Dissertations / Theses on the topic 'Endophytic'

The aims of this study were to find endophytic brown algae in marine macroalgae from New Zealand, isolate them into culture and identify them using morphological as well as molecular markers, to study the prevalence of pigmented endophytes in a representative host-endophyte relationship, and to reveal the ultrastructure of the interface between the obligate parasite Herpodiscus durvillaeae (LINDAUER) SOUTH and its host Durvillaea antarctica (CHAMISSO) HARRIOT. Three species of pigmented endophytic Phaeophyceae were isolated from New Zealand macrophytes. They were distinguished based on morphological characters in culture, in combination with their distribution among different host species and symptoms associated with the infection of hosts. ITS1 nrDNA sequences confirmed the identity of two of the species as Laminariocolax macrocystis (PETERS) PETERS in BURKHARDT & PETERS and Microspongium tenuissimum (HAUCK) PETERS. A new genus and species, Xiphophorocolax aotearoae gen. et sp. ined., is suggested for the third group of endophytic Phaeophyceae. Three genetic varieties of L. macrocystis as well as two varieties each of M. tenuissimum and X. aotearoae were present among the isolates. L. macrocystis and X. aotearoae constitute new records for the marine flora of the New Zealand archipelago, on genus and species level. The red algal endophyte Mikrosyphar pachymeniae LINDAUER previously described from New Zealand is possibly synonymous with Microspongium tenuissimum. The prevalence of infection by Laminariocolax macrocystis was investigated in three populations of Macrocystis pyrifera along the Otago coast. Two of the populations situated inside and at the entrance of Otago Harbour showed high infection rates (average between 95 and 100%), while an offshore population was less infected (average of 35%). The phylogenetic affinities of the parasitic brown alga Herpodiscus durvillaeae, an obligate endophyte of Durvillaea antarctica (Fucales, Phaeophyceae) in New Zealand, were investigated. Analyses combined nuclear encoded ribosomal and plastid encoded RuBisCO genes. Results from parsimony, distance and likelihood methods suggest a placement of this species within the order Sphacelariales. Even though H. durvillaeae shows a reduced morphology, molecular data were supported by two morphological features characteristic for the Sphacelariales: the putative presence of apical cells and the transistory blackening of the cell wall with 'Eau de Javelle'. Ultrastructural sections showed evidence for a symplastic contact between the cells of the parasite H. durvillaeae and its host D. antarctica. Within the host cortex, parasite cells attack the fields of plasmodesmata connecting host cells. In these areas, parasite cells squeeze between the host cells and form secondary plasmodesmata connecting the primary plasmodesmata of the host cells with the cytoplasma of the parasite cell. Moreover, despite being described as lacking pigments, H. durvillaeae possesses a rbcL gene, and its plastids show red autofluorescence in UV light, suggesting the presence of a possibly reduced, but functional photosynthetic apparatus. Vestigial walls between developing spores in the 'secondary unilocular sporangia' of H. durvillaeae confirm the identity of these sporangia as plurilocular gametangia, derived from reduced gametophytes which were entirely transformed into gametangia.

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.

Endophytes are mutualistic fungi living in green tissue of all plants examined so far.Some of these fungi can produce compounds that are beneficial to the host plant, and it isalso known that some pathogenic fungi live parts of their lives as endophytes. Endophyticinteractions have been well characterized in various grasses, but much is unknown abouttheir interactions with trees. One reason for this is that the fungal biodiversity is muchlarger among endophytes in trees than in grasses, another is that screening for endophytestakes a lot of work. The goal of this thesis work was to develop a polymerase chainreaction (PCR) based method that is simple, fast and reliable for detection of endophytesin aspens. Eleven primer pairs were designed, each pair specific for one fungus. Afteroptimization and evaluation four of the primer pairs were found to be both specific andsensitive, and could detect fungus in DNA preparations from leaf samples.

Plants can be colonized by complex communities of endophytic fungi. This thesis presents two studies, both of which investigate biotic filtering in endophytic fungal communities. Chapter 1. Endophytic fungi can be acquired horizontally via propagules produced in the environment such as in plant litters of various species. Given that litters from different plant species harbor distinct endophytic fungal communities and that endophytic fungi may be dispersal-limited, the structure of the endophytic fungal community of a given plant may be determined by proximity to particular inoculum sources. Community assembly may also be affected by biotic filtering by the plant. Therefore, a plant may be able to select particular fungal taxa from among the available pool. In that case, the structure of the endophytic fungal community in the plant could be somewhat independent of the structure of the inoculum community. We tested the hypothesis that biotic filtering of endophytic fungal communities occurs in Bromus tectorum by exposing it to a variety of inoculum sources including litters from several co-occurring plant species. The inoculum sources differed significantly from each other in the structures of the communities of endophytic fungi they harbored. We characterized the structures of the resulting leaf and root endophytic fungal communities in Bromus tectorum using high-throughput sequencing. All tested inoculum sources successfully produced complex communities of endophytic fungi in Bromus tectorum. There was significantly more variation in the structures of the communities of endophytic fungi among the inoculum sources than in the resultant endophytic fungal communities in the leaves and roots of Bromus tectorum. These results suggest that biotic filtering by Bromus tectorum played a significant role in the assembly of the endophytic fungal communities in tissues of Bromus tectorum. Because endophytic fungi influence plant fitness, it is reasonable to expect there to be selective pressure to develop a uniform, desirable endophytic fungal community even from disparate inoculum sources via a process known as biotic filtering. Chapter 2. Frequently one finds that different plant species harbor communities that are distinct. However, the nature of this interspecific variation is not clear. We characterized the endophytic fungal communities in six plant species from the eastern Great Basin in central Utah. Four of the species are arbuscular mycorrhizal (two in the Poaceae and two in the Asteraceae), while the other two species are nonmycorrhizal (one in the Brassicaceae and one in the Amaranthaceae). Our evidence suggests that both host mycorrhizal status and phylogenic relatedness independently influence endophytic fungal community structure.

Endophytic fungi from Cassia fistula or golden shower, a well known medicinal plant in Thailand and Asia, were isolated from trees growing in three geographical separate sites. These locations were Kanchanaburi, Nakhon Ratchasima and Bangkok and were selected to allow comparisons between their endophytic assemblages and to evaluate these data in relation to differences in plant diversity and density and local environment. Kanchanaburi which was the site closest to a natural forest situation provided the highest number of isolates with Bangkok, where the trees were isolated individuals, having the least. Members of the Xylariaceae proved to be common and frequent isolates especially species of Xylaria and Daldinia but Nemania and Hypoxylon were also obtained. Phomopsis was also well represented and clearly was dominant at the Kanchanaburi site. Species of Fusarium, Colletotrichum, Penicillium, Nigrospora, Coprinus and Psathyrella were also identified but were occasional isolates. Differences in endophytic assemblages between samples obtained early in the rainy season (July, 2001) with those sampled towards the end of the rainy season (December, 2001) were found to occur in the Nakhon Ratchasima samples with over twice as many isolates obtained from the December samples. This is likely to be a reflection on the longer exposure period to the potential inoculum of these leaves. A total of 956 endophytic isolates were obtained from the three sites with samples from Kanchanaburi (December 2000) and Bangkok and Nakhon Ratchasima in July 2001 with a further samples from Nakhon Ratchasima in December, 2001. Isolations were also made from different anatomical regions of the leaf, leaf lamina, midrib and veins. There were no appreciable differences in either the number of isolates obtained or an association between leaf area and specific fungal species. Identification of many xylariaceous endophytic isolates is well known to be problematic since Xylaria species rarely produce their anamorphic form in culture and virtually no members of the Xylariaceae develop their teleomorph in culture. Therefore molecular techniques were used to compare DNA sequences of the ITS region from a selection of endophyes with sequences obtained from teleomorphic material, or cultures derived from teleomorphs of identified and authenticated Xylariaceae. Comparisons were also made with data held in GenBank. This enabled the identity of a number of taxa to be made although more sequences from Xylaria species are required for future investigations. A number of non-xylariaceous taxa were also named as a result of DNA sequence comparisons. Secondary metabolites from the xylariaceae were also investigated and their metabolite profiles used to support identifications. The metabolite profiles proved to be a useful tool to confirm doubtful endophytic isolates when their DNA sequences could not place them with certainty in a right group. Together with extracts from other endophytic species, their inhibitory effects on bacteria and fungi were tested. Cassia endophytes were found to show low antimicrobial activity. However, they may later be shown to have other activities when when tested e. g. anti-malarial, anti-cancer and anti-HIV.

Doutoramento Biologia
Os sapais são ecossistemas marinhos altamente produtivos que frequentemente recebem contaminantes de natureza antropogénica. A Ria de Aveiro encontra-se no noroeste de Portugal e contém numerosos sapais. Halimione portulacoides é um dos halófitos mais importantes em sapais Europeus e tem sido amplamente estudada devido ao seu potencial para ser usada em fins de fitorremediação, e como bioindicador de contaminação de sedimentos. Bactérias endofíticas podem apresentar capacidade promotora do crescimento de plantas (PCP), quer diretamente por produção de fito-hormonas e aquisição de nutrientes, quer indiretamente via competição com fitopatogenos. No presente trabalho, a diversidade de bactérias endofíticas da planta de sapal H. portulacoides da Ria de Aveiro é explorada extensivamente. Isolados de bactérias endofíticas foram obtidos e caracterizados quanto à sua taxonomia, capacidade de produzir enzimas e características PCP. As características mais observadas foram atividade celulolítica, xilanolítica e desaminase de 1-aminociclopropano-1-carboxilato, e a produção da auxina ácido indol-3-acético. Os resultados revelaram um enorme potencial da coleção para PCP in vitro e in vivo. A coleção de isolados foi também explorada para procurar diversidade não descrita. Como resultado, dez novas espécies de bactérias foram amplamente caracterizadas e descritas: Microbacterium diaminobutyricum, Saccharospirillum correiae, Altererythrobacter halimionae, Altererythrobacter endophyticus, Zunongwangia endophytica, Salinicola halimionae, Salinicola aestuarina, Salinicola endophytica, Salinicola halophytica e Salinicola lusitana. Consequentemente, o presente trabalho expôs a endosfera de H. portulacoides como um foco de diversidade bacteriana desconhecida. A composição taxonómica da comunidade endofítica foi averiguada via sequenciação do gene 16S rRNA da coleção de isolados, e mais profundamente com a utilização de sequenciação de alto rendimento independente do cultivo. A última abordagem revelou cinco filos principais: Proteobacteria, Planctomycetes, Actinobacteria, Bacteroidetes e Firmicutes. Destes, apenas Planctomycetes não foi obtido na coleção de isolados. As comunidades diferiram de acordo com o local (no ensaio dependente do cultivo, para locais contaminados e não-contaminado) e tecido (em ambos os ensaios) de amostragem. As principais famílias obtidas no endofitoma nuclear foram Oceanospirillaceae em tecidos de parte aérea, e Enterobacteriaceae e Kiloniellaceae em tecidos de raiz. O trabalho apresentado providenciou uma compreensão profunda das bactérias endofíticas presentes no halófito H. portulacoides, e expôs o seu potencial como foco de bactérias não descritas e bactérias promotoras do crescimento de plantas.
Salt marshes are highly productive marine ecosystems that often act as a sink for contaminants of anthropogenic nature. The Ria de Aveiro lagoon is located in the north-west of Portugal and comprises numerous salt marshes. Halimione portulacoides is one of the most important halophytes in European salt marshes and has been widely researched for its potential for phytoremediation, and as a bioindicator of sediment contamination. Endophytic bacteria can present plant growth promotion (PGP) abilities, either directly by production of phytohormones and nutrient uptake, or indirectly via competition with phytopathogens. In the present work, the diversity of endophytic bacteria from the salt marsh plant H. portulacoides from Ria de Aveiro is extensively explored. Endophytic bacterial isolates were obtained and characterized for their taxonomy, ability to produce specific enzymes and PGP traits. The most observed traits were cellulolytic, xylanolytic and 1-aminocyclopropane-1-carboxylate deaminase activities, and the production of the auxin indol-3-acetic acid. The results revealed an enormous potential of the collection for in vitro and in vivo PGP. The collection of isolates was also explored for undescribed diversity. As a result, ten novel bacterial species were thoroughly characterized and described: Microbacterium diaminobutyricum, Saccharospirillum correiae, Altererythrobacter halimionae, Altererythrobacter endophyticus, Zunongwangia endophytica, Salinicola halimionae, Salinicola aestuarina, Salinicola endophytica, Salinicola halophytica and Salinicola lusitana. Consequently, the present work exposes the endosphere of H. portulacoides as a hotspot of unknown bacterial diversity. The taxonomic composition of the endophytic community was assessed via 16S rRNA gene sequencing of the isolate collection, and with more depth using culture-independent high-throughput sequencing. The latter approach revealed five main phyla: Proteobacteria, Planctomycetes, Actinobacteria, Bacteroidetes and Firmicutes. From these, only Planctomycetes was not obtained in the isolate collection. The communities differed according to sampling site (for the culture-dependent assay, for contaminated and non-contaminated sites) and tissue (in both assays). The main families found in the core endophytome were Oceanospirillaceae for aboveground tissues, and Enterobacteriaceae and Kiloniellaceae for belowground tissues. The present work provided a deep understanding of the endophytic bacteria present in the halophyte H. portulacoides, and exposed its potential as a hotspot of undescribed bacteria and plant growth promoting bacteria.

>Magister Scientiae - MSc
The term endophyte is used to define all microorganisms that, during a part of their life cycle, colonize the internal tissues of a plant host. Many endophytes have been found to promote plant growth by acting either as biocontrol agents, biofertilizers or phytohormone producers. This study aimed to characterise the endophytic microbial community diversity associated with sorghum farmed in South Africa. Members of any common endophytic bacterial species identified during the study might in future studies be developed to improve sorghum production. Sorghum tissues (roots, shoots, stems) were sampled in three South African provinces (Free State, Limpopo and North West), each site being characterised by the use of different agricultural practices. Denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analyses were used to characterise the endophytic bacterial communities. The analysis clearly demonstrated that the endophytic bacterial community structure in the three sorghum tissue types differed, suggesting that endophyte colonization is tissue-specific. The endophytic bacterial community structure is quite similar in each tissue when comparing the populations present in the sampling sites. In the sorghum endophytic microbial communities, common bacterial species were identified using molecular tools: The cyanobacterium Synechococcus and Staphylococcus saprophyticus were identified in the root samples. Pantoea sp., Erwinia sp., Enterobacter sp. and Klebsiella sp. were found in all shoot samples. Nocardia fluminea, Bacillus cereus and Microbacterium sp. were isolated as common shoot endophytic bacteria. This study defines, for the first time, the endophytic bacterial species associated with South African sorghum plants. These common endophytic bacterial species can be used to enhance the yield of sorghum crops.

Taxonomic diversity, biology and ecological aspects of fungal endophytes of Tectona grandis (teak) from Chiang Mai Province, Thailand were investigated. It was found that the endophyte assemblages of mature leaves sampled from natural forest and plantation teak were not significantly different. Members of the Xylariaceae, especially Daldinia eschscholzii, Nemania subannulata, Hypoxylon haematostroma and Xylaria cubensis were frequent isolates. Widely reported endophytic fungi such as Phomopsis, Colletotrichum, Cladosporium and Fusarium were also isolated. There is little evidence to support host specificity for the majority of the isolates. Differences in endophyte assemblages between young and mature leaves were shown to occur with a much lower infection percentage in the young leaves. Species of Phomopsis and Colletotrichum were dominant in the young leaves but members of the Xylariaceae dominated in mature leaves. This pattern was the same for both natural forest and plantation samples. However comparison of taxa isolated from leaf lamina, midrib and veins gave conflicting results. Samples from mature leaves from natural forest trees exhibited little variation with greater variation in taxa recovered being found to occur between sampling years than between position of isolation from the leaf. In plantation leaves, although the results were similar to those from natural forest tree leaves for two of the years sampled, in 1997 the overall recovery rate was highest for the lamina, followed by veins and then the midrib. There was no evidence obtained to link individual taxa with specific regions of the leaf. It is now possible to devise a sampling strategy to obtain suitable diversity of endophytic isolates from teak leaves for industrial screening of these fungi. Techniques were developed to overcome current problems of identification of xylariaceous endophytes in the absence of their teleomorph. Inoculation of suitable woody substrata combined with selective incubation was used to induce teleomorph formation in many of the isolates and this together with chemical profiling enabled identification to species of many of these isolates. Rates of development of specific species were obtained and differences in environmental conditions necessary for development of teleomorphs to maturity were noted for members of different genera. Thus species of Daldinia and Hypoxylon required drier conditions than species of Xylaria and Nemania which only developed under wet shaded conditions. Xylariaceae from the natural forest, plantation, and forest surrounding the plantation were surveyed and a number of the Xylariaceae recovered as endophytes were found to be new to science, new records for Thailand or were recorded as endophytes for the first time.

Fungi are rich sources of structurally diverse secondary metabolites, some of which possess biological activities of importance to medicine and agriculture. Research in our group focuses on application of ecological considerations to the selection of underexplored fungi for chemical analysis in search of new natural products with potentially important bioactivities. The work described in this thesis involved studies of members of two different fungal ecological groups, fungicolous and endophytic fungi. Fungicolous fungi colonize other fungi and are viewed as potential sources of antifungal agents due to the negative effects often exerted on the host organisms. Our prior studies of such fungi have proven them to be productive sources of new bioactive natural products. Fractionation of extracts produced from eleven fungicolous fungal isolates resulted in the isolation and identification of 24 different, structurally-diverse natural products, many of which were bioactive. Most of these were previously known, but six were new compounds, with one being a distinctive new peptaibol-type metabolite. Endophytic fungi colonize the inner tissues of host plants, often asymptomatically, and occur widely in most plant species. Some endophytes may benefit the host, possibly through production of protective secondary metabolites, but in most cases, their roles are not well understood. Stenocarpella maydis is a widely occurring fungal endophyte and pathogen of corn and is associated with diplodiosis (a neuromycotoxicosis) of cattle. Investigations of extracts of S. maydis and S. macrospora cultures led to the identification of seven compounds, including diplodiatoxin, diplosporin, chaetoglobosins K, L, M, and O, and (all-E) trideca-4,6,10,12-tetraene-2,8-diol, none of which were previously known from U.S. Stenocarpella isolates. Diplodiatoxin was detected as a major component of S. maydis-rotted maize grain, stalks, and stalk residues, and chaetoglobosin M was detected in extracts of naturally-diseased S. maydis-rotted maize seeds collected in the field. Chaetoglobosin K displayed potent antifungal and antiinsectan activity, while diplodiatoxin displayed phytotoxicity. Because of their well-known cytotoxic effects, we proposed that mixtures of chaetoglobosins are responsible, at least in part, for inducing diplodiosis in livestock. Our prior investigations of another common corn endophyte, Acremonium zeae, led to isolation of the pyrrocidines, which display potent antifungal and antibacterial activities against a range of corn pathogens. Further studies of these extracts led to the isolation and identification of three resorcylic acid lactone (RAL)-type compounds, one of which was new. These RAL's, did not exhibit antifungal activity, but demonstrated mild phytotoxicity to maize leaves in assays. They are also members of a compound class we have encountered among other endophytes that inhibit the heat shock protein Hsp90, and have been proposed to play a role in the colonization process. These chemical investigations provide further knowledge that may be useful in determining the roles that endophytes, in particular S. maydis and A. zeae, play in maize. The metabolites encountered during these investigations represented different biosynthetic pathways, and included polyketides, terpenoids, and compounds of mixed biogenetic origin. Structures were characterized by analysis of 1D and 2D NMR data, mass spectrometry, chemical degradation or derivatization reactions, and/or X-ray diffraction analysis. Absolute configuration assignments were determined using Mosher's method, Marfey's method, or chiral amino acid analysis methods.

Fungi have been an important source of many structurally diverse and biologically active secondary natural products. These secondary metabolites have found applications in pharmaceutical and agriculture industry. Fungi are estimated to be second only to insects in species diversity. It is estimated that there are at least 3.5 million species of fungi of which a less than 5% have so far been explored. Fungi are known to produce many secondary natural metabolites, however their role has still not been clearly understood. However it is possible that fungi which often thrive in competitive environments would experience evolutionary pressure to produce such metabolites for defensive or offensive functions. Our research focusses on isolation and structure elucidation of secondary metabolites from endophytic and mycoparasitic/fungicolous fungi. Mycoparasitic and fungicolous fungi are those that colonize other fungi, and could be potential sources of antifungal agents because of the negative effects exerted on their hosts due to this colonization. Endophytic fungi are those that colonize the inner tissues of plants in a symbiotic or a non-symbiotic manner. They may or may not be necessary for the growth, defense and survival of the host. They may protect the plants from attack by other pathogens by producing secondary metabolites that inhibit the growth of other pathogenic organisms. Many biologically active secondary metabolites have been isolated from various members of these two classes of fungi. During the course of this research fourteen new and several known compounds representing various biosynthetic classes including peptides, polyketides, terpenoids, and compounds with mixed biogenetic origins have been isolated and characterized using various tools such as NMR and MS. Details of the isolation, structure elucidation, and biological activity of these new compounds are presented in this thesis. Structure elucidation was performed mainly by analysis of various MS and NMR data, along with chemical derivatization reactions and/or X-ray diffraction analysis data. Absolute configuration assignments were made using, X-ray crystallography, and/or by ECD spectral analysis in combination with computational analysis.

Fungi continue to serve as valuable sources of a diverse variety of bioactive secondary metabolites, some of which have found applications as important pharmaceuticals and agrochemicals. Our research group employs an ecology-based strategy for the selection of fungi for chemical studies. The research described here involves chemical investigation of fungicolous and endophytic fungi that resulted in the isolation and identification of many new natural products, as well as some previously known metabolites. Mycoparasitic and fungicolous fungi are those that colonize other fungi, and are viewed as potential sources of antifungal agents because of the negative effects exerted on their hosts due to this colonization. Many antifungal compounds have been isolated by members of our research group from such fungi. Fractionation of the ethyl acetate extracts of cultures of thirteen fungal isolates, followed by characterization using various NMR and MS techniques, afforded fifteen new and twenty-six known compounds representing diverse structural classes. Some of these compounds showed antifungal and antiinsectan effects accounting for bioactivities originally observed for the crude extracts. Endophytic fungi are those that inhabit host plants asymptomatically and may or may not cause disease after some time. Studies show that in some cases they may be beneficial to the host plant by providing protection against various pathogens or by improving the resistance of the host in some way. Such advantages may be due, at least in part, to the production of bioactive secondary metabolites by these fungi. Chemical investigations of seven crop endophytes led to the isolation and characterization of eleven new and twenty-eight known compounds. Some of these fungal known fungal metabolites exhibited antifungal, antimicrobial, and/or antiinsectan effects, and may therefore provide insight into the potential protective roles of the corresponding crop endophytes against plant pathogens or insect pests. The fungal secondary metabolites isolated during the course of these studies represent a variety of biosynthetic classes, including peptides, polyketides, terpenoids, and compounds with mixed biogenetic origins. Details of the isolation, structure elucidation, and biological activity of these new compounds are presented in this thesis. Structure elucidation was performed mainly by analysis of various MS and NMR data, although chemical derivatization reactions and/or X-ray diffraction analysis data were employed in some instances. Absolute configuration assignments were made using Mosher's method, X-ray crystallography, and/or by ECD spectral analysis in combination with computational analysis. Details of the isolation, structure elucidation, and biological activity of these new compounds are presented in this thesis.

The plant hormones salicylic and jasmonic acid (SA and JA, respectively) both play a crucial role in the induction of plant defense system pathways and long-term pathogen resistance. Plants, which do not have an active cellular immune system like animals, instead rely on the release of specific molecules to mediate defense. In general, the SA pathway is activated by biotrophic pathogens and primarily induces antimicrobial responses, while JA is activated by necrotrophic pathogens and herbivory, and induces separate chemical responses. SA and JA are reciprocally antagonistic: activation of one pathway inhibits activation of the other. Here we explore how SA-JA inhibitory crosstalk is used by pathogens or herbivores to combat plant defense. We study the effects of hormone crosstalk on bacterial growth in two plant models: Cardamine cordifolia and Arabidopsis thaliana, which we treated to induce defense pathways. These plants were inoculated with endophytic bacteria isolated from field C. cordifolia plants, and the effects of hormone treatment on bacterial growth rates were measured. We show that JA-induced defenses, which are commonly associated with necrotrophic pathogens, affect varied biotrophic Pseudomonas strains both positively and negatively. Notably, we show that JA-induce defenses affect wild P. fluorescens strains more negatively than SA-defenses.

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.

Wheat is the most economically important crop forming one quarter of Australian farm production. The wheat industry is severely affected by diseases, with fungal pathogens causing the most important economic losses in Australia. The application of fungicides and chemicals can control crop diseases to a certain extent, however, it is expensive and public concern for the environment has led to alternative methods of disease control to be sought, including the use of microorganisms as biological control agents. Microorganisms are abundant in the soil adjacent to plant roots (rhizosphere) and within healthy plant tissue (endophytic) and a proportion possess plant growth promotion and disease resistance properties. Actinobacteria are gram-positive, filamentous bacteria capable of secondary metabolite production such as antibiotics and antifungal compounds. A number of the biologically active endophytes belonging to the Actinobacteria phylum were isolated in our laboratory. A number of these isolates were capable of suppressing the wheat fungal pathogens Rhizoctonia solani, Pythium sp. and Gaeumannomyces graminis var. tritici, both in vitro and in planta indicating the potential for the actinobacteria to be used as biocontrol agents. The aim of this research was to investigate the molecular mechanisms underlying this plant-microbe interaction. The indigenous microbial populations present in the rhizosphere and endophytic environment are critical to plant health and disruptions of these populations are detrimental. The culture-independent technique Terminal Restriction Fragment Length Polymorphism (T-RFLP) was used to characterise the endophytic actinobacteria population of wheat roots under different conditions. Soils which support a higher number of indigenous microorganisms result in wheat roots with higher endophytic actinobacterial diversity and level of colonisation. Sequencing of 16S rRNA gene clones, obtained using the same actinobacteria-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity, and identified a number of Mycobacterium and Streptomyces species. It was found that the endophytic actinobacterial population of the wheat plants contained a higher diversity of endophytic actinobacteria than reported previously, and that this diversity varied significantly among different field soils. The endophytic actinobacteria have previously been shown to protect wheat from disease and enhance growth when coated onto the seed before sowing. As the endophytes isolated were recognised as potential biocontrol agents, the impact on the indigenous endophytic microbial population was investigated. Utilising the T-RFLP technique it was established that the use of a commercial microbial inoculant, containing a large number of soil bacterial and fungal strains applied to the soil, disrupts the indigenous endophyte population present in the wheat roots. The hypothesis is that non-indigenous microbes proliferate and dominate in the soil preventing a number of endophytic-competent actinobacterial genera from access to the seed and ultimately endophytic colonisation of the wheat roots. This dramatically reduces diversity of endophytes and level of colonisation. In contrast the use of a single endophytic actinobacteria endophyte inoculant results in a 3-fold increase in colonisation by the added inoculant, but does not significantly affect this indigenous population. Colonisation of healthy plant tissues with fungal endophytes has been shown to improve the competitive fitness with enhanced tolerance to abiotic and biotic stress and improved resistance to pathogens and herbivores. In this study the fungal endophyte population of wheat plants grown in four different soils was analysed using partial sequencing of 18S rRNA gene sequences. Sequence anlaysis of clones revealed a diverse range of fungal endophytes. In this diverse range of fungal endophytes a number sequences were highly similar to those of previously known fungal phytopathogens. A number of sequences detected were similar to fungal species previously identified in soil or plant material but not as endophytes. The remaining sequences were similar to fungal species without a known relationship with plants. Plants have developed an inducible mechanism of defence against pathogens. In addition to local responses plants have developed a mechanism to protect uninfected tissue through a signal that spreads systemically inducing changes in gene expression. In the model plant Arabidopsis thaliana activation of the Systemic Acquired Resistance (SAR) pathway and the Jasmonate (JA)/Ethylene (ET) pathway is characterised by the production of pathogenesis-related (PR) and antimicrobial proteins resulting in systemic pathogen resistance. Endophytic actinobacteria, isolated from healthy wheat roots in our laboratory, have been shown to enhance disease resistance to multiple pathogens in wheat when coated onto the seed before sowing. Real Time RT-PCR was used to determine if key genes in the SAR and JA/ET pathways were induced in response to inoculation with endophytic actinobacteria. Inoculation of wild-type Arabidopsis thaliana with selected strains of endophytic actinobacteria was able to �prime� the defence pathways by inducing low level expression of SAR and JA/ET genes. Upon pathogen infection the defence-genes are strongly up-regulated and the endophyte coated plants had significantly higher expression of these genes compared to un-inoculated plants. Resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora was mediated by the JA/ET pathway whereas the fungal pathogen Fusarium oxysporum triggered primarily the SAR pathway. Further analysis of the endophytic actinobacteria-mediated resistance was performed using the Streptomyces sp. EN27 and Arabidopsis defence-compromised mutants. It was found that resistance to E. carotovora subsp. carotovora mediated by Streptomyces sp. EN27 occurred via a NPR1-independent pathway and required salicylic acid whereas the jasmonic acid and ethylene signalling molecules were not essential. In contrast resistance to F. oxysporum mediated by Streptomyces sp. EN27 occurred via a NPR1-dependent pathway but also required salicylic acid and was JA- and ET-independent. This research demonstrated that inoculating wheat with endophytic actinobacteria does not disrupt the indigenous endophytic population and may be inducing systemic resistance by activating defence pathways which lead to the expression of antimicrobial genes and resistance to a broad range of pathogens.

Drug discovery is reliant on new developments in natural product chemistry as well as advances in chemical synthesis. The interconnectivity and interdependence of natural and synthetic investigation in drug discovery is evident. The chemical exploration reported herein elaborates the relationship between natural product chemistry and chemical synthesis. Of particular interest are chemicals from organisms residing in less accessible environments, particularly Antarctica and endophytic microbial communities. Degradation via reductive ozonolysis of palmerolide A, a macrocyclic polyketide isolated from the Antarctic tunicate Synoicum adareanum, and subsequent synthetic preparation of the resulting polyols (1,2,6-hexanetriol and 1,2,3,6-hexanetetraol) led to a revision in the absolute configuration of the bioactive natural product (7R, 10R, 11R to 7S, 10S, 11S). A partial synthesis of palmerolide A (C3-14) was completed using Grubb’s 2nd generation catalyst to couple fragments formed using the previously developed methodology from the degradation study. Isolation of indole-pyrimidine containing alkaloids meridianins A, B, C, and E from the Antarctic tunicate Synoicum sp. prompted a synthetic investigation of psammopemmin A, a related alkaloid from the Antarctic sponge Psammopemma sp. resulting in reassignment of the structure of psammopemmin A to that of meridianin A. Both meridianin A and psammopemmin A were synthesized through a Suzuki coupling of the same 4-indolol nucleophile to the apposite pyrimidine electrophile. Several synthetic 3-pyrimidylindole analogs were also prepared and investigated for central nervous system, antimalarial, and cytotoxic activity. Chemical investigation of extracts from mangrove fungal endophytes that displayed antimalarial properties in vitro resulted in the isolation of several potent but cytotoxic and cytostatic compounds: cytochalasin D, roridin E, and 12,13-deoxyroridin E.

Mestrado em Biologia Aplicada - Microbiologia Clínica e Ambiental
A contaminação de solos com metais é um problema ecológico grave que requer medidas de resolução urgentes. As metodologias de remediação convencionais revelam-se muitas vezes ineficazes e muito dispendiosas. O processo de fitoremediação surge como uma alternativa promissora para a recuperação de solos, a aplicar de um modo económico e com maior potencial para recuperar os serviços do ecossistema. Os programas de fitoremediação para além de se debruçarem sobre a escolha da espécie vegetal mais adequada para o processo, devem igualmente debruçar-se sobre as comunidades microbianas associadas às raízes das plantas, nomeadamente as bactérias endofíticas e da rizosfera. No presente trabalho, é estudado o potencial da espécie Fraxinus angustifolia para a fitoremediação de solos contaminados com metais, recolhidos na área de exploração de uma mina de urânio abandonada, na Cunha Baixa, Mangualde, Portugal. A estratégia assumida consistiu na exposição das plantas ao solo contaminado, a um solo de referência e a um solo controlo, por um período de aproximadamente 3 meses. Com o fim de avaliar a capacidade de F. angustifolia de resistir ao solo contaminado, durante a exposição aos diferentes solos acompanhou-se o crescimento e o estado fisiológico das plantas medindo um conjunto de parâmetros bio- e fisiológicos (crescimento acima do solo, área foliar, conteúdo hídrico relativo, máxima eficiência e rendimento do fotosistema II e conteúdo foliar em clorofila a, clorofila b, carotenoides, prolina e malondialdeído) ao longo do período de exposição. Ademais, procedeu-se à análise genética dos perfis das comunidades bacterianas (endofíticas e rizosfera) associadas às raízes de F. angustifolia. Esta análise foi realizada através do método de PCR-DGGE, tendo como alvo uma região conservada 16S rDNA, antes e após a exposição aos diferentes solos. Apenas o parâmetro “crescimento acima do solo” se revelou, ao longo de toda a experiência, claramente indicativo do efeito negativo das propriedades do solo proveniente da mina nas plantas. Nos restantes parâmetros, foi observada uma resposta positiva das plantas expostas à contaminação, tendo demonstrado capacidade de manter o seu estado fisiológico ou, após oscilações, retomar ao estado normal. Comparativamente às comunidades bacterianas analisadas pré-tratamento, as alterações dos perfis das comunidades foram notáveis, principalmente aquelas referentes às plantas expostas ao solo contaminado. As comunidades referentes às plantas do solo controlo e referencia apresentaram maior similaridades entre si e entre a análise pré-tratamento. O estudo fisiológico demonstrou que, quando expostas ao solo contaminado, as plantas F. angustifolia apresentam capacidade de resistência e adaptativa às condições adversas, demonstrando potencial passível de ser explorado para fins de fitoremediação. Foi igualmente demonstrado que as alterações exercidas sobre as comunidades bacterianas das raízes expostas ao solo contaminado resultaram em perfis consideravelmente diferentes dos observados nas restantes comunidades. O desempenho das plantas pode estar intrinsecamente relacionado com estas alterações microbianas.
The contamination of soils with metals is a serious ecological problem requiring urgent measures to counteract the impacts. The conventional remediation techniques are, most of the times, inefficient and expensive. Phytoremediation comes as a promising alternative for the cleaning of the soils, to be applied in a more economic and eco-friendly manner, with a great potential for recovering ecosystem services. Besides looking for the best suitable plant species for the task, phytoremediation programs should also focus in the microbial communities associated with the roots of the plants, namely endophytic and rhizosphere bacteria. In this work, it was studied the potential of the species Fraxinus angustifolia for the phytoremediation of metal contaminated soils, collected in the Cunha Baixa uranium mine area (Mangualde, Centre of Portugal). The planned strategy consisted of the exposure of the plants to the contaminated soil, a reference soil and a control soil, for a period of about 3 months. In order to access the ability of F. angustifolia to tolerate the contaminated soil, growth and physiological performance of plants, a set of bio- physiologic parameters (above ground growth, leaf area, relative water content, maximum efficiency and quantum yield of photosystem II and leaf chlorophyll a and b, carotenoid, proline, malondialdehyde contents) were measured during the test. Furthermore, the analysis of the genetic profiles of the bacterial communities (endophytic and rhizosphere) associated with the roots of F. angustifolia was also performed. This analysis was carried out through the PCR-DGGE technique, targeted for a conserved region of 16S rDNA, pre- and post-treatment. Only the “above ground growth” parameter clearly showed to be, throughout the course of the experiment, an indicator of the negative effects of the properties of the mine soil on the plants. Concerning the remaining parameters, a good response of the exposed plants was observed, which showed the ability to maintain their physiologic status or, after some variation, return to a normal state. In comparison with the bacterial communities profiles analyzed pre-treatment, the alterations of the profiles were notable, principally those corresponding to the contaminated soil. The communities related to the plants from the control and reference soils showed more similarity between each other and the pre-treatment analysis. This physiologic study demonstrated that, when exposed to the contaminated soil, F. angustifolia plants had the ability to resist and adapt to the adverse conditions, revealing a potential which can be exploited for phytoremediation. It was also possible to demonstrate that the changes exerted on the bacterial communities from the roots exposed to the contaminated soil resulted in profile considerably different from those observed on the remaining communities. The performance of the plants might be related to these microbial alterations.

Le but de ce travail de thèse était de rechercher de nouvelles molécules inhibitrices de la SOD dans des extraits de substances naturelles. Cette enzyme majeure du stress oxydant étant impliquée dans de nombreux mécanismes de défense des cellules cancéreuses contre l'apoptose représente une voie thérapeutique d'avenir. Nous en avons recherché dans les champignons endophytes de plantes péruviennes, ces micro-organismes produisant de nombreux métabolites de défense des plantes hôtes. Ce travail de thèse a d'abord porté sur l'isolement, la culture, l'identification, l'extraction et la caractérisation des souches de champignons endophytes. Les analyses statistiques effectuées sur ces extraits avec les résultats obtenus en HPLC ont confirmé les problèmes de variabilités qualitative et quantitative pouvant être rencontrés au cours de la culture des endophytes et décrites dans la littérature. La seconde partie expérimentale a porté sur la recherche de furocoumarines dans les extraits obtenus, en raison de leur potentiel effet inhibiteur sur la SOD. Nous avons dérépliqué ces composés dans les extraits d'endophytes au cours de deux stratégies de spectrométrie de masse réalisées en mode d'ionisation négatif. La première approche, à l'aide d'un QTOF, a abouti à l'identification de deux furocoumarines, la 5-Methyl-4H-furo[2,3-b][1]benzopyran-4-one et la déhydropachyrrhizone. La seconde, à l'aide d'un OrbiTrap, a vu l'identification de quatre autres furocoumarines : l'Ochrocarpine A, la Moellendorffiline, l'Anisolactone et l'Anhydrorutarétine .Afin de compléter cette approche métabolomique et identifier les molécules inhibitrices de la SOD, nous avons tenté de mettre au point un test d'activité de cette enzyme, rapide, peu coûteux et réalisable en routine. Nous avons sélectionné le test au pyrogallol, mais celui-ci n'a pas démontré les qualités recherchées tant au niveau de la sensibilité que de la reproductibilité. Nous orientons à présent les recherches vers des approches différentes, la recherche directe d'adduits sur la SOD par LC-MS
Superoxide dismutase is one of the major proteins controlling the oxidizing stress and cellular homeostasis. It is involved in numerous cancer cells proliferation processes. This protein is considered as major anti-cancer target for the development of new anti-cancer drugs. The goal of this work, was to research and identify an inhibitor of the SOD in endophytic fungi from Peruvian plants. These micro-organisms are known to produce numerous metabolites for host plants protection. During the preparation of endophytic extracts and their characterization by HPLC and statistical analyzes, we have pointed out a high quantitative and qualitative variability of the chemical content of endophytic extracts inside a same strain. Considering the literature we have focused our work on the identification of furocoumarins because of their potential inhibitory effect on the SOD. For this purpose two mass spectrometry strategies using negative ionization mode were carried out. With the QTOF mass spectrometer we have identified Methyl-4H-furo [2,3-b] [1] benzopyran-4-one and the déhydropachyrrhizone. With the OrbiTrap, the Ochrocarpine A, Moellendorffiline the Anisolactone and the Anhydrorutarétine were identified. Biological evaluation of the different extracts was performed using pyrogallol test. This investigation did not allow us to identify an inhibitor of the SOD. In the future we may consider seeking SOD inhibitors by looking at the formation SOD-chemical compound adducts using an LC-MS investigation

Some aspects of the presence of systemic endophytic fungi in agriculturally important New Zealand grasses were studied in relation to plant breeding. Seedling resistance to adult Argentine stem weevil feeding in perennial ryegrass, Italian ryegrass and tall fescue was found to be related to the presence of their respective Acremonium endophytes in the seed rather than to plant genetic resistance. In addition a study of perennial ryegrass revealed that this resistance was independent of endophyte viability. The seedling resistance conferred by the endophyte of Italian ryegrass was found to be beneficial for field establishment. This endophyte differs from that in perennial ryegrass and tall fescue in that it does not confer resistance to Argentine stem weevil on mature plants, but only on seedlings. The extent of plant genetic seedling tolerance to adult Argentine stem weevil feeding was limited to broad inter-specific differences, with tall fescue more tolerant than perennial ryegrass and both of these more tolerant than Italian ryegrass. This ranking corresponds with previous observations on feeding preference on mature plants. A study of factors affecting the concentration of endophyte mycelia in infected seed of perennial ryegrass revealed that plant genetic factors had little effect. The major factors studied were: 1) the endophyte concentration in the maternal parent plant directly influenced the endophyte concentration in the seed. 2) nitrogen fertilizer applications to a seed crop reduced the concentration of mycelia in the seed, with earlier applications having a greater effect. 3) application of the fungicide propiconazole (Tilt) to a seed crop reduced the endophyte concentration in the seed. 4) the endophyte concentration in the seed was found to directly influence the endophyte concentration in seedlings, six month old plants and that of seed harvested from a first year seed crop. As there have been no previous reports of tetraploid perennial ryegrass cultivars with endophyte an experiment was conducted to determine if these could be developed by the standard procedure of colchicine treatment. The results revealed that endophyte was retained following colchicine treatment.

This dissertation comprises a series of studies designed to explore the associations between plants and the endophytic fungi they harbor in their above-ground tissues. By viewing endophyte diversity in ecologically and economically important hosts through the lenses of phylogenetic biology, microbiology, and biotechnology, this body of work links plant ecology with newly discovered symbiotic units comprised of endophytic fungi and the bacteria that inhabit them.This work begins with a large-scale survey of endophytic fungi from native and non-native Cupressaceae in Arizona and North Carolina. After isolating over 400 strains of endophytes, I inferred the evolutionary relationships among these fungi using both Bayesian and parsimony analyses. In addition to showing that native and introduced plants contained different endophytes, I found that the endophytes themselves harbor additional microbial symbionts, recovering members of the beta- and gamma-proteobacterial orders Burkholderiales, Xanthomonadales, and Enterobacteriales and numerous novel, previously uncultured bacteria. This work finds that phylogenetically diverse bacterial endosymbionts occur within living hyphae of multiple major lineages of ascomycetous endophytes.A focus on 29 fungal/bacterial associations revealed that bacterial and fungal phylogenies are incongruent with each other and did not reflect the phylogenetic relationships of host plants. Instead, both endophyte and bacterial assemblages were strongly structured by geography, consistent with local horizontal transmission. Endophytes could be cured of their bacterial endosymbionts using antibiotics, providing a tractable experimental system for comparisons of growth and metabolite production under varying conditions. Studies of seven focal fungal/bacterial pairs showed that bacteria could significantly alter growth of fungi at different nutrient and temperature levels in vitro, and that different members of the same bacterial lineages interact with different fungi in different ways.Focusing on one isolate, I then describe for the first time the production of indole-3-acetic acid (IAA) by a non-pathogenic, foliar endophytic fungus (Pestalotiopsis neglecta), suggesting a potential benefit to the host plant harboring this fungus. I show that this fungus is inhabited by an endohyphal bacterium (Luteibacter sp.) and demonstrate that mycelium containing this bacterium produces significantly more IAA in vitro than the fungus alone. I predict that the general biochemical pathway used by the fungal-endohyphal complex is L-tryptophan-dependent and measure effects of IAA production in vivo, focusing on root and shoot growth in tomato seedlings.

As one of the most diverse and ecologically important clades of life, fungi are best known as pathogens, saprotrophs, mycorrhizae, and lichens. Yet an enormous amount of previously unknown diversity occurs among endophytic and endolichenic fungi--species-rich, horizontally transmitted fungi that live within asymptomatic photosynthetic structures such as leaves and lichens. Here, I explore the biodiversity of these understudied symbiotrophs and the ecological and biogeographic factors influencing their communities.To evaluate methods currently used in ecological studies of environmental samples of fungi, I assessed inter- and intraspecific divergence of a fast-evolving locus for four genera commonly found as endophytes, and compared analytical methods for identifying and delimiting OTUs. Then I used the most robust methods to show that after soil contact, seeds of a focal tree species contain diverse fungi that are closely related to endophytes and pathogens.To explore the ecological specificity of symbiotrophic fungi, I examined endophytic, endolichenic, and saprotrophic communities inhabiting physically proximate hosts in a biotically rich area of southeastern Arizona. I found that endolichenic fungi are largely distinct from plant-associated fungi, with the exception of a group of ecologically flexible symbionts that occur in lichens and mosses. Although numerous endophytes were found in non-living leaves, fungi that were highly abundant in leaf litter were seldom found as endophytes.To assess symbiotroph biodiversity and ecological specificity at a broad geographic and phylogenetic scale, I isolated>4100 endophytic and endolichenic fungi from diverse communities of plants and lichens across five climatic regions in North America. I found that the abundance, diversity, and composition of these nearly ubiquitous fungi differ as a function of climate, locality, and host. Differences among communities reflect environmental characteristics more strongly than geographic distance.Last, I addressed a series of hypotheses regarding the ecological specificity of fungi inhabiting living and non-living leaves. I show that like endophytes, saprotrophic communities are structured by environmental characteristics, and at small spatial scales by host and leaf status. Yet, differences in communities between living leaves and leaf litter suggest that most endophytes either rapidly complete their life-cycle or are out-competed by robust saprotrophs once leaves senesce.

Aster tataricus (Asteraceae) is a plant native to Northern Asia and known for its use in the Traditional Chinese and Japanese Medicine. Beside many other secondary metabolites, it contains pentapeptides called astins from which some show an antitumor activity against different human cell lines. Astins are chlorinated, cyclic pentapeptides consisting of proteinogenic and non-proteinogenic amino acids. The astin structure indicates the involvement of non ribosomal peptide synthetases as well as flavin-dependent halogenases. Both enzymes are currently only known from bacteria and fungi. A new endophytic fungus Pelliciarosea asterica was isolated from A. tataricus which produces some of the astins found in the different plant organs. The nearest neighbors of P. asterica are ostropalean fungi from the Stictidaceae lineage (Stictidaceae, Ostropales, Lecanoromycetes, Pezizomycetes, Ascomycota). P. asterica is located in all plant organs of A. tataricus but the highest accumulation of the fungus is found in rhizomes and above-ground organs like leaves or inflorescences. In contrast, the highest astin concentration was found in the roots where nearly no fungus was detectable. P. asterica produces only one of the dichlorinated astins (astin C) in liquid culture, but in A. tataricus all three forms of the dichlorinated astins (A/B and C) were found. This indicates that either the plant is “using” the fungal astin C and metabolize it into one of the other astins or that the fungus, once living inside the plant, is itself producing the other astins. It was also searched for a candidate gene of a halogenase which is essential for the dichlorination of the astins with an antitumor activity. No halogenase could be found by PCR or Southern as well as colony blot, neither in A. tataricus nor in P. asterica. Even the genome sequencing of P. asterica revealed no candidate gene for a halogenase. Endophytes support the plant by suppressing pathogens (antibiosis) or by providing additional nutrients like phosphates or iron to the plant. P. asterica can solubilize different phosphate sources on agar plates. Different fungi are inhibited in growth by P. asterica on agar plates. The endophyte P. asterica from A. tataricus supports its host in different ways and produces secondary metabolites. These secondary metabolites seem to be fungal metabolites either used or degraded by the plant. P. asterica is therefore a good alternative for a possible large-scale production of such antitumor acting astins.

Magister Scientiae - MSc (Biotechnology)
Endophytes are bacteria that have been shown in previous studies to promote plant growth without eliciting any sign of infection or plant disease, as well as promote bio-fortification and has thus gained much attention in agriculture. However, due to the limited confirmatory visual evidence of the in planta presence of bacteria, the significance of this plant-endophyte interaction has not been comprehensively understood. The aim of this study was to synthesise quantum dots to track the translocation of endophytes in a plant. Conventional methods track endophytes by transformation of green fluorescent protein which has the problem of fluorophore bleaching and blinking reducing detectability. Quantum dots (QDs) offer distinct advantages over organic dyes and fluorescent proteins for biological imaging applications because of their brightness, photostability, tunability and their extremely small size ensures little to no interference with the normal functions of the bacterium. This project required the synthesis of cadmium telluride QDs to be capped with mercaptopropionic acid thus making them biocompatible and the subsequent characterization techniques were performed. CdTe and Carbon QDs were transformed into the endophytic bacteria and were thereafter used to treat Brassica napus L plants. Physiological and biochemical assays were done to detect the effect of QDs on the plant and bacteria, respectively.
2020-04-30

>Magister Scientiae - MSc
In this study, the diversity of endophytic bacteria associated with food crops, sorghum (Sorghum bicolor L. Moench), pearl millet (Pennisetum glaucum L.) and groundnut (Arachis villosulicarpa) is investigated using culture-independent techniques: terminal retriction fragment length polymorphism (t- RFLP) and next generation sequencing (NGS). The first objective of this study was to investigate the effect of different DNA extraction protocols on mDNA yield and quality, as well as the diversity of endophytic bacteria retrieved from root and stem tissues (0.1g or 0.3g) of sorghum, pearl millet and groundnut. Protocols used include two classical methods (CTAB- and SDS-based) and five commercial kits: MoBio PowerPlant Pro® DNA Isolation Kit, Qiagen DNeasyR Plant Mini Kit, Fermentas GeneJET Plant Genomic DNA Purification Kit, MoBio PowerSoilTM DNA Purification Kit and MoBio UltraClean® Soil DNA Isolation Kit. Eletrophoresis and the Nanodrop were used to determine DNA yield and purity

Taxonomic diversity, biology and ecology of leaf endophytes were studied from some evergreen plants including Arbutus unedo, Buxus sempervirens, flex aquifolium, Laurus nobilis, Ligustrum vulgare, Prunus lusitanica, Rhododendron ponticum, Rhododendron sp., and Skimmia sp. from England, and some others from China. It was found: 1. A great number of fungal species, including several new species and new British records, have been isolated. Distribution patterns of endophyte assemblages and their variations between plant species and geographical locations are described. 2. Comparisons of leaf endophytes and saprobes of R. ponlicum at the same locality showed they belonged to two different ecological groups. This was further confirmed by study on endophytes and saprobes from a number of plant species growing in the same locality. 3. Host specificity of endophytic fungi at the species level was rare and this was supported by comparisons of endophyte assemblages from both taxonomically related (same family) and unrelated (different families) plant species. Molecular characterisations of Phyllosticta species confirmed this. 4. Infection and colonisation studies during a two year period showed that leaf endophytes of R. ponlicum were horizontally transmitted. Internal bud material was sterile and became infected by aerial spores. The infection and colonisation level of endophytes were strongly affected by environmental conditions. 5. Phylogenetic studies of Phyllosticta based on ITS 1-5.8s rDNA-ITS2 sequences concluded there was no evidence to show that the evolution of host plants of Phyllosticta species and ITS were related. Most Phyllosticta species from the same locality were found to have a broad host range and occurred on many taxonomically unrelated plants in the same locality. P. concentrica was separated into 4 species including P. concentrica on Hedera, P. arxii on Ilex, P. maxima on Rhododendron and P. taxi on Taxus.

The biological control of plant pests with beneficial microbes has become increasingly important over the last decades. Soil microbes such as fungi and bacteria colonise the roots of plants and promote their growth. Some beneficial microbes can trigger a weak plant defence response that enhances the immune response of the plant at subsequent pathogen attacks and therefore increase the resistance of the plant to other invaders. This mechanism is called “priming”. While biocontrol agents are applied against a variety of plant pests fundamental knowledge of the molecular mechanisms of plant-microbe interactions is still lacking. Especially molecular studies on the role of resistance genes in the interaction of plants with beneficial endophytic fungi are rare. In this study it was investigated how the fungal biocontrol agent Acremonium alternatum affects the development of the clubroot pathogen Plasmodiophora brassicae within the plant host Arabidopsis thaliana. Clubroot is a devastating disease in crop plants such as cabbage and rapeseed and causes abnormal root growth that leads to so called “club roots”. P. brassicae develops within the plant roots and forms resting spores that are very durable and stay infective in soils for up to 2 decades. The control of clubroot by chemical means is difficult and the disease continues to spread on all continents and was also found in Saxony, Germany in recent years. In 2 preliminary studies the co-inoculation of clubroot plants with the fungus A. alternatum resulted in reduced clubroot symptoms in Chinese cabbage and Arabidopsis. It was therefore hypothesised that A. alternatum induces resistance mechanisms in the plant and thus enhances immunity. The focus of this study was to test this hypothesis by carrying out expression analyses on root tissue of infected Arabidopsis plants. For this the plants were inoculated with spores of P. brassicae and A. alternatum before RNA was extracted from the roots, followed by cDNA synthesis and quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR). A microarray of root tissue of infected Arabidopsis plants was carried out to depict the events at the stage of initial root hair infection with the clubroot pathogen. The findings from the gene expression analyses were verified for 2 genes with Arabidopsis mutants that are defective in the respective gene and with 2 overexpressor lines. Clubroot symptoms were assessed by rating the root galls according to their stage of development. The overall plant health was further evaluated by recording the developmental stage of the plants (generative vs. vegetative), stem lengths and plant biomass. In addition, 2 local varieties of the economically important crop plant rapeseed (Brassica napus var. Ability and var. Visby) were investigated with qRT-PCR and by recording the disease parameters just described. A second goal of this study was to assess the general biocontrol potential of the yet relatively unknown endophyte A. alternatum in terms of enzymatic activity and competitive behaviour against other phytopathogenic fungi. The potential of this fungus for the use in integrative pest management was investigated. The results presented here are novel findings for this fungus and have not been studied before. The microarray from Arabidopsis roots revealed that the clubroot pathogen P. brassicae suppresses its recognition by pathogen receptors of the plant and thus prevents the host to induce resistance mechanisms. The fungus A. alternatum boosted the level of the pathogen recognition-related genes BAK1 and FLS2 and thus helped to establish early plant defence responses. PCR analyses confirmed that these early responses led to salicylic acid-dependent resistance in the plants which was maintained for several days as shown by elevated levels of the PATHOGENESIS-RELATED gene PR1. Marker genes for an alternative resistance pathway that is mediated over the plant signals jasmonate and ethylene were not activated in Arabidopsis. The co-inoculation of Arabidopsis plants with the endophyte A. alternatum resulted in a significant reduction of clubroot symptoms by up to 24%. In rapeseed the reduction of disease symptoms was 19% and 28% when the plants were treated with a crude cell wall extract of A. alternatum before inoculation with the clubroot pathogen. PCR analyses from Arabidopsis showed a strong response of pathogen recognition genes to the cell wall extract and spores of the endophytic fungus. In rapeseed all of the investigated pathogen recognition genes were upregulated after the endophyte treatment but not with the clubroot pathogen. Together with the PCR results from the microarray these findings suggest that A. alternatum primes its host plant and enhances the resistance of the plant towards P. brassicae. In addition, the fungus increased biomass, stem lengths and survival rates of clubroot-infected plants. In vitro tests revealed that the endophyte can solubilise phosphate and is not very competitive against other phytopathogenic fungi such as Aspergillus or Fusarium which is likely an effect of the relatively slow growth of the endophyte on agar plates. From this study it can be concluded that i) the fungus Acremonium alternatum induces resistance mechanisms in Arabidopsis and 2 Brassica napus cultivars and facilitates the recognition of the clubroot pathogen Plasmodiophora brassicae; ii) that Arabidopsis and Brassica react differently to this beneficial microbe, a fact that has been observed for Plasmodiophora and other microorganisms as well; iii) living spores are not necessary for clubroot biocontrol in rapeseed as a crude cell wall extract reduces symptoms more efficiently. Overall the endophyte A. alternatum is a very promising candidate for the use in integrative pest management in plant strengtheners or as biocontrol agent
Die biologische Kontrolle von Pflanzenkrankheiten gewinnt zunehmend an Bedeutung. Bodenbewohnende Mikroben wie Pilze oder Bakterien kolonisieren die Wurzeln von Pflanzen und fördern deren Wachstum. Einige dieser förderlichen Mikroben aktivieren eine schwache Abwehrreaktion in der Pflanze die sich verstärkt bei einer weiteren Infektion mit einem Krankheitserreger. Dieser Mechanismus, den man “Priming” nennt, führt zu einer verbesserten Resistenz der Pflanze gegenüber Pflanzenpathogenen. Obwohl natürliche Schädlingsbekämpfer bereits gegen eine Vielzahl an Krankheiten eingesetzt werden, weiss man über grundsätzliche molekulare Mechanismen dieser Pflanzen-Mikroben-Interaktionen nur wenig. Besonders die Rolle von Resistenzgenen ist bisher wenig erforscht, welche bei der Beziehung zwischen Pilzen und Pflanzen eine Rolle spielen. In der hier vorliegenden Arbeit wurde untersucht, wie der endophytische Pilz Acremonium alternatum die Entwicklung des Krankheitserregers Plasmodiophora brassicae in der Pflanze Arabidopsis thaliana beeinflusst. Die Kohlhernie, ausgelöst von P. brassicae, ist eine verheerende Krankheit die u. a. bei Kohl und Raps auftritt und Wurzelgallen, so genannte “Hernien”, hervorruft. Der Krankheitserreger entwickelt sich im Wurzelsystem der Pflanze und bildet Dauersporen, die bis zu 20 Jahre lang im Boden infektiös überdauern können. Ein Eindämmen der Krankheit mit Pflanzenschutzmitteln ist durch den komplexen Lebenslauf des Erregers sehr schwierig, das führte zu einer weltweiten Verbreitung der Kohlhernie. Auch in Sachsen wurden in den letzten Jahren Fälle von Kohlhernie gemeldet. Wie 2 Studien zeigen, führt die Ko-Inokulation von Kohlhernie-erkrankten Pflanzen mit A. alternatum zu einer Verringerung der Symptome in Chinakohl und Arabidopsis. Es wurde daher die Hypothese aufgestellt, dass der Pilz Resistenzmechanismen in der Pflanze anschaltet und damit ihre Immunität erhöht. Um diese Hypothese zu testen, wurden in der hier vorliegenden Studie Genexpressionsanalysen an infizierten Arabidopsiswurzeln durchgeführt. Dafür wurden die Pflanzen zunächst mit Sporen des Kohlhernieerregers und des Pilzes inokuliert, es wurde RNA aus den Wurzeln extrahiert, in cDNA umgeschrieben und diese mittels quantitativer Reverse-Transkriptase-Polymerasenkettenreaktion (RT-qPCR) untersucht. Ein Microarray von Wurzeln infizierter Pflanzen wurde durchgeführt um die Ereignisse abzubilden, die sich zeitnah nach der Infektion in den Wurzeln abspielen. Die Ergebnisse der Genexpressionsanalysen wurden dann an Arabidopsismutanten, die einen Gendefekt im jeweiligen Gen haben, und an Überexprimierer-Pflanzen verifiziert. Kohlherniesymptome an Pflanzen wurden durch eine Kategorisierung der Schadsymptome erfasst. Die allgemeine Pflanzengesundheit sowie der Entwicklungsstand der Pflanze, Stengellängen und das Frischgewicht wurden bestimmt. Zusätzlich wurden 2 Rapssorten, die in Sachsen angebaut werden, untersucht im Hinblick auf die Krankheitsenwicklung und die Reguation von Abwehrgenen. Ein weiteres Ziel dieser Arbeit war es das Biokontrollpotential des bisher schlecht untersuchten Pilzes A. alternatum zu bestimmen. Dazu wurde in vitro die Enzymaktivität des Pilzes getestet sowie seine Konkurrenzfähigkeit gegenüber anderen pflanzenpathogenen Pilzen. Das Potential des Pilzes für die Anwendung im integrierten Pflanzenschutz wurde getestet. Die hier präsentieren Ergebnisse stellen neue Erkenntnisse dar, die für diesen Pilz noch nie untersucht wurden. Der Microarray von Arabidopsiswurzeln zeigte, dass der Kohlhernieerregers die Erkennung durch die Pflanze verhindert und damit Abwehrmechanismen verhindert. Der Pilz A. alternatum förderte die Aktivität der pflanzlichen Erkennungsrezeptoren FLS2 und BAK1 und setzte damit die Erkennung von P. brassicae in Gang. PCR-Analysen ergaben, dass diese früh induzierten Abwehrmechanismen zu einer systemischen Resistenz in der Pflanze führte durch die Aktivierung des Pathogenese-relevanten Gens PR1. Genmarker, die die Aktivität eines alternativen, von Jasmonat und Ethylen vermittelten Abwehrweges anzeigen, waren nicht ativiert. Die Ko-Inokulation von Arabidopsis mit dem Endophyten führte zu einer signifikanten Reduktion der Krankheitssymptome um 24%. In Raps betrug die Reduktion 19% und 24% wenn die Pflanzen vor der Kohlhernie-Infektion mit einem Zellwandextrakt des Pilzes behandelt wurden. Mittels PCR konnte gezeigt werden, dass Gene für das Erkennen von Pathogenen in der Wurzel von Arabidopsis auf den Zellwandextrakt und Sporen des Pilzes reagieren. In Raps wurden alle der untersuchten Erkennungsgene aufreguliert nach der Infektion mit A. alternatum, nicht jedoch bei der Infektion mit P. brassicae. Zusammenfassend lässt sich sagen, dass der endophytische Pilz A. alternatum die Wirtspflanze auf eine folgende Infektion vorbereitet (Priming) und systemische Abwehr-mechanismen in der Pflanze induziert, wenn diese mit Kohlhernie infiziert ist. Außerdem treibt der Pilz das Sprosswachstum voran, erhöht die Biomasse und fördert das Überleben von Kohlhernie-infizierten Pflanzen. In vitro-Tests ergaben, dass der Endophyt Kalziumphosphat löslich machen kann und wenig kompetitiv gegenüber Pflanzenpathogenen wie Aspergillus oder Fusarium ist. Dies ist vermutlich mit dem langsameren Wachstum des Endophyten im Gegensatz zu den anderen Pilzen zu erklären. Aus den Ergebnissen dieser Arbeit lassen sich folgende Schlüsse ziehen: i) der endophytische Pilz Acremonium alternatum induziert Resistenzmechanismen in Arabidopsis und Raps und und fördert die Erkennung des Kohlhernieerregers Plasmodiophora brassicae; ii) Arabidopsis und Raps reagieren unterschiedlich auf diesen förderlichen Pilz, ein solcher Unterschied wurde bereits für Plasmodiophora und andere Mikroben beschrieben; iii) lebende Sporen des Pilzes sind nicht notwendig um Krankheitssymptome der Kohlhernie in Raps zu verringern, ein Zellwandextrakt von A. alternatum ist dafür besser geeignet. Ganz allgemein lässt sich sagen, dass der endophytische Pilz Acremonium alternatum ein sehr vielversprechender Kandidat ist für den Einsatz im integrierten Pflanzenschutz in Pflanzenstärkungsmitteln oder als Biokontrollorganismus

A temporal long-term study of the population structure and dynamics of the red alga Chondrus crispus at Lilstock, Somerset (Bristol Channel), the British Isles, and an investigation of its associated green and brown endophytic algal flora were undertaken from January 1994 to September 1996. The population of C. crispus was composed of plants which consisted of a basal holdfast from which arose numerous fronds in varying stages of development, with or without reproductive fronds (cystocarps or tetrasporangial sori). Plants were found with only gametophyte (haploid) or tetrasporophyte (diploid) fronds, whereas other plants described as mixed clumps consisted of gametophyte and tetrasporophyte fronds associated with the same basal disc. New plants entered the population and others were lost throughout the study. Individual plant longevity ranged from <46 days to >927 days. The monthly gametophyteitetrasporophyte ratio did not significantly deviate from 1:1, and within the population there was a stable long-lived core of plants, with a gametophyte:tetrasporophyte ratio of 1:1. Gametophyte plants with cystocarps had a significantly greater dry mass and contained a significantly greater number of fronds than tetrasporophyte plants with sori. Significantly higher numbers of tetrasporophytes had reproductive structures (sori) than gametophyte plants (cystocarps) during summer months (namely May, June and July). Both recruitment from spores and frond regeneration from remnant basal discs played an important role in maintaining the population structure. At least 6 green algal taxa including Acrochaete heteroclada, A. operculata, A. repens, A. viridis, Enteromorpha sp(p) and Pseudoendoclonium sp., and at least one brown algal taxon assigned to the genus Streblonema were found in association with gametophyte and tetrasporophyte C. crispus plants with cystocarps and sori, respectively. Spatial distribution of these algae within host tissues ranged from epiphytic through epiendophytic to endophytic. Of the C. crispus plants collected the frequency of plants infected with endophytic algae was reported to reach 95%. Infection by brown algae was rarely found without an associated green algal infection. It is hypothesised that a self-purging natural purification process of infected C. crispus fronds helps rid the plants and population of diseased fronds. It is suggested that it is primarily through the retention of the basal discs that C. crispus is an ecologically successful species.

Remediation of trichloroethylene (TCE) is a major priority for many contaminated sites all over the industrialized world including Hill Air Force Base, UT (HAFB). Phytoremediation as part of a cleanup strategy is an appealing option, and trees at HAFB have been studied to this end. Trees have also been used to delineate groundwater plumes because the passive uptake of organic contaminants by trees generally results in a direct relationship between tree and groundwater TCE concentrations if the trees are using the contaminated groundwater. However, the concentrations of plant-produced TCE metabolites can vary greatly. It was hypothesized that the endophytic microbial community present may be affecting the fate of TCE within these trees. This study was designed to determine if the microbial community present within hybrid poplar trees would change in response to TCE exposure. Trees were grown in a greenhouse to reduce environmental variability. Concentrations of TCE, its degradation products, and its metabolites were then measured in these trees. DNA was extracted from the tree tissues and amplified to determine the quantity of microbial DNA. Diversity of this DNA was determined by fragment analysis. Data were analyzed to determine if there was an effect of TCE treatment on the microbial community composition in the trees. Results showed that all tissues of dosed trees contained TCE. Metabolism of TCE to trichloroacetic acid (TCAA) and trichloroethanol (TCEtOH) in tree tissues was observed by the accumulation of these metabolites. Microbial DNA results revealed that TCE treatment reduced both the quantity and diversity of endophytic bacteria and fungi in several cases. Multivariate statistical analyses also showed that the endophytic microbial community shifted in response to TCE treatment. The endophytic microbial communities present in the hybrid poplar trees of this study were high in concentration and diversity, both of which were affected by TCE treatment. Endophytic bacterial concentrations were observed at over 109 copies/g with diversities of 70+ genetically distinct organisms. Decreases in these values with the addition of TCE showed that the community dramatically changed in some cases, but was able to more quickly adapt to TCE addition in other cases. The effects of these endophytic microorganisms associated with plants should therefore be included when phytoremediation is considered.

Masters of Science
Bacterial endophytes are able to improve the growth of their hosts through a number of different mechanisms such as nutrient uptake regulation, plant hormone production and regulation, siderophore production and phosphate solubilisation. They have also been shown to be able to provide protection to plants against various abiotic stressors, through various means such as oxidative stress protection. The purpose of this study was therefore to isolate endophytic bacteria from the stems of different weeds, to characterize their ability to use some of the most important growth promoting mechanisms including the ability to produce IAA, siderophores and ACC deaminase, what effect they had on the nutrient uptake in their hosts and to determine to what extent they could promote growth in the roots, stems and leaves of Brassica napus plants. In addition to this the endophytes were tested to see to what extent they could protect Brassica napus from the negative effects of vanadium stress and how this affected the plant physiologically in terms of morphology, overall biomass, the plants nutrient profile, lipid peroxidation and levels of cell death. The effect of vanadium stress on the oxidative state of Brassica napus was also monitored by determining the levels of stress induced reactive oxygen species (ROS) and the corresponding antioxidants that are responsible for regulating these reactive oxygen species. Six different endophytes (P1, P2, P3, P4, P5, P6) were isolated from different weed samples. Each endophyte was found to be able to significantly improve germination and growth in their host plant. Each isolate was able to improve the uptake of certain macronutrients and micronutrients in their respective hosts, while all of the isolates were shown to be capable of producing siderophores and ACC deaminase. One isolate had high levels of IAA production, with the remaining isolates producing small amounts of IAA. All isolates were also unable to solubilize phosphate. The five best performing endophytes (P1, P2, P3, P5, P6) in the preliminary growth trials were used in the follow up vanadium stressed growth trials, with endophyte P4 being left out of the remaining experiments. All of the endophytes showed improvements in growth promotion in comparison to the control, with endophyte treated plants showing both increased growth and biomass in both the non-stressed and vanadium stressed treatments of the vanadium stressed growth trial; however, the leaves of the vanadium stressed plants were significantly smaller than their non-stressed counterparts. When looking at the oxidative state it was found that vanadium stress caused a significant increase in the development of O2 -, H2O2 and •OH in the control and in addition to this it was shown that treatment with endophytes was able to cause a significant decrease in the levels of stress induced H2O2 and •OH in all of the treatments and O2 - for plants treated with endophyte P5. The noted change in the oxidative state of endophyte treated plants was attributed to an increase in the antioxidant activity of these plants, as it was found that endophyte treated plants showed a combination of increased activity for Superoxide dismutase, catalase and ascorbate peroxidase. This study has shown that endophytic bacteria from plant stems can be used to improve crop growth and yield, while simultaneously producing more nutrient dense crops from the same amount of land. It has also determined that endophytes P1, P2, P3, P5 and P6 are able to successfully provide protection to crop plants from the harmful effects of exposure to vanadium stress. This has great potential for improving food security locally and around the world, by allowing those who cannot gain access to large amounts of food to take in more nutrients from the same amount of food. Furthermore, it also presents the opportunity to use endophyte treatments to grow crops on land that has been previously contaminated with certain heavy metals.
2023-12-01

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

Mycorrhizas are ubiquitous symbiotic relationships between soil-inhabiting fungi and the roots of over 90 % of terrestrial plants. Mycorrhizal colonization of Wollemia nobilis Jones, Hill & Allen (Wollemi Pine), a newly discovered and extremely rare conifer native to southeastern Australia, is a largely unexplored subject. The objectives of this work were: (1) to assess mycorrhizal colonization of Wollemi Pines following fungal inoculation, (2) to identify endophytic fungi associated with Wollemi Pine roots growing in the field and in horticultural substrates, and (3) to evaluate the allelopathic activity of Wollemi Pines. In both laboratory and field environments, juvenile trees were inoculated with several different arbuscular mycorrhizal fungi and one fungus known to form ectendomycorrhizas on members of the Pinaceae. Mycorrhizal colonization was not evident in any of the inoculated plants; however, endophytic fungal structures formed by Cylindrocarpon pauciseptatum and Phialocephala fortinii were consistently present in the roots of plants grown in the field or in growth chambers. Preliminary evidence of allelopathic chemical production by Wollemi Pines was also found. Extracts of soil used to grow Wollemi Pines suppressed germination of leek seeds but not sorghum seeds. This thesis presents the results from the first mycorrhizal fungal inoculation of Wollemi Pine, the first identification of fungal endophytes colonizing Wollemi Pine roots, and the first evidence for the production of allelopathic chemicals by Wollemi Pine.

Magister Scientiae - MSc (Biotechnology)
Endophytic bacteria are known to have an endosymbiotic relationship with plants and provide them with many beneficial properties. These bacteria stimulate plant hormones, provide protection from pathogens and increase nutrient availability in the environment. In this study some of these potential growth factors were tested. Endophytic bacteria have the potential to be of great value for the increase of crop production. They offer a variety of processes that aid in plant growth promotion in an ecofriendly manner. The use of endophytic bacteria provides a cheaper and cleaner approach compared to industrial made fertilizers. They also have potential uses in bioremediation to clean the environment polluted by industrial processes. Endophytes were isolated and showed significant growth improvement. Each isolate displayed different morphologies. Isolates were tested for classical growth promotion mechanisms such as the ability to solubilize phosphate, Indole-3-acetic acid and siderophore production. Inductively Coupled Plasma Optical Emission Spectrometry was performed to measure the effect of the isolates on the plants nutrient profile. The isolates were then tested again while the plants were under heavy metal stress to determine if they were still capable of growth promotion. The plants were then assayed for cell death using Evans blue and biomass was measured to determine the effect of vanadium stress. Inductively Coupled Plasma Optical Emission Spectrometry was performed again to assess the change in nutrient profile while under vanadium stress.
2021-08-31