Endophytic fungi inhabit the living tissue of a host plant for at least a portion of their life cycle. While some researchers have shown that various endophytic fungi participate in litter decomposition, we do not know whether such fungi are actually saprotrophic, meaning that they can obtain energy from litter. Therefore, I determined if endophytic fungi are saprotrophs using leaf litter as the energy source. All 49 tested isolates were found to be saprotrophic. To compare the saprotrophic capacities of fungi from different habitats, which produce different types of litter, a universal litter proxy needs to be used. I hypothesized that pure cellulose would be an adequate proxy for litter for in vitro studies because of its abundance in litter. This was tested in the first study. Saprotrophic capacity on pure cellulose was not highly correlated with that on leaf litter. I conclude, therefore, that cellulose may not be a good proxy for leaf litter. Some endophytic fungi are biotrophs, presumably acquiring energy from photosynthate produced by the host plant. This suggests that the level of exposure to sunlight by the plant should influence the competitive ability of such fungi. If saprotrophic endophytic fungi do exist, they ought to be less competitive against biotrophic endophytic fungi in leaves receiving full sunlight than in shaded leaves. I, therefore, hypothesized that the frequency of saprotrophy will be influenced by the level of sun exposure of the leaf from which the fungi were isolated. This was tested in the second study. Moreover, because closely related organisms ought to be more similar to each other than more distantly related organisms, I also hypothesized that saprotrophic capacity has a strong phylogenetic component, which was also tested in the second study. Unexpectedly, isolate identity within genus accounted for far more variability in saprotrophic capacity than genus identity, and sun exposure did not have a significant effect on saprotrophy. These results suggest that saprotrophic capacity may not be highly consequential in the ecology of these organisms.

Orientador: Lourdes Campaner dos Santos / Resumo: Os microrganismos são considerados uma fonte promissora de substâncias com potencial biológico e de alta capacidade biossintética para produção de novas substâncias com aplicação em diversas áreas tais como agricultura, medicina e alimentos. Os fungos endofíticos colonizam o interior das plantas e tem demonstrado grande importânica na produção de metabólitos secundários bioativos. Dessa forma, este trabalho foi idealizado objetivando a bioprospecção dos extratos produzidos por fungos endofíticos associados à Paepalanthus planifolius, espécie vegetal do Cerrado, e a avaliação epigenética em Aspergillus terreus, isolado de espécie vegetal do Deserto de Sonora, na obtenção de novas substâncias. Os fungos endofíticos isolados de P. planifolius (15 linhagens) foram cultivados em escala reduzida em meio líquido de batata e dextrose (PDB), a 25 oC, sob modo estático para obtenção dos respectivos extratos brutos em AcOEt. A avaliação das atividades antimicrobiana e citotóxica foram realizadas, sendo que a bioprospecção inicial conduziu a seleção de dois fungos endofíticos identificados como Paraphaeosphaeria sporulosa e Cochliobolus eragrostidis. Estes, foram cultivados em diferentes meios líquidos e sólidos (escala reduzida) para novamente avaliar a atividade biológica dos extratos e selecionar os mais promissores para isolamento e determinação/elucidação estrutural dos metabólitos secundários em escala ampliada. Do cultivo de P. sporulosa no meio sólido de arroz foram isoladas seis... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Microorganisms are considered a promising source of substances with biological potential and high biosynthetic capacity to produce new substances with application in various areas such as agriculture, medicine and food. Endophytic fungi colonize the interior of plants and have shown great importance in the production of bioactive secondary metabolites. Thus, this work was conceived aiming the bioprospection of extracts produced from endophytic fungi associated with Paepalanthus planifolius, a Cerrado plant species, and the epigenetic evaluation in Aspergillus terreus, isolated from Sonora Desert plant species, to obtain new substances. The isolated endophytic fungi of P. planifolius (15 strains) were cultivated in small scale in potato and dextrose liquid medium (PDB) at 25 oC, under static mode to obtain the respective EtOAc crude extracts. Antimicrobial and cytotoxic activities were evaluated and the initial bioprospection led to the selection of two endophytic fungi identified as Paraphaeosphaeria sporulosa and Cochliobolus eragrostidis. These were cultivated in different liquid and solid media (reduced scale) to again evaluate the biological activity of the extracts and select the most promising ones for isolation and structural determination/elucidation of the secondary metabolites on larger scale. From the cultivation of P. sporulosa in the solid medium of rice, six new substances, sporulosaldeins A-F (1-6) were isolated, with good antifungal activity (MIC <100 µg/mL) a... (Complete abstract click electronic access below) / Doutor

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Strains MS455 and MS389, endophytic bacteria, were isolated from healthy soybean plant growing adjacent to a patch of plants affected by charcoal rot disease, caused by the fungal pathogen Macrophomina phaseolina. The complete genomes of both strains were sequenced and identified as Burkholderia species Strain MS455 exhibits broad-spectrum antifungal activities against economically important pathogens, including Aspergillus flavus. Random and site-specific mutations were employed in discovery of the genes that share high homology to the ocf gene cluster of Burkholderia contaminans strain MS14, which is responsible for production of the antifungal compound occidiofungin. RNA-seq analysis demonstrated ORF1, a homolog to the ambR1 LuxR-type regulatory gene, not only regulates occidiofungin biosynthesis in MS455, but also involved in expression of multiple genes, especially those involved in ornibactin biosynthesis. Plate and corn kernel assays showed that growth of A. flavus and aflatoxin production were reduced significantly by MS455 as compared with buffer control and the ORF1 mutant. Strain MS389 shows significant antifungal and antibacterial activities as well. Mutagenesis study identified that the TatC gene, an important unit of twin-arginine translocase (Tat) secretion system, and the LysR-type transcriptional regulatory gene were essential for the antifungal activity of strain MS389. RNA-seq analysis implied that the pyrrolnitrin biosynthesis gene cluster and an uncharacterized NRPS / PKS gene cluster were involved in antifungal activity. By comparing several endophytic bacteria of Burkholderia, including MS455 and MS389, to pathogenic Burkholderia species, endophytic bacteria were observed to harbor multiple antimicrobial biosynthesis genes but lack certain pathogenic or virulence genes. The potential endophytic behavior related genes and characteristics related to antibiotic resistance, secretion system, and CRISPR-Cas profiles were well established. The research findings on strains MS455 and MS389 have provided important genetic clues for understanding their molecular mechanism of antimicrobial activities and exhibited their potential possibility as biocontrol agents.

Climate change is expected to increase the frequency of mild winters and warm springs, which can induce premature plant development. This premature development results in a high risk of exposure of young plant tissues to cold stress leading to severe reductions in plant growth and agricultural production. Plants are associated with complex bacterial communities that can activate acclimation processes and positively affect plant performance at low temperatures. Beneficial effects of plant colonization by cold-tolerant bacteria include the modulation of cold-related genes and the reduction in cellular damage under cold stress, but scarce information is available on mechanisms stimulated by bacterial endophytes in tomato plants against cold stress. The aims of this work were i) to analyze the taxonomy and potential functions of plant-associated microbial communities in cold regions, ii) to understand metabolic changes stimulated by cold-tolerant endophytic bacteria in tomato plants exposed to cold stress, and iii) to identify possible genomic traits of cold-tolerant endophytic bacteria responsible for plant growth promotion and cold stress mitigation. The first chapter includes an introduction on cold stress and acclimation processes in plants, and the second chapter defines the aims of the project. In the third chapter, the taxonomic and functional characterization of plant-associated microbial communities of alpine, Arctic, and Antarctic regions was reviewed, highlighting the main environmental factors affecting their taxonomic structure. e. The key findings of this chapter are the functional roles of microbial communities in plant growth and survival in cold environments, and the suggestion of potential biotechnological applications of ubiquitous and endemic cold-tolerant microorganisms. In the fourth chapter, metabolic changes stimulated by cold-tolerant endophytic bacteria in tomato plants exposed to cold stress were studied by metabolomic analyses, and compounds possibly associated with cold stress mitigation were found. 14 Tomato seeds were inoculated with two bacterial endophytes isolated from Antarctic Colobanthus quitensis plants (Ewingella sp. S1.OA.A_B6 and Pseudomonas sp. S2.OTC.A_B10) or with Paraburkholderia phytofirmans PsJN, while mock-inoculated seeds were used as control. The metabolic composition of tomato plants was analyzed immediately after cold stress exposure (4°C for seven days) or after two and four days of recovery at 25°C. Under cold stress, the content of malondialdehyde, phenylalanine, ferulic acid, and p-coumaric acid was lower in bacterium-inoculated compared to mock-inoculated plants, indicating a reduction of lipid peroxidation and the stimulation of phenolic compound metabolism. The content of two phenolic compounds, five putative phenylalanine-derived dipeptides, and three further phenylalanine-derived compounds was higher in bacterium-inoculated compared to mock-inoculated samples under cold stress. Thus, the presented work suggests that psychrotolerant endophytic bacteria can reprogram polyphenol metabolism and stimulate the accumulation of secondary metabolites, like 4-hydroxybenzoic and salicylic acid, which are involved in cold stress mitigation, and phenylalanine-derived dipeptides possibly involved in plant stress responses. In the fifth chapter, functional and genomic traits of Ewingella sp. S1.OA.A_B6 and Pseudomonas sp. S2.OTC.A_B10 were studied. In the framework of the present study, Ewingella sp., Pseudomonas sp., and the bacterial consortium showed plant growth-promoting activity on tomato seedlings at low temperatures. Ammonia was produced by both bacterial isolates and their consortium, while indole-3-acetic acid and proteases were produced by Ewingella sp. and Pseudomonas sp., respectively. Ewingella sp. and Pseudomonas sp. genomes (51.57% and 60.63% guanine-cytosine, 4,148 and 5,983 predicted genes, respectively) encompassed genes related to amino acid metabolism, plant hormone metabolism (auxin, cytokinins, ethylene, and salicylic acid), nitrogen metabolism, lytic activities (amylases, cellulases, and proteases). Traits related to plant growth promotion included genes for iron transport, phosphate metabolism, potassium transport, siderophore metabolism and 15 transport, and zinc transport. Moreover, Ewingella sp. and Pseudomonas sp. encompassed genes related to cold tolerance, such as cold shock and heat shock-related proteins, lipid desaturases, and genes related to polyamine metabolism, proline metabolism, proline and glycine betaine transport, reactive oxygen species detoxification, and trehalose metabolism. Thus, in this chapter, it was discovered that Antarctic cold-tolerant endophytes include multiple genomic and functional traits to survive under cold conditions and some of them can contribute to promote the host plant growth at low temperatures. These findings indicate that plant-associated bacteria of cold regions have a great biotechnological potential to mitigate cold stress in crop plants. In particular, Antarctic bacterial endophytes encompass genomic traits responsible for plant growth promotion and protection against cold stress, and they can mitigate cold stress in tomato plants by a complex reprogramming of plant metabolism. Although further metabolomic and functional studies are required to verify compound annotations and to better clarify the role of phenylalanine-derived compounds and phenylalanine-derived dipeptides in cold stress mitigation, these results provided a better understanding of metabolic changes stimulated by psychrotolerant endophytic bacteria in cold-stressed tomato plants. Thus, the validation of cold stress mitigation activated by psychrotolerant endophytic bacteria under field conditions will pave the way for the further development of endophytic bacterial inoculants as sustainable products to protect crops against cold stress.

Eucalyptus benthamii tem se mostrado especialmente vantajoso como alternativa ao cultivo em regiões frias, justificando esforços para o estabelecimento de protocolos para sua micropropagação. Porém, as matrizes são preferencialmente selecionadas quando adultas (material apresenta menor competência morfogênica), tornando a micropropagação dependente de maior número de subcultivos e maior tempo para se reverter o material ao rejuvenescimento. Assim, a redução das perdas in vitro tem merecido atenção, como por exemplo, as manifestações endofíticas, que exigem maximização da eficiência da cultura e adequações no protocolo, visando minimizá-las, possibilitando melhorar o entendimento das relações estabelecidas e mantidas entre os endófitos e seu hospedeiro durante a micropropagação. Dessa maneira, foram utilizadas minicepas provenientes de duas fontes de miniestacas coletadas a partir do brotamento de gemas epicórmicas de megaestacas da base da copa e de brotamentos do anelamento da base do tronco, de uma matriz de E. benthamii com 13 anos de idade, estabelecidas em minijardim clonal sob condição de casa de vegetação, com o objetivo de avaliar como se dá a multiplicação, sob diferentes condições de cultivo, das duas fontes de explantes (minicepas); analisar se a frequência e intensidade das manifestações endofíticas são afetadas pelas diferentes condições de cultivo; investigar a ocorrência de alterações na comunidade bacteriana endofítica devido à alteração das condições de cultivo e fase da micropropagação (in vivo = minicepas, e in vitro = microcepas, material alongado e enraizado). Visando atender estes objetivos, a pesquisa se dividiu em duas partes. Na primeira (capitulo 3) o desenvolvimento, os aspectos morfofisiológicos, histoquímicos e a manifestação endofítica foram avaliados na multiplicação das duas fontes de explante sob diferentes meios e condições de cultivo. Na segunda (capítulo 4) as comunidades bacterianas endofíticas foram analisadas por meio de PCR-DGGE, baseada na região V6 do gene 16S DNAr. Os resultados mostraram que as microcepas provenientes de megaestaca tiveram melhor desenvolvimento independentemente do tratamento e maior frequência de manifestações endofíticas, comparando-se com as de anelamento. As comunidades bacterinas endofíticas foram distintas entre as amostras in vivo e in vitro, e se alteraram ao longo dos subcultivos e nas amostras alongadas e enraizadas. As diferenças existentes no desenvolvimento das microcepas podem ser inerentes à totipotencialidade do material, mas também podem ser afetadas, tanto pela ocorrência de manifestação, quanto pela comunidade bacteriana endofítica mais ou menos sensível ao processo de micropropagação, auxiliando ou prejudicando o desenvolvimento in vitro de seus hospedeiros. Cabe destacar, ainda, que mesmo em um sistema asséptico e ambientalmente controlado, os microrganismos endofíticos que resistiram a todo processo de desinfestação e cultivo, não estão \"adormecidos\", muito pelo contrário podem se alterar em quantidade à medida que seu hospedeiro é submetido a um novo sistema de cultivo (introdução) ou uma nova fase dentro da micropropagação (multiplicação &rarr; alongamento e enraizamnento) ou, ainda, ao longo dos subcultivos. Sendo assim, a complexa rede de relações das plantas com seus endófitos não cessa durante o cultivo in vitro, ao contrário mantém-se dinâmica. / Eucalyptus benthamii has proven to be especially advantageous as an alternative culture in cold regions, justifying efforts to establish protocols for micropropagation. However, the matrices are preferably selected when adults (material with lower morphogenic efficiency), making micropropagation more dependent of subcultures and too longer to reverse the material to rejuvenation. Thus, reduction of losses in vitro has deserved attention, such for example the endophytic manifestations that require the maximization of efficiency culture and adjustments to the Protocol, in order to minimize them, enabling better understanding of the relations established and maintained between endophytes and its host along micropropagation. For this, mini-stumps were used from two sources of mini-cuttings collected from the epicormic shoots of mega-cuttings from the treetop base and shoots from girdling from the trunk base, both of one E. benthamii matrix with 13 years of age established in clonal mini garden under greenhouse condition, aimed to evaluate how is the multiplication of two sources of explants (mini-stumps) under different growing conditions; analyze how the endophytic manifestations frequency and intensity are affected by different conditions; investigate the changes to occurrence in the endophytic bacterial community due to the variation of culture conditions and micropropagation phase (in vivo = mini-stumps, and in vitro = micro-stumps, elongated and rooted materials). In order to meet these objectives, the research was divided in two parts. In the first (chapter 3) the development, morphophysiological aspects, histochemical and endophytic manifestation were evaluated in the multiplication of the two explants sources from different media and culture conditions. In the second (chapter 4) endophytic bacterial communities were analyzed by PCR-DGGE based on the V6 region of 16S rDNA gene. The results showed that micro-stumps from mega-cuttings had better development regardless of treatment and increased frequency of endophytic events, comparing with the girdling. Endophytic bacterial communities were different between samples in vivo and in vitro, and have changed over the subcultures and the elongated and rooted samples. The differences in the development of micro-stumps can be explained by the totipotentiality inherent to the material, but may also be affected by both the manifestation occurrence and the endophytic bacterial community more or less sensitive to the micropropagation, helping or harming the in vitro development of their hosts. We also highlight that even in an aseptic and environmentally controlled system, the endophytic microorganisms that resisted the whole process of disinfection and cultivation, are not \"asleep\", quite the opposite may change in quantity when your host is subjected to a new cultivation system (in vitro establishment) and a new phase within the micropropagation (multiplication &rarr; stretching and enraizamnento), or even along the subcultures. This way, the complex network of relationships of the plant with their endophyte does not cease during the in vitro culture, unlike remains dynamic.

Eucalyptus benthamii tem se mostrado especialmente vantajoso como alternativa ao cultivo em regiões frias, justificando esforços para o estabelecimento de protocolos para sua micropropagação. Porém, as matrizes são preferencialmente selecionadas quando adultas (material apresenta menor competência morfogênica), tornando a micropropagação dependente de maior número de subcultivos e maior tempo para se reverter o material ao rejuvenescimento. Assim, a redução das perdas in vitro tem merecido atenção, como por exemplo, as manifestações endofíticas, que exigem maximização da eficiência da cultura e adequações no protocolo, visando minimizá-las, possibilitando melhorar o entendimento das relações estabelecidas e mantidas entre os endófitos e seu hospedeiro durante a micropropagação. Dessa maneira, foram utilizadas minicepas provenientes de duas fontes de miniestacas coletadas a partir do brotamento de gemas epicórmicas de megaestacas da base da copa e de brotamentos do anelamento da base do tronco, de uma matriz de E. benthamii com 13 anos de idade, estabelecidas em minijardim clonal sob condição de casa de vegetação, com o objetivo de avaliar como se dá a multiplicação, sob diferentes condições de cultivo, das duas fontes de explantes (minicepas); analisar se a frequência e intensidade das manifestações endofíticas são afetadas pelas diferentes condições de cultivo; investigar a ocorrência de alterações na comunidade bacteriana endofítica devido à alteração das condições de cultivo e fase da micropropagação (in vivo = minicepas, e in vitro = microcepas, material alongado e enraizado). Visando atender estes objetivos, a pesquisa se dividiu em duas partes. Na primeira (capitulo 3) o desenvolvimento, os aspectos morfofisiológicos, histoquímicos e a manifestação endofítica foram avaliados na multiplicação das duas fontes de explante sob diferentes meios e condições de cultivo. Na segunda (capítulo 4) as comunidades bacterianas endofíticas foram analisadas por meio de PCR-DGGE, baseada na região V6 do gene 16S DNAr. Os resultados mostraram que as microcepas provenientes de megaestaca tiveram melhor desenvolvimento independentemente do tratamento e maior frequência de manifestações endofíticas, comparando-se com as de anelamento. As comunidades bacterinas endofíticas foram distintas entre as amostras in vivo e in vitro, e se alteraram ao longo dos subcultivos e nas amostras alongadas e enraizadas. As diferenças existentes no desenvolvimento das microcepas podem ser inerentes à totipotencialidade do material, mas também podem ser afetadas, tanto pela ocorrência de manifestação, quanto pela comunidade bacteriana endofítica mais ou menos sensível ao processo de micropropagação, auxiliando ou prejudicando o desenvolvimento in vitro de seus hospedeiros. Cabe destacar, ainda, que mesmo em um sistema asséptico e ambientalmente controlado, os microrganismos endofíticos que resistiram a todo processo de desinfestação e cultivo, não estão \"adormecidos\", muito pelo contrário podem se alterar em quantidade à medida que seu hospedeiro é submetido a um novo sistema de cultivo (introdução) ou uma nova fase dentro da micropropagação (multiplicação &rarr; alongamento e enraizamnento) ou, ainda, ao longo dos subcultivos. Sendo assim, a complexa rede de relações das plantas com seus endófitos não cessa durante o cultivo in vitro, ao contrário mantém-se dinâmica. / Eucalyptus benthamii has proven to be especially advantageous as an alternative culture in cold regions, justifying efforts to establish protocols for micropropagation. However, the matrices are preferably selected when adults (material with lower morphogenic efficiency), making micropropagation more dependent of subcultures and too longer to reverse the material to rejuvenation. Thus, reduction of losses in vitro has deserved attention, such for example the endophytic manifestations that require the maximization of efficiency culture and adjustments to the Protocol, in order to minimize them, enabling better understanding of the relations established and maintained between endophytes and its host along micropropagation. For this, mini-stumps were used from two sources of mini-cuttings collected from the epicormic shoots of mega-cuttings from the treetop base and shoots from girdling from the trunk base, both of one E. benthamii matrix with 13 years of age established in clonal mini garden under greenhouse condition, aimed to evaluate how is the multiplication of two sources of explants (mini-stumps) under different growing conditions; analyze how the endophytic manifestations frequency and intensity are affected by different conditions; investigate the changes to occurrence in the endophytic bacterial community due to the variation of culture conditions and micropropagation phase (in vivo = mini-stumps, and in vitro = micro-stumps, elongated and rooted materials). In order to meet these objectives, the research was divided in two parts. In the first (chapter 3) the development, morphophysiological aspects, histochemical and endophytic manifestation were evaluated in the multiplication of the two explants sources from different media and culture conditions. In the second (chapter 4) endophytic bacterial communities were analyzed by PCR-DGGE based on the V6 region of 16S rDNA gene. The results showed that micro-stumps from mega-cuttings had better development regardless of treatment and increased frequency of endophytic events, comparing with the girdling. Endophytic bacterial communities were different between samples in vivo and in vitro, and have changed over the subcultures and the elongated and rooted samples. The differences in the development of micro-stumps can be explained by the totipotentiality inherent to the material, but may also be affected by both the manifestation occurrence and the endophytic bacterial community more or less sensitive to the micropropagation, helping or harming the in vitro development of their hosts. We also highlight that even in an aseptic and environmentally controlled system, the endophytic microorganisms that resisted the whole process of disinfection and cultivation, are not \"asleep\", quite the opposite may change in quantity when your host is subjected to a new cultivation system (in vitro establishment) and a new phase within the micropropagation (multiplication &rarr; stretching and enraizamnento), or even along the subcultures. This way, the complex network of relationships of the plant with their endophyte does not cease during the in vitro culture, unlike remains dynamic.

Although the molecular mechanisms underlying the onset and progression of cancer has been unraveled to a great extend, cancer continues to remain a leading cause of death around the world. Clinical efficacy of the existing anticancer drugs are largely compromised by the inherent and acquired resistance of cancer cell types and the severe side effects evoked by chemotherapeutic agents. Hence, the search for novel anticancer drugs with minimum side effects remains an active area of cancer research. Although molecular targeted drugs are preferred over the conventional cytotoxic chemotherapy, the screening of natural compounds with cytotoxic potentialities continues as they can serve as lead structures for the development of tumor selective anticancer drugs. Plants and microorganisms have been the prominent sources of therapeutic agents. Microorganisms being readily renewable, inexhaustible sources of diverse bioactive secondary metabolites are preferred over plants as sources of bioactive compounds. Endophytes are microorganisms that reside within the living tissue of host plant and they enhance the survival value of the host plant by mediating various stress tolerance mechanisms. Endophytic fungi have gained attention as potential sources of bioactive secondary metabolites following the discovery of a taxol producing endophytic fungus Taxomyces adrenae, from Taxus brevifolia. Moreover, endophytes occupy a unique biological niche in which they maintain a balanced interaction with the host organism and other co-inhabiting microorganisms. All these factors contribute to the chemical diversity of the metabolites they produce. Plants restricted to extreme or unique habitats or those with ethnobotanical value are likely to lodge endophytes that possess a unique hoard of secondarymetabolites. Saraca asoca is a traditionalmedicinal plant with its occurrence restricted to countries such as India, Sri Lanka, Burma and Malaysia. The purpose of the present study is to explore the endophytic fungal population associated with S. asoca in search of novel anticancer lead structures. S. asoca was found to house a diverse endophytic fungal population belonging to 37 different species. Identification of the fungal isolates was based on ITS (internal transcribed spacer region) sequence analysis as well as colony and spore characteristics. The organic extracts of all fungal species were assessed for their in vitro cytotoxicities in three human cancer cell lines, HeLa, HepG2 and PC3 byMTT assay. 18 species exhibited remarkable cytotoxic activities, among which Pestalotiopsis sp. 6 exhibited themost significant cytotoxicity. The strain with second highest activity was Lasiodiplodia theobromae. In order to identify the active principle present in the organic extracts of Pestalotiopsis sp. 6 and L. theobromae, the organic extracts were chromatographed on TLC plates and individual compounds were recovered by scraping off from the TLC plates and extracting with methanol. The cytotoxicity assay of the TLC purified compounds suggested the cytotoxic activity of Pestalotiopsis sp.6 to be a synergetic effect of two or more compounds whereas the cytotoxicity of L. theobromae organic extract was largely due to a single compound. Hence the active principle present in L. theobromae organic extract was purified by bioassay - guided column chromatography. Repeated chromatography of the crude extract using three silica gel columns resulted in the isolation of anticancer compound. Based on the analysis of ESI-MS, IR, NMR and UV spectral data, the isolated compound was identified as a novel steroidal saponin, cholestan-3-O-¯-Dglucopyranoside (cholestanol glucoside - CG). The in vitro cytotoxic effects of CG towards seven human cancer cell lines, HeLa, HepG2, PC3, U251,MCF 7, OVCAR3 and A549 were examined. Among the cell lines screened, HeLa cells weremost vulnerable to CG treatment, with an IC50 value of 3.2 ¹M. Hence themode of cell death induction in HeLa cells by CG was further investigated. Analysis of cell cycle progression by propidium iodide (PI) staining revealed that CG arrests the cells in S phase of cell cycle prior to the induction of cell death. The morphological and biochemical features of apoptosis were investigated by nuclear staining, DNA fragmentation assay and Annexin V-FITC/ PI dual staining. All these results suggested that CG effectively induced apoptosis in HeLa cells in a concentration dependent manner. It was also found that CG treatment induced remarkable ROS generation and mitochondrial membrane potential loss. The pretreatment of cells with an antioxidant, N-acetyl cysteine (NAC), blocked CG induced ROS generation, mitochondrialmembrane depolarization and apoptotic cell death. Hence it could be concluded that CG kills the cancer cells by augmenting their basal oxidative stress and hence is less likely to be toxic to normal cells. Moreover, a high Bax to Bcl-2 ratio, high levels of Apaf-1 and p53, activation of procaspase-3 and procaspase-9 and cleavage of PARP were observed in CG treated HeLa cells. Taken together, our results suggested that CG induced apoptosis in HeLa cells via ROS mediated mitochondria dependent pathway. Biosynthesis of secondarymetabolites by filamentous fungi is influenced by the availability of nutrient factors. Therefore, it is essential to optimize the culturemedium components to ensure a maximum and consistent yield of desired metabolite by the fungal isolate. We designed a chemically defined production medium for CG production by L. theobromae. Carbon source, nitrogen source and microelements in the production medium were further optimized in stationary flask cultures to improve the mycelial growth and yield of CG by L. theobromae. The conventional one-factor at a time (OFAT)method was employed for the optimization of carbon and nitrogen sourceswhose contribution effects towards the final yield are large. Response surface methodology (RSM) was employed for the optimization of microelements. Optimization of culturemedium enhanced the yield of CG from 10mg L¡1 to 50mg L¡1. Various secondarymetabolites are produced by organisms in response to different stress conditions. This knowledge has been exploited in plant cell culture systems to increase the yield of particular secondary metabolites by artificial implementation of stress conditions. We investigated the effect of oxidative, osmotic and heat shock stresses on the production of CG by L. theobromae. Heat shock and osmotic stresses in liquid cultures were found to enhance the yield of CG by 1.2-fold, relative to the controls. Oxidative stress by both menadione and H2O2 enhanced the yield by 1.8-fold compared to the controls. Thus oxidative stress proved to be an efficient enhancer of CG production by L. theobromae. These findings ensure a large scale, cost-effective production of CG.

Thesis (MScAgric.)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Phomopsis cane and leaf spot disease of grapevine is an economically important disease in many of the vine-growing areas of the world. Four different Phomopsis spp. have previously been associated with this disease. The present study investigates the taxonomic significance of the different taxa found on grapevines in South Africa, as well as the endophytic growth and fungicide sensitivity of Phomopsis viticola isolates. The thesis is compiled of several different parts, which deal with specific, but related topics, and hence some duplication has been unavoidable. Understanding the epidemiology of a disease is important for the correct timing of disease control. To investigate the endophytic growth of P. viticola, asymptomatic shoots were collected at eight different growth stages. Nodes, internodes, leaf petioles, leaves, tendrils and bunch peduncles were investigated. Two Phomopsis spp., taxon 1 and 2 were identified in this study. The Phomopsis viticola-complex had a relative importance of 9% and accounted for 3% of the isolations. P. viticola (taxon 2) is mainly isolated from the nodes and internodes. Inoculations of healthy, young vine tissue confirmed taxon 2 to be a virulent pathogen, suggesting that it is a latent pathogen rather than an endophyte. In contrast, taxon 1 appeared to be a true endophyte, and did not seem to be an important pathogen on vines. The true identity of the causal organism of Phomopsis cane and leaf spot disease was investigated by collecting samples from 58 different vineyards in the grapevine growing areas of the Western Cape. P. viiicola occurred in grapevine material collected from Lutzville to Swellendam, but was not found in the Oudtshoorn and Orange River grapevine areas. Diaporthe perjuncta (taxon 1), P. vutcola (taxon 2), taxon 3 and a Phomopsis species commonly associated with shoot blight of peaches in the U.S.A., P. amygdali, were identified among the South African grapevine isolates. Examination of the Australian culture designated as taxon 4 found it to be a species of Libertella, thus excluding it from the P. viticola-complex. An Italian isolate was found to represent a species of Phomopsis not previously known from grapevines, and this was subsequently described as taxon 5. Species delimitation was based on morphological and cultural characteristics, stem inoculations and the formation of the teleomorph in vitro. The identity of each morphological taxon was confirmed by means of phylogenetic analyses of the nuclear ribosomal DNA internal transcribed spacers (ITS 1 and ITS2) and the 5' end partial sequence of the mitochondrial small subunit (mtSSU). P. amygdali, associated with peach shoot blight in the U.S.A., was isolated once only and appeared to be of lesser importance in this disease complex. Furthermore, taxa 1 (Diaporthe perjuncta) and 3 were also rarely encountered and proved to be non-pathogenic, indicating their non-functional role in Phomopsis cane and leaf spot disease. Taxon 2 (Phomopsis viticolas was common and widely distributed in diseased vineyards. This taxon was associated with the typical disease symptoms and proved to be pathogenic. Morphologically taxon 2 corresponded best with P. viticola, which was also neotypified in this study. Taxon 2 was mostly isolated from buds and nodes, indicating that these are important sites in which the fungus survives during winter. Molecular data indicated that taxon 3 and P. amygdali were not host specific to grapevine. The currently used foliar fungicides were compared to the new strobilurin fungicides. The effects of nine fungicides (azoxystrobin, flusilazole, folpet, fosetyl- Al+mancozeb, kresoxim-methyl, mancozeb, penconazole, spiroxamine and trifloxystrobin) were tested in vitro on inhibition of mycelial growth. The following EC50 (ug/ml) values were obtained: azoxystrobin (0.350), flusilazole (0.007), folpet (4.489), fosetyl-Al+mancozeb (3.925), kresoxim-methyl (1.665), mancozeb (2.891), penconazole (0.023), spiroxamine (0.321) and trifloxystrobin (0.051). Additionally, azoxystrobin, folpet, kresoxim-methyl, mancozeb, propineb and trifloxystrobin were tested for their ability to inhibit spore germination in vitro. The subsequent EC50 (ug/ml) values were obtained: azoxystrobin 0.123), folpet (0.510), kresoxim-methyl (0.0037), mancozeb (0.250), propineb (0.156) and trifloxystrobin (0.003). The results reported in part 4 showed that the strobilurin fungicides inhibited the mycelial growth and spore germination of P. viticola. However, further trials need to be conducted to verify these findings under field conditions. In the present study taxa 1, 3 and P. amygdali were infrequently isolated, suggesting that they played a less prominent role in the P. viticolacomplex. / AFRIKAANSE OPSOMMING: Streepvleksiekte van wingerd is 'n ekonomies belangrike siekte wat in die meeste wingerdproduserende gebiede van die wêreld voorkom. Vier Phomopsis spesies is in die verlede met dié siekte geassosieer. Hierdie studie ondersoek die taksonomiese belangrikheid van die verskillende taksa wat op wingerd in Suid Afrika gevind word, asook die endofietiese groei en fungisiedsensitiwiteit van die Phomopsis vitico/a isolate. Hierdie tesis bestaan uit verskeie dele met spesifieke, maar verwante onderwerpe wat tot onafwendbare duplisering lei. Dit is belangrik om die epidemiologie van 'n siekte te verstaan sodat korrekte en tydsberekende siektebeheer toegepas kan word. Die endofietiese groei van P. vitico/a is ondersoek deur simptoomlose lote by agt verskillende groei stadiums te versamel. Nodusse, internodusse, blaarstele, blare, rankies en trosstele is ondersoek. Twee Phomopsis spp., takson 1 en 2 is geïdentifiseer. Die Phomopsis vitico/a-kompleks het 3% van die isolasies uitgemaak en 'n relatiewe belangrikheid van 9% getoon. P. vitico/a (takson 2) is meestal uit die nodus en internodus geïsoleer. lnokulasies van gesonde, jong wingerdweefsel het bevestig dat takson 2 'n virulente patogeen is en dat die takson eerder 'n latente patogeen as 'n endofiet is. In teenstelling hiermee is takson 1 'n ware endofiet en 'n onbelangrike patogeen op wingerd. Die ware identiteit van die veroorsakende organisme van streepvlek is ondersoek deur plantmateriaal vanaf 58 verskillende wingerde in die wingerproduserende gebiede van die Wes-Kaap te versamel. P. vitico/a is in wingerdmateriaal vanaf Lutzville tot Swellendam aangetref, maar nie in die Oudtshoorn en Oranjerivier wingerd produserende gebiede nie. Diaporthe perjuncta (takson 1), P. vitico/a (takson 2), takson 3 en P. amygdali is in die Suid Afrikaanse wingerdisolate geïdentifiseer. P. amygdali word met lootverskroeiing van perske bome in die V.S.A. geassosieer. Die Australiese isolaat wat benoem is as takson 4, is met die huidige ondersoek gevind om 'n spesie van Libertella te wees. Takson 4 is daarvolgens uit die P. vitico/a-kompleks gelaat. 'n Italiaanse isolaat het 'n nuwe spesie van Phomopsis op wingerd verteenwoordig en is vervolgens as takson 5 beskryf. Spesie-onderskeiding is op morfologiese en kulturele eienskappe, staminokulasies en die vorming van die teleomorf in vitro gebaseer. Die identiteit vanelke morfologiese takson is met behulp van filogenetiese analises van die nukleêre ribosomale DNS intern transkriberende spasieerders (ITS 1 en ITS2) en die 5' punt gedeeltelike nukleotied volgorde van die mitochondriale klein subeenheid (mtSSU) bevestig. P. amygdali is slegs een keer geïsoleer en blyk van minder belang in die siektekompleks te wees. Takson 1 (Diaporthe perjuneta) en takson 3 het ook min voorgekom en is nie-patogenies, wat hul nie-funksionele rol in streepvleksiekte aandui. Takson 2 (P. viticola) is algemeen geïsoleer en kom wyd verspreid voor. Hierdie takson is geassosieer met die tipiese siektesimptome en is ook patogenies. Morfologies stem takson 2 met P. viiicola ooreen en is ook geneotipifiseer in hierdie studie. Takson 2 is meestal vanaf die ogies en nodusse geïsoleer, wat daarop dui dat hierdie belangrike setels is waar die swam tydens die winter oorleef. Die molekulêre data toon aan dat takson 3 en P. amygdali nie gasheerspesifiek tot wingerd is nie. Die swamdoders wat tans teen streepvlek gebruik word, is met die nuwe strobilurin swamdoders vergelyk. Die effek van nege swamdoders (azoksistrobin, flusilasool, folpet, fosetyl-Al + mancozeb, kresoxirn-metiel, mankozeb, penconasool, spiroksamien en trifloksistrobin) is in vitro op die inhibisie van miseliumgroei getoets. Die volgende EKso-waardes (g/ml) is verkry: azoxystrobin (0.350), flusilasool (0.007), folpet (4.489), fosetiel-Al + mankozeb (3.925), kresoxirn-metiel (l.665), mankozeb (2.891), penkonasool (0.023), spiroksamien (0.321) en trifloxystrobin (0.051). Azoxystrobin, folpet, kresoxim-rnetiel, mankozeb, propineb en trifloksistrobin is ook in vitro getoets vir hul inhibisie op spoorontkieming. Die volgende EKso-waardes is verkry: azoxystrobin (0.123), folpet (0.510), kresoxim-metiel (0.0037), mankozeb (0.250), propineb (0.156) en trifloxystrobin (0.003). Die resultate vervat in deel 4 toon dat die strobilurin swamdoders die miseliumgroei en spoorontkieming van P. viticola inhibeer. Toetsing in die veld word egter benodig om die effektiwiteit van die middels te bevestig. In hierdie studie is taksa I, 3 en P. amygdali selde geïsoleer, wat aangedui het dat hierdie taksa 'n minder belangrike rol in die P. viticola-kompleks speel.

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.