Animal pollination represents one of the key innovations of the flowering plants, and constitutes an essential ecological service in most ecosystems. While pollinators are the main drivers of flower evolution, some floral traits are puzzling when viewed only in the context of this mutualistic interaction. In particular, the pollen of plants belonging to several families has spines or compounds with toxic effects on insects. Little is known about the causes and consequences of these enigmatic floral traits. Yet, pollen defences might play an important role in pollination given that pollen is the main source of food of the principal pollinators in most ecosystems: bees. My thesis investigates why plants sometimes have seemingly defended pollen and how these putative defences affect host-plant use by bees. Given the potential role of flower-colonizing microbes in pollination, I also investigate the potential for these microorganisms to influence flower evolution. I found that pollinators are unlikely to act as potential agents of selection on the concentration of defence compounds in the pollen of Lupinus argenteus. Rather, physiological spillover or pleiotropy from tissues highly defended against herbivores might be responsible for a baseline level of defence compounds in pollen, while such compounds could also mediate the interaction between plants and pollen-colonizing microbes. However, I did not find evidence that flower-colonizing microbes drive the evolution of floral traits in an experimental study. I also found that pollen chemical and mechanical defences likely restrict pollen-host use by Osmiini, a group of solitary bees exhibiting high interspecific variability in their pollen diet. Bees tolerated the defences of their pollen hosts, but were often harmed by the pollen defences of co-occuring plants exploited by other Osmiini species. This pattern provides a striking parallel with the evolution of host-use in herbivorous insects feeding on vegetative tissues, and suggests that pollen defences might play an important role in structuring plant–bee interactions. Overall, my thesis contributes to our understanding of the causes of the presence of chemical defences in pollen and their consequences for the pollination mutualism.

Capillary electrophoresis (CE) is an analytical chemistry approach that allows for the efficient separation by charge of diverse classes of compounds for analysis, including secondary metabolites. The goal of this work was to optimize a buffer system for plant tissue analysis using micellar electrokinetic chromatography (MEKC), and by doing so to understand the role of buffer components in the performance of this form of capillary electrophoresis. In this experiment we implemented a factorial design to optimize buffer composition for separating plant tissue and secondary metabolites. The results of this experiment will be used to optimize a universal buffer for MEKC analysis that can be used on any variety of plant tissues. To determine the feasibility of this, a diverse set of plant secondary metabolite chemical standards in solution were tested as well as Helianthus annuus tissue to confirm the separation in a real biological sample. The results of this optimization yield insights into the utility of buffer components like electrolyte and pH for MEKC separation.

No description available.

The pharmacologically important alkaloids morphine and codeine are found in latex of opium poppy (Papaver somniferum). Latex is harbored in laticifers, a specialized vascular cell-type. Isolation and characterization of latex-specific genes may provide a useful tool to metabolically engineer increased alkaloid production. Previous research in the Nessler laboratory identified genes that exhibit latex-specific gene expression. Latex-specific genes were an 2-oxoglutarate-dioxygense (DIOX), involved in hydroxylation, desaturation and epoxidation reactions, and two of the major latex proteins, MLP146 and MLP149. MLP-like proteins function in fruit ripening in various species that do not have the laticifer cell type. The latex-specific promoters (LSPs) for the three genes were sequenced. The 2.5 kb DIOX promoter was fused to the reporter gene Β-glucuronidase (GUS) to characterize its expression pattern. To assess the functional sites within the DIOX promoter, deletions were made 1.5 kb and 0.14 kb upstream of the ATG start codon, fused to GUS, and transformed into opium poppy, Arabidopsis thaliana, and tobacco (Nicotiana tabacum). The 2.5 kb DIOX:GUS and 1.5 kb EcoRIDIOX:GUS reporter gene constructs showed vascular specific expression in opium poppy, Arabidopsis, and tobacco. The 0.14 kb SpeIDIOX promoter deletion construct showed no activity in opium poppy, and limited expression in the shoot apical meristem and root hypocotyl axis in Arabidopsis. These results indicate that the minimum active DIOX promoter is greater than 0.14 kb. Over 1 kb of the LSPs were sequenced and analyzed for regulatory elements using the Plant cis-acting regulatory DNA elements database, PLACE (http://www.dna.affrc.go.jp/PLACE). Knowledge of the cis-elements and regulatory regions of LSPs would serve as a tool for metabolic engineering of poppy alkaloids. Sixty-five elements were conserved among 2 of the 3 LSPs. Among the cis-elements identified, some are associated with basic functions such as: light regulation, carbon metabolism and plant defense. Other elements include: WRKY elements that are binding sites of transcription factors known for signaling plant defense genes, a vascular cis-element, and a fruit specific element. The presence of plant defense and vascular cis-elements in the LSPs, correlate with the concept that latex is a protective defense mechanism found in the vascular system. The latex-specific promoters isolated and cis-elements identified in this research are potential tools for driving increased alkaloid production in opium poppy. / Master of Science

Biofouling is the undesired biofilm formation on surfaces at a liquid interface that interferes with the affected substrate’s function. It is a ubiquitous problem in many engineered systems in industry. Biofouling causes contamination, essential damage to materials, and impedances to crucial industrial processes. These adverse effects lead to health hazards, gross increase in energy consumption, and significant decrease in overall productivity, all of which result in higher operational costs and environmentally destructive consequences. Interest in discovering effective alternatives to conventional antimicrobial agents has gained momentum. Current anti-biofouling strategies have significant disadvantages, such as the generation of toxic by-products, indiscriminate corrosion of surrounding materials and the environment, and promotion of resistance development. Alternative methods of controlling biofouling are in high demand because present-day solutions are far from sustainable. Plant secondary metabolites are promising candidates as novel biocides because they are (i) highly effective in killing microbes while being non-toxic to humans at antimicrobially active concentrations, and (ii) safer and non-damaging to the natural environment. Herein, antimicrobial efficacies of five plant-derived compounds were assessed against various species of planktonic bacteria as well as biofilms at various maturity stages. Allyl isothiocyanate (AIT) and cinnamaldehyde (CNAD) displayed the greatest inhibitory effects against all planktonic species tested. The minimum inhibitory concentration is defined as the lowest concentration of a substance that inhibits visible microbial growth, and the MBC is defined as the lowest concentration at which 99.9% of the population is killed. AIT yielded MICs of 156.25 mg/L and MBCs of 156.25 to 312.5 mg/L, and CNAD yielded MICs of 78.125 to 156.25 mg/L and MBCs of 78.125 to 312.5 mg/L. Furthermore, 312.5 mg/L AIT and 625 mg/L CNAD successfully reduced > 80% of biofilm adhesion as compared to negative controls. AIT and CNAD were therefore further evaluated extensively. Hindered by their volatile nature and immiscibility, plant secondary metabolites typically do not reach their maximum antimicrobial capacity due to low bioavailability. Thus, they would benefit from being protected and delivered in nano-sized carriers. In this study, mesoporous silica nanoparticles (MSNs) were evaluated as carriers for AIT and CNAD delivery. In one, employment of MSNs as carriers doubled the antibacterial efficacy of free form AIT and increased kill rate of free form CNAD by six times. Furthermore, free form AIT caused ~70% of 60 day-old biofilm to detach, whereas AIT-loaded MSNs essentially removed all of the biofilm. As for CNAD, its free form had no significant effect, whereas CNAD-loaded MSNs caused ~80% reduction in biofilm biomass. MSNs were further engineered to incorporate lactose pore caps to achieve specific, on-command delivery. These MSNs were designed to respond to external stimuli intelligently, with gatekeepers that degrade only in the vicinity of certain target bacteria that are able to metabolise lactose. Capped AIT-loaded MSNs reduced bacterial viability by ~85% as compared to the negative control, while capped CNAD-loaded versions reduced viability by ~40%. This stimuli-triggered MSN delivery technology would be more sustainable than current methods because resistance development would be lowered, and the delivery vehicles could be recycled and reused. Herein, the complete AIT- or CNAD-loaded, lactose-capped MSNs delivery complex proved to be an effective and environmentally conscientious system for killing unwanted bacteria.

1 Abstract: Secondary metabolism of Gram-positive soil bacteria from the genus Streptomyces is a inestimable source of natural products including manumycins, which belong to a polyketide group. These products possess weak antimicrobial, but important antiinflammatory, and antitumor activities. Streptomyces sp. offers broad amounts of yet undiscovered antibiotics, potentially utilizable in clinical medicine. This fact makes out of these organisms a promising solution to our present problem with rising antibiotic resistance among microorganisms. Two main ways are applied in this research: There are efforts of prepairing new derivates based on known products and creating various modifications in their structure. Next, new producers are discovered by "genome mining" methods, activation of silent gene clusters, followed by improvements of antibiotic production. One of those silent clusters was found in the Saccharothrix espanaensis DSM44229 strain. The genetic information has been transferred to a heterologous host in order to characterize its product. Cluster activation and production of novel manumycin-type metabolites occurred in the host after the transfer.

Manumycin antibiotics represent an important class of secondary metabolites produced by Streptomyces bacteria. They belong to a big class of polyketide metabolites and posses significant antimicrobial, anti-inflammatory, antitumor, and many other biological activities. They are characterized by two short polyketide chains, which are attached to a central subunit. Polyketide chains are synthesized by enzymes of the iterative type II polyketide-synthase. Mechanism of regulation of the polyketide chains length has not been known yet. Understanding mechanism can lead to biosynthesis of novel manumycin antibiotics with predetermined chain lengths what may improve their biological activities in favour of a practical use of these compounds. We prepared a mutant strain of asukamycin producer Streptomyces nodosus ssp. asukaensis with deletion of genes coding for type I/II β-ketoacylsynthase and protein AsuC14, which is a potential factor affecting lower polyketide chain length, for the identification of the chain length factor in manumycin antibiotics producers. Next, the genes for type I/II β-ketoacylsynthase and potential chain length-affecting factor C14 from strains producing manumycins with variable length of the lower polyketide chains were expressed in this mutant strain. Our results demonstrate...

Bacteria of the genus Xenorhabdus live as the natural symbionts of the entomopathogenic nematodes of the family Steinernematidae. They produce a wide range of toxic secondary metabolites of different chemical structure and effect. The synthesis of particular products is specific for each strain of Xenorhabdus. The thesis is focused on evaluating the impact of bacterial metabolites on facultatively entomoparasitic nematodes Oscheius myriophila, the free-living nematode Caenorhabditis elegans and the phytophagous potato cyst nematode Globodera rostochiensis. Target species of nematodes were exposed to 37 strains of Xenorhabdus bacteria, isolated from various species of Steinernematidae. Testing the impact of bacterial metabolites on O. myriophila and C. elegans nematodes was performed both by direct cultivation of target species on solid medium with live bacterial cultures as well as in sterilized liquid bacteria cultures. The effect of toxic substances on G. rostochiensis was evaluated only in selected sterilized bacteria cultures.

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.

As espécies patogênicas do gênero Colletotrichum apresentam importância mundial, pois causam danos a várias culturas de interesse agronômico. Diversas medidas de controle são empregadas, mas estas nem sempre são eficazes devido à ocorrência de linhagens resistentes. Desta forma, se faz necessário a busca por novos compostos que possam ser utilizados no manejo integrado desta doença. Os produtos naturais isolados de micro-organismos podem ser uma alternativa para o desenvolvimento de novos defensivos agrícolas. Dentre os micro-organismos, as actinobactérias são conhecidas pela produção de inúmeros compostos antimicrobianos. Neste contexto, o presente estudo teve como objetivo o isolamento e a identificação de compostos antifúngicos produzidos por actinobactérias da rizosfera de guaranazeiros. Para isto, a seleção de actinobactérias foi baseada em dois ensaios. No primeiro, as 65 actinobactérias foram avaliadas em ensaio de cultivo pareado frente ao fitopatógeno Colletotrichum gloeosporioides. Destas, os isolados mais promissores foram AM1 (43,78 % de inibição do crescimento micelial), AM3 (43,98 %), AM18 (37,86 %), AM25 (43,17 %), AM30 (47,12 %), AM61 (40,12 %) e AM68 (47,94 %). No segundo ensaio, estes isolados foram cultivados em meio BD e, após o cultivo, o meio metabólico foi submetido a três métodos de extração: (a) partição líquido-líquido com n-butanol; (b) partição líquido-líquido com acetato de etila e (c) coluna sílica gel C18. As frações obtidas a partir das três metodologias foram avaliadas pelo método de difusão em disco de papel contra C. gloeosporioides. Neste ensaio de difusão em disco foram selecionadas as linhagens AM1(n-butanol), AM3 (acetato de etila) e AM25 (C18) para o estudo de bioprospecção. Estas foram identificadas por técnicas moleculares como pertencentes ao gênero Streptomyces. A partir do extrato bruto da Streptomyces sp. AM1 foi isolado um composto análogo do ácido proclavamínico, o qual apresentou atividade mínima inibitória (MIC) de 1,25 mg mL-1 contra o fitopatógeno C. gloeosporioides. Da linhagem Streptomyces sp. AM3 foi isolado o composto streptimidona que apresentou MIC de 1,25 mg mL-1. Já no estudo de Streptomyces sp. AM25 um composto não identificado apresentou MIC de 2,50 mg mL-1. Estes três compostos apresentaram atividade superior aos fungicidas Captan SC® (Captana) e Dithane NT® (Mancozeb), e inferior ao Score® (Difenoconazol). A atividade antifúngica destes compostos ao C. gloeosporioides está sendo relatada pela primeira vez. / The pathogenic species of the genus Colletotrichum present importance worldwide because they cause damage to numerous crops of agronomic interest. Several control methods are employed, but they are not always effective due to the occurrence of resistant strains. Thus, it is necessary searching for new compounds that can be used in the integrated management of this disease. Natural products isolated from microorganisms can be an alternative for the development of new agricultural pesticides. Among microorganisms, actinobacteria are known to produce numerous antimicrobial compounds. In this context, the present study aimed to isolate and identify antifungal compounds produced by actinobacteria from guarana rhizosphere. For this, the selection of actinobacteria was based on two tests. In the first one, the 65 actinobacteria were evaluated in paired cultivation test against the plant pathogen Colletotrichum gloeosporioides. Among them, the most promising isolates were AM1 (43.78% inhibition), AM3 (43.98%), AM18 (37.86%), AM25 (43.17%), AM30 (47.12%), AM61 (40.12%) and AM68 (47.94%). In the second assay, these isolates were cultured in BD medium and, after culturing, the metabolic medium was subjected to three extraction methods: (a) liquid-liquid partition with n-butanol; (B) liquid-liquid partition with ethyl acetate and (c) silica gel column C18. The fractions obtained from the three methodologies were evaluated by paper disc diffusion method against C. gloeosporioides. In this disk diffusion assay, the strains AM1 (n-butanol), AM3 (ethyl acetate) and AM25 (C18) were selected for the bioprospecting study. These were identified by molecular techniques as belonging to the genus Streptomyces. From the crude extract of Streptomyces sp. AM1 the analogous compound proclavaminic acid was isolated, which presented minimal inhibitory activity (MIC) of 1.25 mg mL -1 against the plant pathogen C. gloeosporioides. From Streptomyces sp. AM3, the compound streptimidone was isolated, which presented MIC of 1.25 mg mL-1. In the study of Streptomyces sp. AM25 an unidentified compound had MIC of 2.50 mg mL-1. These three compounds presented superior activity to the fungicides Captan SC® (Captan) and Dithane NT® (Mancozeb), and inferior to the Score® (Difenoconazole). The antifungal activity of these compounds to C. gloeosporioides is being reported here for the first time.