Tall fescue, a cool-season grass native to Europe, central Asia, and northern Africa, has been widely distributed throughout the U.S. for use as turf and forage. Following its widespread planting, its ability to associate with a toxic fungal endophyte, Neotyphodium coenophialum, was discovered. Research has linked this fescue-endophyte association with increased biotic and abiotic stress resistance in endophyte-infected (E+) versus endophyte-free (E-) plants, and these differences may affect the ability of land managers to eradicate tall fescue and restore native grasslands. I conducted three studies to examine whether E+ tall fescue plants respond differently to management than E- plants, and whether the success of planted native species might be impacted via indirect soil effects. My overall hypotheses were that E+ plants would recover from restoration/eradication efforts better than E- plants, and that E+ fescue would reduce microbial symbionts in the soil needed by planted native species. I first conducted a field study of a tall fescue pasture consisting of four sub-units being restored with different combinations of prescribed burns and/or herbicide applications, as well as an unmanaged control. I found no evidence of E+ plants preferentially surviving restoration management; however this field had unusually low endophyte infection rates to begin with. The second study was a greenhouse experiment in which I measured growth of E+ and E- plants exposed to different watering regimes (wet, dry) and prescribed burn treatments (none, one, or two burns). Watering regime significantly affected all measured growth parameters (wet>dry), but few endophyte effects were found and when present were opposite the hypothesis (E->E+). All burned plants quickly re-grew tiller lengths comparable to the unburned control, with recovery occurring faster following the second burn compared to the first. My final study examined growth and arbuscular mycorrhizal colonization of native species planted by seed into soil from beneath E+ and E- tall fescue. I observed few differences in mycorrhizal colonization or biomass for seedlings between soil from E+ and E- tall fescue. Taken together, my results indicate endophyte status of tall fescue pastures being restored to native grassland species may not be important in governing restoration success.

Tall fescue (Lolium arundinaceum (Schreb.) Darbysh.) is a cool-season perennial grass used in pastures throughout the Southeastern United States. The grass can harbor a fungal endophyte (Epichloë coenophiala) thought to provide the plant with enhanced resistance to biotic and abiotic stress. However, the alkaloids produced by the common variety of the endophyte cause severe animal health issues resulting in a considerable amount of research focused on eliminating the toxic class of alkaloids while retaining the positive abiotic and biotic stress tolerance attributes of the other alkaloids. In doing so, very little attention has been paid to the direct influence the fungal-plant symbiosis has on rhizosphere processes. Therefore, my objectives were to study the influence of this relationship on plant biomass production, root exudate composition, and soil biogeochemical processes using tall fescue cultivars PDF and 97TF1 without an endophyte (E-), or infected with the common toxic endophyte (CTE+), or with two novel endophytes (AR542E+, AR584E+). I found that root exudate composition and plant biomass production were influenced by endophyte status, tall fescue cultivar, and the interaction of cultivar and endophyte. Cluster analysis showed that the interaction between endophyte and cultivar results in a unique exudate profile. These interactions had a small but perceptible impact on soil microbial community structure and function with an equally small and perceptible impact on carbon and nitrogen cycling in soils from rhizobox and field sites. These studies represent the first comprehensive analysis of root exudate chemistry from common toxic and novel endophyte infected tall fescue cultivars and can be used to help explain in part the observed changes in C and N cycling and storage in pastures throughout the Southeast U.S..

This 2-yr experiment evaluated productivity of wild-type, endophyte-infected tall fescue (E+) and novel endophyte-infected tall fescue (NE) summer stockpiled (SS) pastures and the performance of fall-calving beef cow/calf pairs stocked on each cultivar. Fescue cultivars used were KY-31 and MaxQ for E+ and NE treatments, respectively. Pregnant Simmental x Angus cows (128 total, 64 each yr) were stratified by BW, BCS, and expected calving date and then allotted to 1 of 10 pasture groups within each yr (20 groups total, 10 per treatment). Forage growth was stockpiled from April until the initiation of strip-grazing on August 31 of 2017 and 2018. Cows grazed treatment pastures for 52 d from 23 ± 14 d prepartum to 29 ± 14 d postpartum, and calved on treatment pastures. Forage quadrats were clipped from the grazed and ungrazed portions of each pasture to determine weekly forage mass. Total ergot alkaloid (TEA) concentrations were analyzed for all pastures at the beginning of the experiment and every subsequent 2 wk for E+ tall fescue. Cow BW was recorded on 2 consecutive d and BCS determined at the start and end of the experiment. In yr 2, ultrasound 12th rib fat thickness (FT) was measured at the beginning and end of the treatment period Milk production was estimated using the weigh-suckle-weigh technique at 29 ± 14 d postpartum. Initial TEA concentrations for NE (Yr. 1 = 112 µg/kg; Yr. 2 = 632 µg/kg) were decreased (P ≤ 0.01) compared to E+ (Yr. 1 = 1831 µg/kg; Yr. 2 = 2903 µg/kg). TEA concentrations for E+ pastures did not differ (P < 0.23) by sample date. However, average TEA concentrations were greater for yr 2 than yr 1(P ≤ 0.01). Fescue cultivars were not different (P ≥ 0.06) in forage CP, Ash, Ether Extract, and grazed or ungrazed forage mass. However, differences were observed (P ≤ 0.02) for ADF, NDF, and TDN by fescue cultivar. Cow BW, BCS, and FT at the beginning and end of grazing were not different (P ≥ 0.41) by treatment. Milk production was greater (P < 0.01) for cows grazed on E+. Calving date, calf BW, calf ADG were not different (P ≥ 0.65) by treatment. Neither AI nor overall conception rates differed (P ≥ 0.23) between cultivars. Performance of fall-calving cows pre-exposed to E+ was not hindered when grazed on E+ relative to NE in a SS system. / Master of Science / Wild-type, endophyte-infected tall fescue (E+) is the predominant forage of use for producers within the southeastern United States. Endophyte-infected tall fescue gained notoriety due to its drought and pest resistance as well as climate adaptability. These advantages are the result of a symbiotic relationship with an endophytic fungus that has been shown to decrease in animal performance through the production of toxic ergot alkaloids. Development of improved fescue cultivars provided producers with an alternative forage, known as novel endophyte-infected tall fescue (NE), that maintains the agronomic advantages noted with E+ fescue without negative impacts on animal gain and reproductive performance. In adopting NE tall fescue, producers are faced with the financial challenge of renovating existing stands of E+ with NE fescue; leaving pastures unusable during times of normal grazing behavior. This constraint has highlighted the need to explore forage utilization and strategies that can extend the grazing period that do not require the significant cost of total renovation with NE. Fall stockpiled tall fescue has provided producers an option to extend grazing from late November through February when pasture growth ceases and hay supplementation is normally needed. However, a 60 to 90 d period between summer and fall stockpiled grazing leaves producers looking for an additional strategy to provide standing forage for their herds. Summer stockpiling (SS), is a novel grazing strategy that bridges summer and fall stockpiled grazing through proper accumulation of 25 % of total pasture to help extend producers grazing season. The objective of the current experiment is to evaluate both E+ and NE SS pastures and its effect on animal performance, forage availability, and nutritive value. Cow BW and BCS were measured at the initiation, conclusion, and before artificial insemination. Calf BW and ADG were assessed 48 h post-calving and at weaning. Ungrazed and grazed forage mass was collected weekly, while measurements of ADF, NDF, CP, TDN, ether extract, and ash were measured every 2 weeks. After a 52 d treatment period, forage mass was similar across both cultivars, with ADF, NDF, and TDN favoring E+ tall fescue. Additionally, animal performance across both E+ and NE pastures were similar, however increased milk production was observed for cows grazed on E+. This experiment helps shed light on the concept of strategic renovation. Strategic renovation can be best utilized by producers who are interested in maximizing pasture utilization through rotationally grazing E+ and NE tall fescue pastures. By following this renovation strategy, proper allocation of NE tall fescue during times of enhanced ergotism and E+ tall fescue during low thresholds will develop a more specific rotation thus decreasing renovation costs for producers when ergotism is lowest.

Au cours de cette thèse, un isolat de sol de désert, Saccharothrix algeriensis NRRL B-24137, a été évalué pour ses propriétés bioactives contre le champignon phytopathogène Botrytis cinerea, pour sa colonization sur Vitis vinifera L., et Arabidopsis thaliana ainsi qu’en vue d’étudier les méchanismes de résistance systémique induite (ISR) contre B. cinerea. Les résultats obtenus nous ont permis premièrement de montrer que Sa. algeriensis NRRL B-24137 peut présenter des activités antifongiques contre B. cinerea et que des métabolites peuvent être responsables de cette activité antifongique. Bien que ces métabolites soient encore en cours d’étude et que cette étude mérite d’être approfondie, nous avons démontré ensuite les propriétés de colonisation de l’isolat du sol du désert chez la vigne. Les résultats ont permis de montrer que la souche peut former des populations rhizosphèriques ainsi que des sous-populations endophytiques chez des plants de vigne (Cabernet Sauvignon sur porte-greffe 44-53 M) à des étapes précoces de colonisation. Puis nous avons démontré que la souche bénéfique peut induire une résistance systémique contre B. cinerea. Bien que les mécanismes impliqués ne soient pas encore compris, des parties préliminaires de ces travaux démontrent que les expressions de gènes responsables de la production de glucanase, chitinase ainsi qu’un inhibiteur de polygalacturonase ne semblent pas potentialisés pendant le phénomène de résistance systémique. Enfin nous avons démontré l’interaction entre Sa. algeriensis NRRL B-24137 et Arabidopsis thaliana qui résulte dans une association intime dûe également à colonisation rhizosphèrique et endophytique de la plante modèle. La souche bénéfique peut églement induire un phénomène de résistance systémique sur A. thaliana contre B. cinerea et les analyses de plantes mutées ont permis de determiner des parties des mécanismes impliqués dans l’ISR aini que des nouveaux mécanismes impliqués qui peuvent être induits par des microbes bénéfiques / In this thesis, the desert soil isolate, Saccharothrix algeriensis NRRL B-24137, has been evaluated for its bioactive properties towards the phytopathogenic fungus Botrytis cinerea, for its colonization of Vitis vinifera L., and Arabidopsis thaliana as well as to study the mechanisms of induced systemic resistance (ISR) towards B. cinerea. The results obtained allowed us firstly to show that Sa. algeriensis NRRL B-24137 can exhibit strong antifungal properties towards B. cinerea and that some metabolites can be responsible of this antifungal activity. Although these metabolites are still under consideration and that this study needs further works, we have demonstrated then the colonization properties of the desert soil isolate with grapevine plants. The results showed that the strain can form rhizospheric as well as endophytic subpopulations with grapevine plants (Cabernet Sauvignon cultivar graffed on 44-53 M rootstock) at early step of colonization. Then we have demonstrated that the beneficial strain could induce a systemic resistance towards B. cinerea. Although the mechanisms are not yet well understood, preliminary parts of this work demonstrated that the genes responsible of glucanase production, chitinase as well as inhibitor of polygalacturonase activity do not seems to be primed during the systemic resistance phenomenon. Finally we demonstrated that the interaction between Sa. algeriensis NRRL B-24137 and Arabidopsis thaliana plants results in a close association due also to a rhizo- and endophytic colonization of the model plant. The beneficial strain can also induce a systemic resistance in A. thaliana towards B. cinerea and analyzes of plant mutants have allowed to determine parts of the mechanisms involved in ISR as well as new mechanisms that could be trigerred by beneficial microbes

Natural products as sources of novel therapeutic agents experienced a steady increase from around the turn of the twentieth century until it peaked in the 1970s and 1980s. However since this time pharmaceutical research in natural products has experienced a decline. Despite this trend the natural products industry now seems to be experiencing a revival of sorts. This thesis represents a continuation of the work on the isolation and structure elucidation of potential drug leads from terrestrial fungal sources that the natural products group at the University of Canterbury is engaged in. The known compound, pseurotin A (2.7) and two novel diastereomers, pseurotin A2 (2.8) and pseurotin A3 (2.9) were isolated from the extract of a Penicillium sp. of fungus collected from the foreshore of a beach in Vancouver, Canada. The absolute stereochemistry of pseurotin A2 and proposed absolute stereochemistry for A3 were elucidated using a combination of X-ray crystallography (A2 only), circular dichrosim, oxidative cleavage reactions, and J2-resoved 2D NMR experiments. The extract of an as yet unidentified endophytic fungus has yielded eight novel compounds related to the spirobisnaphthalene class of compounds. These eight compounds fall into to distinct groupings. The spiro-mamakones, distinguished by a structurally unprecedented oxygenated spiro-nonene skeleton, comprise five compounds, spiro-mamakones A-E (3.11, 3.15-3.18). In addition to these naturally occurring compounds, the semi-synthetic compounds, 4-oxo-spiro-mamakone A (3.12) and O-acetyl-spiro-mamakone A (3.21), were also synthesised. spiro-Mamakone A was found to be racemic, while X-ray crystallography and optical rotation revealed spiro-mamakone C (3.15) to be present as an enantiomeric mixture (4S*, 5S*, 9R*). Unfortunately the enantiomeric excess was unable to be elucidated. NOE experiments revealed spiro-mamakone B (3.16) to have the relative stereochemistry 4S*, 5S*, 9S*. The relative stereochemistry of spiro-mamakones D (3.17) (4S*, 5S*, 8S*, 9S*) and E (3.18) (4S*, 5S*, 8S*, 9R*) was proposed from comparison of coupling constant calculations from energy-minimised models with those of the experimentally determined values. The second group, comprising three novel compounds named the mamakunoic acids, mamakunoic acid A-C (3.8, 3.7, 3.10), are characterised by their acid substituted dihydro benzofuran system. The low yield obtained of these compounds, unfortunately prevented their stereochemical elucidation. In addition to structure elucidation, biosynthetic studies on spiro-mamakone A and mamakunoic acid B were also carried out. Analysis of the NMR spectra derived from spiro-mamakone A, labelled with isotopic acetate, revealed a situation complicated by the presence of isotopomers and racemisation, resulting in NMR spectra that were somewhat anomalous in appearance. These irregularities however, were resolved leading to the proposal that spiro-mamakone A was derived from a dihydroxynaphthalene (DHN) intermediate, which proceeds through to spiro-mamakone via an epoxide intermediate. Despite problems with purity and low yields of isotopically labelled mamakunoic acid B, it was proposed that like spiro-mamakone A, it proceeded via a DHN intermediate. The extract derived from a Malaysian Scleroderma sp. was found to contain a new dichlorinated pulvinic acid derivative, methyl-3',5'-dichloro-4,4'-di-O-methylatromentate (4.14), the structure of which was confirmed by X-ray crystallography. In addition three previously reported compounds, 4,4'-dimethoxyvulpinic acid (4.11), methyl-3'-chloro-4,4'-di-O-methylatromentate (4.12) and methyl-4,4'-dimethoxyvulpinate (4.13), were also isolated. The extract of another, as yet unidentified endophytic fungus was found to contain the new acetogenin, 1,5-dihydroxy-6-(2-hydroxyethyl)-3-methoxyacetophenone (5.7), differing from the known compound, 2,4-dihydroxy-6-(2-hydroxyethyl)-3-methoxyacetophenone (5.8) only by virtue of the substitution pattern. The structure of 5.7 was confirmed by X-ray crystallography. The implementation of efficient dereplication procedures is paramount for those working in the field of natural products. The recent advances that have been made in the dereplication process in the natural products group at the University of Canterbury are given using examples from this research and where necessary from other group members.

La Maladie de la Feuille Cassante du Palmier dattier (Phoenix dactylifera L.) constitue un cas d'émergence d'une maladie à fort impact économique causée par un agent étiologique inconnu. Notre stratégie a visé à élaborer une approche sans à priori de l'émergence pouvant être transposée à n'importe quelle situation de ce type. En nous appuyant sur des caractérisations successives des compartiments viraux, bactériens et fongiques de tissus sains et malades, nous avons cherché à mettre en évidence des différences de composition spécifiques et de distribution de ces flores sur 2 campagnes de prélèvements réalisées en 2010 et 2012. Alors que la microscopie électronique à transmission nous a permis de visualiser des structures d'origine virale probable au niveau des chloroplastes du parenchyme chlorophyllien, une étude moléculaire de séquençage de gènes ribosomaux nous a permis de corréler l'apparition de ces structures a de profondes modifications qualitative et quantitative de la microflore endophyte. Ainsi il nous est apparu que la symptomologie de la maladie était corrélée à une modification profonde de la distribution spécifique de la microflore endophyte, visible à la fois au niveau du compartiment fongique et bactérien, suggérant la complète disparition de la pression de sélection exercée par le palmier sain sur sa flore et mise en évidence dans les 2 cas, par un shift d'une répartition de type Poisson vers une répartition normale. Dans le compartiment fongique, une claire déplétion des Pleosporaceae, associées à la plante saine pouvait ainsi être liée à une apparition de nouvelles familles (Trichocomaceae et Mycosphaerellaceae). De même, parmi les bactéries, une disparition des Rhizobium et Ensifer sp associés au compartiment racinaire de la plante saine a ainsi pu être mise en évidence, ces espèces pouvant servir ultérieurement d'indicatrices de bonne santé des palmiers. Dans une deuxième partie de notre travail nous avons cherché à utiliser des éléments de la flore endophyte mais également de substances naturelles dans la lutte biologique contre d'autres pathogènes du palmier. Ainsi, un antagonisme a été mis en évidence entre une souche endophyte d'Arthrobacter agilis et un pathogène majeur, Fusarium oxysporum sp Albedinis / The Brittle Leaf Disease of the Date Palm Tree (Phoenix dactylifera L.) constitutes a case study of an emerging disease of economic impact caused by a yet uncharacterized etiologic agent. Our strategy was to develop an approach that could be indistinctly transposed to any situation of this type. While basing our investigations on the successive characterization of the diversity of viral, bacterial and fungal endophytic compartments of healthy and diseased Palm trees, we aimed at enlightening differences in species composition but also distribution over two sampling campaigns performed in 2010 and 2012. While transmission electronic microscopy allowed us to visualize structures of probable viral origin affecting chloroplasts of the chlorophyllic cell layer, a molecular approach based on ribosomal gene sequencing allowed us to evidence correlations between the occurrence of such structures and deep modifications of the structure of the palm date tree associated endophytic flora suggesting a strong depletion of the ability of the palm to control its associated endophytes. This was evidenced in both fungal and bacterial compartments by a shift from a Poisson like diversity distribution towards a Gaussian distribution in the flora associated to MFC affected palms. In the fungal compartment, Pleosporaceae, that dominated in healthy palms were replaced by an opportunistic flora of Trichocomaceae and Mycosphaerellaceae. Among bacteria, the disappearance of Rhizobium and Ensifer species, typically associated to the root compartment of healthy palms was enlighten, suggesting that these species could indeed be used as biomarkers of healthy plant status. In a second part of this study, we investigated the potential use of cultivable palm endophytes, but also natural compounds for biocontrol applications. In particular, we evidence the antagonistic potential of Arthrobacter agilis, a palm endophyte, against a major palm date disease agent, Fusarium oxysporum sp. Albedinis.

Os fungos endofíticos têm sido reconhecidos pela sua grande importância para as plantas hospedeiras, pois podem conferir proteção contra insetos herbívoros e patógenos, promover o crescimento vegetal, além de produzir metabólitos secundários com atividades biológicas diversas, entre outros. A cana-de-açúcar é uma cultura de grande importância social e econômica no Brasil, especialmente para o estado de São Paulo. Ultimamente esta cultura vem recebendo especial atenção devido ao crescente aumento da demanda de matéria prima, principalmente em função do acréscimo no consumo de etanol como biocombustível. Fungos do gênero Penicillium habitam os tecidos e a rizosfera de cana-de-açúcar, onde podem estabelecer associações mutualísticas com a planta e conferir diversos benefícios. Dentro deste contexto, estudos que avaliem a interação de Penicillium spp. com cana-de-açúcar são bastante promissores para geração de conhecimentos que auxiliem na otimização da agricultura. Dessa forma, o presente trabalho teve como objetivos a avaliação do potencial biotecnológico dos endofíticos de raiz e da rizosfera, do gênero Penicillium, pertencentes à comunidade fúngica de cana-de-açúcar, por meio de ensaios de antagonismo, produção de enzimas, solubilização de fosfato inorgânico e produção de ácido indol acético; assim como o estudo da interação de um isolado de P. pinophilum com cana-de-açúcar a partir do desenvolvimento de um sistema de transformação genética mediada pela bactéria Agrobacterium tumefaciens. Tanto a análise da atividade antimicrobiana como a produção de metabólitos apresentaram extensa variação fisiológica entre os isolados avaliados. Um isolado da espécie P. pinophilum (linhagem 44) foi escolhido para ser usado na transformação genética por mostrar-se superior estatisticamente em relação aos demais isolados nos ensaios anteriores. Para aumento da eficiência deste sistema de transformação foram avaliados diferentes parâmetros, dentre eles: tempo de co-cultivo (24 e 48 horas), concentração do indutor acetoseringona (200 M e 400 M) e tipos de membrana (papel filtro e náilon). O sistema de agrotransformação apresentou alta eficiência (482 transformantes por 107 conídios), gerando uma elevada quantidade de transformantes resistentes à higromicina B e expressando GFP. Dentre os parâmetros avaliados, a combinação que deu origem aos melhores resultados de transformação envolveu o co-cultivo por 48 horas sobre membrana de náilon, em meio de cultura contendo 200 M de acetoseringona. A interação fungo-planta foi avaliada a partir da inoculação de P. pinophilum linhagem selvagem e transformantes, em plântulas de cana-de-açúcar, seguida da análise por microscopia óptica de epifluorescência e reisolamento. Os resultados revelaram a natureza não patogênica desse fungo, uma vez que ele foi capaz de colonizar endofiticamente cana-de-açúcar e persistir nas raízes desta planta, sem levar ao desenvolvimento de qualquer sintoma de doença. Além disso, os ensaios de agrotransformação deram origem a uma biblioteca com mil e cem transformantes insercionais, o que constitui uma ferramenta importante para o estudo molecular do metabolismo secundário desse fungo endofítico e poderá contribuir para o entendimento da interação do complexo fungo-cana-de-açúcar, possibilitando no futuro a sua aplicação no melhoramento vegetal e exploração do seu potencial biotecnológico. / Endophytic fungi have been recognized for its great importance for the host plants, they may provide protection against herbivores and pathogens, promote plant growth, and produce secondary metabolites with biological activity, among other benefits. Sugarcane is a socially and economically important crop in Brazil, especially for the state of São Paulo. Lately, this culture has received special attention due to the growing demand for raw materials, mainly due to the increase in consumption of ethanol as a biofuel. Fungi Penicillium inhabit the tissues and rhizosphere of sugarcane, where they can establish mutualistic associations with the plant and provide several benefits. Within this context, studies evaluating the interaction of Penicillium spp. with sugar cane are very promising to generate knowledge in order to assist in the agriculture optimization. Thus, this study aimed to evaluate the biotechnological potential of endophytic Penicillium from root and rhizosphere, belonging to the fungal community of sugarcane, through tests of antagonism, enzyme production, solubilization of inorganic phosphate and indole acetic acid production, as well as studying the interaction of an isolate of P. pinophilum with sugarcane using the development of a system for genetic transformation mediated by Agrobacterium tumefaciens. Both the analysis of antimicrobial activity and the production of metabolites showed extensive physiological variation among isolates. An isolate of the species P. pinophilum (strain 44) was chosen to be used in genetic transformation for being statistically superior than the other strains in previous trials. Different parameters were evaluated to increase the efficiency of this transformation system, among them: co-culture time (24 and 48 hours), concentration of the inducer acetosyringone (200 µM and 400 µM) and types of membrane (filter paper and nylon). Agrotransformation system showed high efficiency, generating a high amount of hygromycin B resistant transformants that expressed GFP. Among the factors evaluated, the combination that showed the best results involved the transformation with a co-cultivation for 48 hours on a nylon membrane, in culture medium containing 200 µM of acetosyringone. The plant-fungus interaction was assessed from the inoculation of wild type and transformants P. pinophilum in seedlings of sugarcane followed by analysis by epifluorescence microscopy and reisolation. Results revealed the non-pathogenic nature of this fungus, since it was capable of endophytically colonize sugarcane and persisted in the roots of this plant, without developing any symptoms of illness. In addition, agrotransformation tests gave rise to a library with a thousand and one hundred insertional transformants, which is an important tool for molecular study of secondary metabolism of endophytic fungus, and may contribute to the comprehension of the complex interaction of fungus-sugarcane, allowing its future application in plant breeding and exploitation of their biotechnological potential.

Plant growth-promoting bacteria (PGPB) have been studied in many agriculturally interesting plants, but never in pitcher plants. Sarracenia oreophila (the green pitcher plant) is an endangered species in Georgia, Alabama, and North Carolina (Rice 2010). With the help of Dr. Jim Spain's lab, a previous student in Dr. Gerald Pullman's lab discovered evidence that nitrogen-fixing bacteria (Burkholderia spp.) live within these pitcher plants. This study aims to determine whether these nitrogen-fixing bacteria confer a benefit to their host plants by providing fixed nitrogen. To do this, pitcher plants were inoculated with the Burkholderia and grown on a control medium, a medium without sugar (as the sugar causes the bacteria to grow until they hinder the plants), various media that are missing nitrogen-containing compounds usually provided in growth media, and a medium completely lacking nitrogen. These plants were compared to control plants on the same media that had not been inoculated with Burkholderia. The plants' biomass and root growth were measured. The data suggest that Burkholderia may stimulate plant biomass growth when sufficient nitrogen is present and there may be a nitrogen-threshold that needs to be met in order to sustain the Burkholderia-Sarracenia symbiosis. Also, the Burkholderia has a negative effect on roots grown in high-nitrogen media, possibly due to competition for nutrients.

Natural products as sources of novel therapeutic agents experienced a steady increase from around the turn of the twentieth century until it peaked in the 1970s and 1980s. However since this time pharmaceutical research in natural products has experienced a decline. Despite this trend the natural products industry now seems to be experiencing a revival of sorts. This thesis represents a continuation of the work on the isolation and structure elucidation of potential drug leads from terrestrial fungal sources that the natural products group at the University of Canterbury is engaged in. The known compound, pseurotin A (2.7) and two novel diastereomers, pseurotin A2 (2.8) and pseurotin A3 (2.9) were isolated from the extract of a Penicillium sp. of fungus collected from the foreshore of a beach in Vancouver, Canada. The absolute stereochemistry of pseurotin A2 and proposed absolute stereochemistry for A3 were elucidated using a combination of X-ray crystallography (A2 only), circular dichrosim, oxidative cleavage reactions, and J2-resoved 2D NMR experiments. The extract of an as yet unidentified endophytic fungus has yielded eight novel compounds related to the spirobisnaphthalene class of compounds. These eight compounds fall into to distinct groupings. The spiro-mamakones, distinguished by a structurally unprecedented oxygenated spiro-nonene skeleton, comprise five compounds, spiro-mamakones A-E (3.11, 3.15-3.18). In addition to these naturally occurring compounds, the semi-synthetic compounds, 4-oxo-spiro-mamakone A (3.12) and O-acetyl-spiro-mamakone A (3.21), were also synthesised. spiro-Mamakone A was found to be racemic, while X-ray crystallography and optical rotation revealed spiro-mamakone C (3.15) to be present as an enantiomeric mixture (4S*, 5S*, 9R*). Unfortunately the enantiomeric excess was unable to be elucidated. NOE experiments revealed spiro-mamakone B (3.16) to have the relative stereochemistry 4S*, 5S*, 9S*. The relative stereochemistry of spiro-mamakones D (3.17) (4S*, 5S*, 8S*, 9S*) and E (3.18) (4S*, 5S*, 8S*, 9R*) was proposed from comparison of coupling constant calculations from energy-minimised models with those of the experimentally determined values. The second group, comprising three novel compounds named the mamakunoic acids, mamakunoic acid A-C (3.8, 3.7, 3.10), are characterised by their acid substituted dihydro benzofuran system. The low yield obtained of these compounds, unfortunately prevented their stereochemical elucidation. In addition to structure elucidation, biosynthetic studies on spiro-mamakone A and mamakunoic acid B were also carried out. Analysis of the NMR spectra derived from spiro-mamakone A, labelled with isotopic acetate, revealed a situation complicated by the presence of isotopomers and racemisation, resulting in NMR spectra that were somewhat anomalous in appearance. These irregularities however, were resolved leading to the proposal that spiro-mamakone A was derived from a dihydroxynaphthalene (DHN) intermediate, which proceeds through to spiro-mamakone via an epoxide intermediate. Despite problems with purity and low yields of isotopically labelled mamakunoic acid B, it was proposed that like spiro-mamakone A, it proceeded via a DHN intermediate. The extract derived from a Malaysian Scleroderma sp. was found to contain a new dichlorinated pulvinic acid derivative, methyl-3',5'-dichloro-4,4'-di-O-methylatromentate (4.14), the structure of which was confirmed by X-ray crystallography. In addition three previously reported compounds, 4,4'-dimethoxyvulpinic acid (4.11), methyl-3'-chloro-4,4'-di-O-methylatromentate (4.12) and methyl-4,4'-dimethoxyvulpinate (4.13), were also isolated. The extract of another, as yet unidentified endophytic fungus was found to contain the new acetogenin, 1,5-dihydroxy-6-(2-hydroxyethyl)-3-methoxyacetophenone (5.7), differing from the known compound, 2,4-dihydroxy-6-(2-hydroxyethyl)-3-methoxyacetophenone (5.8) only by virtue of the substitution pattern. The structure of 5.7 was confirmed by X-ray crystallography. The implementation of efficient dereplication procedures is paramount for those working in the field of natural products. The recent advances that have been made in the dereplication process in the natural products group at the University of Canterbury are given using examples from this research and where necessary from other group members.

Os fungos endofíticos têm sido reconhecidos pela sua grande importância para as plantas hospedeiras, pois podem conferir proteção contra insetos herbívoros e patógenos, promover o crescimento vegetal, além de produzir metabólitos secundários com atividades biológicas diversas, entre outros. A cana-de-açúcar é uma cultura de grande importância social e econômica no Brasil, especialmente para o estado de São Paulo. Ultimamente esta cultura vem recebendo especial atenção devido ao crescente aumento da demanda de matéria prima, principalmente em função do acréscimo no consumo de etanol como biocombustível. Fungos do gênero Penicillium habitam os tecidos e a rizosfera de cana-de-açúcar, onde podem estabelecer associações mutualísticas com a planta e conferir diversos benefícios. Dentro deste contexto, estudos que avaliem a interação de Penicillium spp. com cana-de-açúcar são bastante promissores para geração de conhecimentos que auxiliem na otimização da agricultura. Dessa forma, o presente trabalho teve como objetivos a avaliação do potencial biotecnológico dos endofíticos de raiz e da rizosfera, do gênero Penicillium, pertencentes à comunidade fúngica de cana-de-açúcar, por meio de ensaios de antagonismo, produção de enzimas, solubilização de fosfato inorgânico e produção de ácido indol acético; assim como o estudo da interação de um isolado de P. pinophilum com cana-de-açúcar a partir do desenvolvimento de um sistema de transformação genética mediada pela bactéria Agrobacterium tumefaciens. Tanto a análise da atividade antimicrobiana como a produção de metabólitos apresentaram extensa variação fisiológica entre os isolados avaliados. Um isolado da espécie P. pinophilum (linhagem 44) foi escolhido para ser usado na transformação genética por mostrar-se superior estatisticamente em relação aos demais isolados nos ensaios anteriores. Para aumento da eficiência deste sistema de transformação foram avaliados diferentes parâmetros, dentre eles: tempo de co-cultivo (24 e 48 horas), concentração do indutor acetoseringona (200 M e 400 M) e tipos de membrana (papel filtro e náilon). O sistema de agrotransformação apresentou alta eficiência (482 transformantes por 107 conídios), gerando uma elevada quantidade de transformantes resistentes à higromicina B e expressando GFP. Dentre os parâmetros avaliados, a combinação que deu origem aos melhores resultados de transformação envolveu o co-cultivo por 48 horas sobre membrana de náilon, em meio de cultura contendo 200 M de acetoseringona. A interação fungo-planta foi avaliada a partir da inoculação de P. pinophilum linhagem selvagem e transformantes, em plântulas de cana-de-açúcar, seguida da análise por microscopia óptica de epifluorescência e reisolamento. Os resultados revelaram a natureza não patogênica desse fungo, uma vez que ele foi capaz de colonizar endofiticamente cana-de-açúcar e persistir nas raízes desta planta, sem levar ao desenvolvimento de qualquer sintoma de doença. Além disso, os ensaios de agrotransformação deram origem a uma biblioteca com mil e cem transformantes insercionais, o que constitui uma ferramenta importante para o estudo molecular do metabolismo secundário desse fungo endofítico e poderá contribuir para o entendimento da interação do complexo fungo-cana-de-açúcar, possibilitando no futuro a sua aplicação no melhoramento vegetal e exploração do seu potencial biotecnológico. / Endophytic fungi have been recognized for its great importance for the host plants, they may provide protection against herbivores and pathogens, promote plant growth, and produce secondary metabolites with biological activity, among other benefits. Sugarcane is a socially and economically important crop in Brazil, especially for the state of São Paulo. Lately, this culture has received special attention due to the growing demand for raw materials, mainly due to the increase in consumption of ethanol as a biofuel. Fungi Penicillium inhabit the tissues and rhizosphere of sugarcane, where they can establish mutualistic associations with the plant and provide several benefits. Within this context, studies evaluating the interaction of Penicillium spp. with sugar cane are very promising to generate knowledge in order to assist in the agriculture optimization. Thus, this study aimed to evaluate the biotechnological potential of endophytic Penicillium from root and rhizosphere, belonging to the fungal community of sugarcane, through tests of antagonism, enzyme production, solubilization of inorganic phosphate and indole acetic acid production, as well as studying the interaction of an isolate of P. pinophilum with sugarcane using the development of a system for genetic transformation mediated by Agrobacterium tumefaciens. Both the analysis of antimicrobial activity and the production of metabolites showed extensive physiological variation among isolates. An isolate of the species P. pinophilum (strain 44) was chosen to be used in genetic transformation for being statistically superior than the other strains in previous trials. Different parameters were evaluated to increase the efficiency of this transformation system, among them: co-culture time (24 and 48 hours), concentration of the inducer acetosyringone (200 µM and 400 µM) and types of membrane (filter paper and nylon). Agrotransformation system showed high efficiency, generating a high amount of hygromycin B resistant transformants that expressed GFP. Among the factors evaluated, the combination that showed the best results involved the transformation with a co-cultivation for 48 hours on a nylon membrane, in culture medium containing 200 µM of acetosyringone. The plant-fungus interaction was assessed from the inoculation of wild type and transformants P. pinophilum in seedlings of sugarcane followed by analysis by epifluorescence microscopy and reisolation. Results revealed the non-pathogenic nature of this fungus, since it was capable of endophytically colonize sugarcane and persisted in the roots of this plant, without developing any symptoms of illness. In addition, agrotransformation tests gave rise to a library with a thousand and one hundred insertional transformants, which is an important tool for molecular study of secondary metabolism of endophytic fungus, and may contribute to the comprehension of the complex interaction of fungus-sugarcane, allowing its future application in plant breeding and exploitation of their biotechnological potential.