may be a tetramer composed of four subunits with a molecular weight of 58 kD. The pH optimum of the purified tannase was 5.5 and the temperature optimum for activity was 30 C. The enzyme was separated into six bands in the pH range of 4.6 to 5.1 which may represent isoenzymes or post-translational modifications. / Ph. D.

Results of inoculations of grafts, seedlings, excised stems, and intact trees of large, surviving American chestnut indicated that some have heritable blight resistance. Some degree of resistance in American chestnut possibly would assist spread of hypovirulence (H) factors. The pathogenicity of the H isolate but not the V (virulent) isolate appeared to influence the size and sporulating characteristics of biocontrolled H+V cankers. The histopathology of canker development on blight-resistant Chinese and blight-resistant and blight-susceptible American chestnut was examined after inoculation with V and H E-parasitica. Rapid formation and continual growth of mycelial fans distinguished chestnut blight cankers incited by V E-parasitica from those incited by H E-parasitica, and distinguished cankers on susceptible chestnut from cankers on resistant chestnut. Differences among such cankers in wound periderm and other pathological alterations of bark and wood anatomy probably were results of differences in rates of fan formation and growth. Wound periderm formation began at the deepest point of a wound or canker and progressed outward to the bark surface. Superficial cankers arose in resistant trees or with hypovirulent fungus when slow-forming mycelial fans expanded through areas where wound periderm had not formed. Rapid epidemics of chestnut blight occur in recently clearcut areas but not in areas with large trees of species other than American chestnut, most probably because chestnut sprouts released after cutting become larger than they do when overtopped by large forest trees. The size of chestnut sprouts appeared to influence the rate of blight progress by setting a maximum limit on canker size and thus canker sporulation. It may be possible to foster spread of H factors in the Appalachians if blight epidemics in clearcuts can be prolonged. This might be done by restricting inoculum production. Cutting and removing some blighted sprouts, cutting some large unblighted sprouts, or increasing blight resistance in the chestnut population are three potential means of restricting inoculum production. / Ph. D.

Le système d'incompatibilité végétative a été décrit chez tous des champignons (Eumycètes) comme intervenant dans la limitation des fusions somatiques entre conspécifiques. Chez les champignons la fusion somatique est uniquement possible entre individus de même GCV (Groupe de Compatibilité Végétative). Comme tous les systèmes de reconnaissance du soi, le fonctionnement du système d'incompatibilité végétative des champignons est basé sur une grande diversité allélique. Cette thèse propose d'étudier la relation qui semble exister entre cette diversité des gènes impliqués dans l’incompatibilité végétative des champignons et la pression parasitaire exercée par des éléments cytoplasmiques délétères (ou DCE) transmis lors des fusions somatiques. Trois problématiques ont été abordées, avec trois approches différentes : (1) une approche conceptuelle générale portant sur l’évolution des systèmes de reconnaissance du soi, (2) une approche de modélisation sur le maintien de la diversité en GCV de la population de champignon par un DCE et (3) une approche expérimentale, pour étudier d’une part la perméabilité de la barrière d‘incompatibilité végétative et d’autre part l’interaction C. parasitica/CHV et les liens existant entre transmission et virulence du CHV. Ces études ont permis de montrer l'importance de la perméabilité de la barrière d'incompatibilité végétative à la fois au niveau du maintien de la diversité génétique de la population d'hôte et au niveau de la prévalence des DCE. Il semblerait donc que les DCE évoluent vers des niveaux de virulence faible du fait de la limitation de leur transmission par le système d'incompatibilité végétative de leur hôte. Nos résultats expérimentaux suggèrent que lorsque la diversité en GCV de la population d'hôte est faible, la virulence des DCE pourrait évoluer suivant le modèle du trade-off impliquant une évolution vers un niveau de virulence intermédiaire optimal. Ces travaux permettent donc de mieux comprendre les mécanismes agissant sur l'écologie et l'évolution des interactions champignon/DCE qui, au vu de cette étude, apparaissent comme de bon modèles pour l’étude des systèmes hôtes/parasites. / Vegetative incompatibility systems have been described in Fungi as controlling somatic fusion between conspecifics. For fungi, only fungi of the same vc type can fuse together. As other self recognition systems, this system involved high allelic diversity at specific genes. The issue of this work is to study the cause and effect relationship between the evolution of vegetative incompatibility systems and the selective pressure drove by cytoplasmic deleterious elements, transmitted during somatic fusion. Three problematics with three different approach were done : (1) a conceptual general framework on the evolution of self recognition systems (2) a theoretical work on the maintenance of vc type diversity by DCE and (3) an experimental work on the study of relationship between transmission and virulence in the C. parasitica/CHV host-parasite system. Ours results showed the key role of the permeability of the vegetative incompatibility barrier both for vc type diversity maintenance and on DCE prevalence. DCE would evolve toward avirulence in response to the transmission limitation by host incompatibility systems. Experimental work suggested a positive link between virulence and transmission in some population of CHV when host present a low vc type diversity, which could allow the evolution of the DCE toward an intermediate optimal virulence. This study would shed some light on mechanisms acting on the ecology and the evolution of fungi/DCE interaction which, according to our results, would be good study models for works on host-parasite systems.

Choisya ternata est une plante ornementale souvent touchée par la maladie de la pourriture racinaire provoquée par Phytophthora. Cette maladie peut induire de pertes allant jusqu’à 80 %, ce qui implique l’utilisation intensive de produits phytosanitaires. Afin de limiter l’utilisation de ces produits toxiques, une meilleure connaissance des acteurs de la défense des plantes est nécessaire. Pour cela, les objectifs de cette thèse sont I) d’identifier les espèces du genre Phytophthora pathogènes de C. ternata, II) d’étudier des acteurs de défense au niveau racinaire et III) de développer une approche de lutte alternative aux phytosanitaires. L’identification des espèces de Phytophthora par l’amplification et le séquençage de la région ITS, montre la présence de Phytophthora parasitica dans la production de C. ternata en pépinière. Ils mettent aussi en évidence, pour la première fois en France et chez Choisya, la présence de Phytophthora tropicalis. Deux cultivars de C. ternata sont utilisés pour étudier les acteurs de la défense racinaire contre P. parasitica, Aztec pearl (moins sensible à la pourriture racinaire) et Goldfinger (plus sensible). Les arabinogalactane-protéines (AGPs) sont des glycomolécules de la paroi cellulaire qui constitue une barrière physique face aux pathogènes. Des études ont montré le rôle des AGPs dans l’interaction plante-pathogène et plus particulièrement avec les oomycètes. Toutefois, le rôle des AGPs racinaires de Choisya dans l’interaction avec P. parasitica n’est pas étudié. Nos résultats montrent des différences biochimiques au niveau de la composition monosaccharidique des AGPs racinaires d’Aztec pearl en comparaison avec les AGPs de feuilles de ce cultivar et les AGPs de feuilles et de racines de Goldfinger. Contrairement aux autres fractions, ces AGPs n’ont pas augmenté la croissance du mycélium de P. parasitica. Ces résultats suggèrent que l’oomycète n’est pas capable de dégrader cette fraction pour l’utiliser comme une source d’énergie. Pendant l’infection de la plante par P. parasitica, ces AGPs peuvent freiner la pénétration du pathogène, et par conséquent diminuer la sévérité de la maladie. Les plantes ont aussi développé des molécules chimiques nommées métabolites secondaires (MS) capables de les protéger contre leurs agresseurs. Des études ont montré que la partie foliaire de Choisya est riche en MS tels que les alcaloïdes dont plusieurseffets pharmacologiques sont connus. Cependant, leur composition racinaire et leur rôle dans la protection de la plante contre P. parasitica ne sont pas élucidés. Nos résultats montrent que les racines des deux cultivars sont riches en alcaloïdes furoquinoliques. Certains alcaloïdes sont présents en plus grande quantité chez Aztec pearl, mais suite à l’inoculation de zoospores de P. parasitica cette différence n’est plus détectable. De plus, l’extrait contenant les alcaloïdes totaux d’Aztec pearl ont été capables d’inhiber la croissance du mycélium de l’oomycète, contrairement à l’extrait issu de Goldfinger. Ces résultats montrent le rôle potentiel des alcaloïdes furoquinoliques dans la protection de la plante contre cet oomycète. / Choisya ternata is an ornamental plant that suffers from root rot disease due to Phytophthora. This disease can lead to severe production losses (up to 80 %), which require intensive use of phytosanitary products. A better understanding of plants defenses in required in order to reduce the use of these products. Therefore, the objectives of this thesis are I) identifying Phytophthora spp. pathogens of C. ternata, II) studying roots defense actors and III) developing an alternative control approach. Phytophthora spp. identification by ITS region amplification and sequencing highlighted the presence of Phytophthora parasitica on Choisya. Furthermore, Phytophthora tropicalis was identified for the first time in France and on Choisya culture. Two C. ternata cultivars were used to study the plants root defense actors against P. parasitica, Aztec pearl (less susceptible to root rot) and Goldfinger (more susceptible to root rot). Arabinogalactan proteins (AGPs) are glycomolecules of the cell wall which constitutes a physical barrier to pathogens. Studies showed the role of AGPs in the plant-pathogen interaction and more specifically in the case of oomycetes. However, Choisya root AGPs role in the interaction with P. parasitica is not studied. Our results showed biochemical differences in the monosaccharide composition of Aztec pearl root AGPs and the other AGPs fractions (Aztec pearl leaves and Goldfinger roots and leaves). Contrary to other fractions Aztec pearl root AGPs did not increase P. parasitica mycelium growth. These results suggest that oomycete was not able to degrade this fraction and use it as energy source. When P. parasitica infects the plant, these AGPs may be able to slow the infection and reduce disease severity. Plants have also developed chemical molecules known as secondary metabolites (SM) capable of protecting them against attackers. Studies showed that C. ternata leaves are rich with SM as alkaloids that have many pharmacological activities. Nevertheless, roots alkaloids composition and role in plant protection are not studied. Our results showed that roots of the two cultivars are riche with furoquinoline alkaloids. Some these alkaloids were more concentrated in Aztec pearl. But after inoculation with P. parasitica zoospores, the difference was not detectable anymore. Moreover, the total alkaloids extract of Aztec pearl inhibited P. parasitica mycelium growth, unlike the extract of Goldfinger. Another strategy of plants protection is the use of beneficial soil microorganisms that limits pathogens development and stimulate plant defenses. These microorganisms are known as biological control agents (BCA) and are sometimes used in horticulture as an alternative control strategy. In our study, treatments of C. ternata by different BCA were evaluated by a developed real time PCR (qPCR) targeting ypt1 and by symptoms annotation. This evaluation showed that combined treatments by Glomus intraradices with Gliocladium catenulatum and G. intraradices with Trichoderma atroviridae (respectively mycorrhizal fungi and filamentous fungi) offer a better protection against P. parasitica.

L'agriculture mondiale a connu de profonds changements qui lui ont permis de faire face à l'augmentation constante de la demande alimentaire. Cependant, les conséquences de ces nouvelles pratiques agricoles sur l'environnement et la santé humaine font l'objet de préoccupations croissantes. Notamment, les politiques sanitaires actuelles visent à réduire l'utilisation des produits phytosanitaires. Aussi de nouvelles stratégies de protection des cultures doivent-elles être développées. Une meilleure compréhension des échanges moléculaires qui contribuent au succès des bioagresseurs est nécessaire. Ces échanges impliquent notamment la sécrétion de protéines qui interfèrent avec le métabolisme de l'hôte : les effecteurs. Certains d'entre elles sont accumulés au cours de la pénétration des premières cellules végétales, une étape décisive pour le succès de la tentative d'infection. Les travaux menés au cours de cette thèse se sont concentrés sur 3 de ces effecteurs, sécrétés par l'oomycète Phytophthora parasitica. L'analyse des lignées de surexpression chez Arabidopsis thaliana a permis de mettre en évidence des perturbations du développement et de la physiologie de certaines hormones végétales en réponse à l'accumulation de ces effecteurs. Ces données confirment l'importance de la manipulation des voies hormonales dans le cadre des interactions plantes-pathogènes et soutiennent l'hypothèse récente selon laquelle des effecteurs sécrétés par les agents pathogènes interfèrent avec un petit nombre de cibles clefs du métabolisme de l'hôte. Ces cibles constituent des candidats de choix pour développer des variétés plus résistantes.

The environmental impacts of genetically modified (GM) crop plants such as Bt (Bacillus thuringiensis) maize have not yet been fully assessed in South Africa. Bt maize designed to express Bt endotoxin for control of Busseola fusca (Fuller) (Lepidoptera: Noctuidae) and Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) is planted on approximately 1.103 million hectares in South Africa. The monitoring of GM crops after release is important in order to assess and evaluate possible environmental effects. No risk assessment for Bt maize was done in South Africa before its release in 1998 and no targeted post-release monitoring of possible resistance development or impact on non-target species have been done. Awareness has risen in South Africa through research highlighting the possible effects GM crops may have. The aim of this study was to determine, through feeding experiments, the effects of Bt maize on selected non-target Lepidoptera, Coleoptera and Diptera species that occur in maize agro-ecosystems in South Africa. Results provide information for use in future risk assessment studies on Bt maize and indicate which species could possibly be of importance in post-release monitoring of Bt maize. Priority insect species were identified and laboratory- and semifield experiments were conducted to evaluate the effect of Bt maize on these species. In the light of the reportedly lower toxicity of Bt maize to certain noctuid borers, the effect of Bt maize was evaluated on Sesamia calamistis (Hampson), Agrotis segetum (Denis & Schiffermüller), and Helicoverpa armigera (Hubner). Feeding studies were also conducted to determine the effect of Bt maize on non-target Coleoptera, i.e. Heteronychus arator Fabricius (Coleoptera: Scarabaeidae) and Somaticus angulatus (Fahraeus) (Coleoptera: Tenebrionidae). The effect of indirect exposure of the stem borer parasitoid Sturmiopsis parasitica (Curran) (Diptera: Tachinidae) to Bt toxin was evaluated to determine if there is any effect when it parasitizes Bt-resistant B. fusca larvae that have fed on Bt maize. Results from the study conducted with S. calamistis indicated that Bt maize of both events (Bt11 and MON810) were highly toxic to S. calamistis. The behavioural characteristic of S. calamistis to feed behind leaf sheaths and to enter stems directly did not result in escape of exposure to the toxin. Larval feeding on leaf sheaths therefore resulted in the ingestion of sufficient toxin to kill larvae before they entered maize stems. Results showed that the effect of Cry1Ab toxin on the biology of A. segetum larvae and moths were largely insignificant. Whorl leaves were observed to be an unsuitable food source for H. armigera larvae and larval growth was poor. No larvae survived to the pupal stage on any of the Bt maize treatments. When feeding on maize ears H. armigera larval mass increased on non-Bt maize whereas no increase occurred on Bt maize. The feeding study conducted with Coleoptera showed that the effect of Bt maize on H. arator and S. angulatus was insignificant and no differences were observed in any of the parameters measured for the two species. Although not always significant, the percentage parasitism of Bt-consuming host larvae by S. parasitica was always higher compared to host larvae that fed on non-Bt maize. It could be that Bt toxin affects B. fusca fitness to such an extent that the immune systems of host larvae were less effective. The different parameters tested for S. parasitica indicated only one case where fly maggots originating from diapause host larvae feeding on non-Bt maize had a greater mass compared to host larvae that fed on Bt maize. The same applied to S. parasitica pupal length. For other parameters tested there were no significant differences. Sesamia calamistis is stenophagous and occurs in mixed populations with other borer species. It was therefore concluded that the ecological impact of local extinctions of S. calamistis caused by Bt maize is not expected to be great. Bt maize will most likely not have any significant effect on the control of A. segetum under field conditions. The feeding study conducted with H. armigera quantified the effects of Bt maize on this species and provided important information on the potential of Bt maize as protection against this polyphagous pest. However, the likelihood of H. armigera becoming an important secondary pest is high. It can be concluded that the Cry1Ab toxin targeting lepidopteran pests will not have adverse effects on H. arator or S. angulatus. Although some adverse effects were observed on S. parasitica mass and pupal length it is most likely that this will not contribute to adverse effects in the field, but that there rather be synergism between Bt maize and S. parasitica. An ecological approach was followed in which the potential effects of exposure of priority species to Bt toxin in maize was investigated. A series of selection matrixes were developed in which each of the above mentioned species was ranked for its maximum potential exposure to Bt toxin by assessing it occurrence, abundance, presence and linkage in the maize ecosystem. Through the use of these selection matrixes, knowledge gaps were identified for future research and to guide the design of ecologically realistic experiments. This study contributes to knowledge regarding the possible effects of Bt maize on the most economically important non-target pests in South Africa. There is, however, a need to evaluate other non-target species in feeding studies, as well as in field studies. From this study it can be concluded that some species can be eliminated from further testing since Bt maize had no adverse effect while more research have to be conducted on other species. / Thesis (Ph.D. (Environmental Science)--North-West University, Potchefstroom Campus, 2010.

The environmental impacts of genetically modified (GM) crop plants such as Bt (Bacillus thuringiensis) maize have not yet been fully assessed in South Africa. Bt maize designed to express Bt endotoxin for control of Busseola fusca (Fuller) (Lepidoptera: Noctuidae) and Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) is planted on approximately 1.103 million hectares in South Africa. The monitoring of GM crops after release is important in order to assess and evaluate possible environmental effects. No risk assessment for Bt maize was done in South Africa before its release in 1998 and no targeted post-release monitoring of possible resistance development or impact on non-target species have been done. Awareness has risen in South Africa through research highlighting the possible effects GM crops may have. The aim of this study was to determine, through feeding experiments, the effects of Bt maize on selected non-target Lepidoptera, Coleoptera and Diptera species that occur in maize agro-ecosystems in South Africa. Results provide information for use in future risk assessment studies on Bt maize and indicate which species could possibly be of importance in post-release monitoring of Bt maize. Priority insect species were identified and laboratory- and semifield experiments were conducted to evaluate the effect of Bt maize on these species. In the light of the reportedly lower toxicity of Bt maize to certain noctuid borers, the effect of Bt maize was evaluated on Sesamia calamistis (Hampson), Agrotis segetum (Denis & Schiffermüller), and Helicoverpa armigera (Hubner). Feeding studies were also conducted to determine the effect of Bt maize on non-target Coleoptera, i.e. Heteronychus arator Fabricius (Coleoptera: Scarabaeidae) and Somaticus angulatus (Fahraeus) (Coleoptera: Tenebrionidae). The effect of indirect exposure of the stem borer parasitoid Sturmiopsis parasitica (Curran) (Diptera: Tachinidae) to Bt toxin was evaluated to determine if there is any effect when it parasitizes Bt-resistant B. fusca larvae that have fed on Bt maize. Results from the study conducted with S. calamistis indicated that Bt maize of both events (Bt11 and MON810) were highly toxic to S. calamistis. The behavioural characteristic of S. calamistis to feed behind leaf sheaths and to enter stems directly did not result in escape of exposure to the toxin. Larval feeding on leaf sheaths therefore resulted in the ingestion of sufficient toxin to kill larvae before they entered maize stems. Results showed that the effect of Cry1Ab toxin on the biology of A. segetum larvae and moths were largely insignificant. Whorl leaves were observed to be an unsuitable food source for H. armigera larvae and larval growth was poor. No larvae survived to the pupal stage on any of the Bt maize treatments. When feeding on maize ears H. armigera larval mass increased on non-Bt maize whereas no increase occurred on Bt maize. The feeding study conducted with Coleoptera showed that the effect of Bt maize on H. arator and S. angulatus was insignificant and no differences were observed in any of the parameters measured for the two species. Although not always significant, the percentage parasitism of Bt-consuming host larvae by S. parasitica was always higher compared to host larvae that fed on non-Bt maize. It could be that Bt toxin affects B. fusca fitness to such an extent that the immune systems of host larvae were less effective. The different parameters tested for S. parasitica indicated only one case where fly maggots originating from diapause host larvae feeding on non-Bt maize had a greater mass compared to host larvae that fed on Bt maize. The same applied to S. parasitica pupal length. For other parameters tested there were no significant differences. Sesamia calamistis is stenophagous and occurs in mixed populations with other borer species. It was therefore concluded that the ecological impact of local extinctions of S. calamistis caused by Bt maize is not expected to be great. Bt maize will most likely not have any significant effect on the control of A. segetum under field conditions. The feeding study conducted with H. armigera quantified the effects of Bt maize on this species and provided important information on the potential of Bt maize as protection against this polyphagous pest. However, the likelihood of H. armigera becoming an important secondary pest is high. It can be concluded that the Cry1Ab toxin targeting lepidopteran pests will not have adverse effects on H. arator or S. angulatus. Although some adverse effects were observed on S. parasitica mass and pupal length it is most likely that this will not contribute to adverse effects in the field, but that there rather be synergism between Bt maize and S. parasitica. An ecological approach was followed in which the potential effects of exposure of priority species to Bt toxin in maize was investigated. A series of selection matrixes were developed in which each of the above mentioned species was ranked for its maximum potential exposure to Bt toxin by assessing it occurrence, abundance, presence and linkage in the maize ecosystem. Through the use of these selection matrixes, knowledge gaps were identified for future research and to guide the design of ecologically realistic experiments. This study contributes to knowledge regarding the possible effects of Bt maize on the most economically important non-target pests in South Africa. There is, however, a need to evaluate other non-target species in feeding studies, as well as in field studies. From this study it can be concluded that some species can be eliminated from further testing since Bt maize had no adverse effect while more research have to be conducted on other species. / Thesis (Ph.D. (Environmental Science)--North-West University, Potchefstroom Campus, 2010.