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10 Abstract All plants live in symbiosis with fungal endophytes - they can form mutualistic, commensal or parasitic symbioses. Symbiosis of root endophytes, called for their darkly pigmented and septated hyphae dark septate endophytes (DSE), and plants has been often overlooked although its role for plant communities can be very important. Despite their ubiquitous presence in roots of terrestrial and also aquatic plants the influence of DSE on their host plants is still unresolved. Results of previous studies are inconsistent - some reported that DSE have positive effects on their host plant growth and some negative. The main reason for this inconsistency might be their complicated taxonomy and difficult identification of different cryptic species which are morphologically indistinguishable. We were able to complete a unique collection of the most common DSE species, mainly members of the Phialocephala fortinii - Acephala applanata species complex. The collection includes the most common DSE species isolated from roots of forest plant communities. The goal of my thesis was to describe behaviour of DSE in roots of typical forest plants and elucidate their physiological influence on host plants. In vitro resynthesis experiments were used to observe root colonization patterns. Nutrient flow between the plant...

Neotyphodium species, endophytic fungi associated with cool-season grasses, enhance host fitness and stress tolerance, but also produce biologically active alkaloids including ergot alkaloids associated with fescue toxicosis in grazing animals. One approach to reduce fescue toxicosis is to manipulate genes in the ergot alkaloid pathway. The gene, dmaW, encoding the first pathway-specific step in ergot alkaloid biosynthesis, was cloned previously from Claviceps spp. and its function was demonstrated by expression in yeast. Putative homologs have been cloned from Neotyphodium coenophialum (from tall fescue) and Neotyphodium sp. Lp1 (from perennial ryegrass). In order to confirm the function of dmaW in ergot alkaloid production, dmaW in Neotyphodium sp. isolate Lp1 was knocked out by gene replacement. The dmaW knockout mutant produced no detectable ergovaline or simpler ergot alkaloids. Complementation with Claviceps fusiformis dmaW restored ergovaline production. These results confirmed that the cloned endophyte gene was dmaW, and represented the first genetic experiments to show the requirement of dmaW for ergot alkaloid biosynthesis. Neotyphodium coenophialum, endophyte of the grass tall fescue (Lolium arundinaceum) has two homologs of dmaW. Considering the possible field applications in future, the Cre/lox site-specific recombination system was chosen because of the potential to sequentially knock out both homologs and obtain marker-free dmaW mutants of N. coenophialum. One homolog, dmaW-2, was disrupted by marker exchange, and the marker was eliminated by Cre, thus demonstrating the application of Cre/lox system in N. coenophialum to eliminate a marker gene. The dmaW-2 knockout did not eliminate ergovaline production, indicating that the dmaW-1 was probably also active in N. coenophialum. A putative ergot alkaloid biosynthesis gene cluster was identified in Claviceps purpurea and C. fusiformis. C. purpurea and C. fusiformis produce different subsets of ergot alkaloids. Identification of nine common genes between them suggests the possible role of these genes in the early part of the ergot alkaloid biosynthetic pathway.

Os microrganismos endofíticos vêm sendo estudados na busca de novas espécies e pela sua importância nas aplicações biotecnológicas. Podem ser isolados de sementes, raízes, folhas e em frutos de diversas espécies de plantas. Além disso, pouco se sabe a respeito desses microrganismos associados as folhas e frutos da melancia. Diante disso, o objetivo deste trabalho foi isolar e identificar bactérias e leveduras endofíticas e associadas às folhas e frutos de melancia, assim como avaliar o potencial biotecnológico dos isolados de leveduras. As amostras foram obtidas de frutos oriundos de Brasília-DF e de plantas de Formoso do Araguaia-TO. Para o isolamento dos microrganismos foram adotados diferentes procedimentos para folhas: fragmentação e trituração, testando-se quatro condições de desinfestação superficial e para os frutos: trituração, enriquecimento e filtração em membrana. A identificação e caracterização molecular foi realizada pela técnica de PCR colony para bactérias e pela extração do DNA genômico de leveduras e bactérias. O DNA dos isolados foi amplificado utilizando oligonucleotídeos P027F e R1357 para bactérias, e NL1 e NL4 para leveduras. Para os isolados de leveduras foram realizados os testes de antagonismo contra oito fungos fitopatogênicos e a capacidade de produzir ácido indolacético (AIA). De acordo com resultados, foram obtidos 380 isolados de bactérias e 240 leveduras endofíticas e associadas, com maior número de isolados no tecido foliar, sendo identificadas 71 bactérias e 92 leveduras. Foram identificados 12 gêneros de bactérias, com o gênero Pantoea sp. isolado em maior quantidade. Para os isolados de leveduras, foram identificados 5 gêneros, com destaque para a espécie Sporidiobolus pararoseus, obtida de folhas e frutos de melancia. Os isolados de leveduras associadas TAIL 04 (99% Saccharomyces cerevisiae) e TAIL 63 (99% Pseudozyma antarctica) apresentaram atividade de antagonismo para os fungos Sclerotinia sclerotirum, Alternaria sp., Curvularia lunata, Rhizoctonia solane e Pyricularia oryzae. Este também foi inibido pelos isolados TAIL 95 (99% Hannaella sinensis) e TAIL 66 (100% Sporidiobolus pararoseus) e apresentou maior inibição de 32,82% com o isolado S. cerevisiae (TAIL 04). Simultaneamente, os isolados S. cerevisiae (TAIL 04) e P. antarctica (TAIL 04) mostraram capacidade em produzir ácido indolacético (AIA) nas concentrações de 21,14 μg/mL e 15,79 μg/mL, respectivamente, em após 7 dias de incubação. Conclui-se que folhas e frutos de melancia possuem importantes bactérias e leveduras endofíticas e associadas de interesse biotecnológico, como promotores de crescimento da planta e como antagonistas, possibilitando o seu uso no desenvolvimento de bioprodutos. / Endophytic microorganisms have been studied in the identification of new species and for their importante in biotechnological applications. Can be isolated them from seeds, roots, leaves and in fruits of several plant species. Besides, the relation between these microorganisms and watermelon leaves and fruits is far from being well known. Therefore, the main of this work was to identify both endophytic and associated bacteria and yeasts to leaves and fruits of watermelon, as well as evaluate biotechnological potential applications of yeast isolate. The samples were obtained of fruits from Brasília-DF and plants from Formoso do Araguaia-TO. For the isolation of the microorganisms were used different procedures for the leaves: fragmentation and crushing, by testing four surface disinfection conditions and for the fruits: crushing, enriching and filtration in membrane. Molecular identification and characterization was realized by the colony PCR technique for bacteria and the extraction of genomic DNA from yeasts and bactéria. The DNAs of the isolates was amplified using oligonucleotides P027F and R1357 for bacteria and NL1 and NL4 for yeast. For the yeasts isolate tests of antagonisms for phytopathogenic and producers of indoleacetic acid (AIA) were carried out. A total of 380 bacteria and 243 endophytic and associated yeasts were isolated, obtaining a higher number of isolates from the watermelon leaf tissue and 71 bacteria and 92 yeasts were identified. Twelve genus of bacterias were identified, with the gener Pantoea sp. isolated in larger quantity. For yeasts isolated were identified five genus, with featured for the specie Sporidiobolus pararoseus obtained of leaves and fruits of watermelon. Associated yeasts isolates TAIL 04 (99% Saccharomyces cerevisiae) and TAIL 63 (99% Pseudozyma Antarctica) showed antagonistic activity for the fungi Sclerotinia sclerotirum, Alternaria sp., Curvularia lunata, Rhizoctonia sp., Didymella sp. and Pyricularia oryzae. This also was inhibied by TAIL 95 (99% Hannaella sinensis) and TAIL 66 (100% Sporidiobolus pararoseus) isolated and showed greater inhibition of 32,82% with S. cerevisiae (TAIL 04). Simultaneously, S. cerevisiae (TAIL 04) and P. Antarctica (TAIL 63) showed capacity to producer indoleacetic acid (IAA) in concentrations of 21,14 μg/mL and 15,79 μg/mL, respectively, in 7 days of incubation. It is concluded that watermelon leaves and fruits have importants endophytics and associated bacterias and yeasts of biotechnological interest as grownth promoters and antagonistics, making possible their use in the development of bioproducts.

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.

Plant microbiomes are increasingly appreciated as major drivers of plant health and ecosystem services, and are of ever-greater interest for their potential in human applications. However, plant-associated microorganisms often live in complex associations in nature. Here, I characterize one of these microbial associations: the symbiosis between foliar fungal endophytes and their bacterial endosymbionts (endohyphal bacteria, EHB). EHB influence fungal phenotypes and can shape the outcomes of plant-fungal interactions. EHB are thought to form facultative associations with many foliar fungi in the species-rich Ascomycota, but little is known about how these symbioses are initiated and maintained, or how EHB shape the ecology of their fungal hosts. In this study, I assessed factors mediating the relationships between two foliar fungi (Microdiplodia sp., Dothideomycetes; Pestalotiopsis sp. Sordariomycetes) and their EHB. I first established methods for introducing EHB into axenic mycelia of their fungal hosts, providing an important step forward for understanding the establishment of EHB associations and a critical tool for experimental tests of the effects of EHB on fungal phenotypes. Through experiments in vitro, I found that both the identity of the fungal host and the conditions under which fungi and bacteria are grown influence the establishment of EHB/fungal associations. Moreover, I showed EHB of foliar fungi can be transferred across fungal classes, thus creating experimental strains that could be used for the first time to examine the contribution of each symbiont to important fungal traits. Using these strains I evaluated how EHB influence the capacity of foliar fungi to degrade plant material as saprotrophs. I found that the presence and identity of EHB significantly influenced fungal growth on particular media, cellulase and ligninase activity, and mass loss from senescent tissue of their native host plant species in a partnership-specific manner. Because EHB can be acquired horizontally, they may help shape plant-fungal interactions, resultant ecosystem services, and the functional diversification of plant-associated fungi along the saprotroph-endophyte continuum. By manipulating EHB/fungal interactions in new ways, we can potentially influence fungal phenotypes for diverse human applications.

Significant phylogenetic codivergence between plant or animal hosts (H) and their symbionts or parasites (P) indicate the importance of their interactions on evolutionary time scales. However, valid and realistic methods to test for codivergence are not fully developed. One of the systems where possible codivergence has been of interest involves the large subfamily of temperate grasses (Pooideae) and their endophytic fungi (epichloae). Here we introduce the MRCALink (most-recent-common-ancestor link) method and use it to investigate the possibility of grass-epichloё codivergence. MRCALink applied to ultrametric H and P trees identifies all corresponding nodes for pairwise comparisons of MRCA ages. The result is compared to the space of random H and Ptree pairs estimated by a Monte Carlo method. Compared to tree reconciliation the method is less dependent on tree topologies (which often can be misleading), and it crucially improves on phylogeny-independent methods such as ParaFit or the Mantel test by eliminating an extreme (but previously unrecognized) distortion of node-pair sampling. Analysis of 26 grass species-epichloё species symbioses did not reject random association of H and P MRCA ages. However, when five obvious host jumps were removed the analysis significantly rejected random association and supported grass-endophyte codivergence. Interestingly, early cladogenesis events in the Pooideae corresponded to early cladogenesis events in epichloae, suggesting concomitant origins of this grass subfamily and its remarkable group of symbionts.

Pinus radiata is the most planted tree in the southern hemisphere. The planted trees are especially susceptible to pathogens, but even the native population, nowadays limited tomerely five locations, are threatened by diseases caused by arthropods, fungi and dehydration. Endophytes are bacteria or fungi that reside inside healthy plant tissue, and often have a beneficial effect on their hosts. Endophytes can help plants adapt to abiotic stress such as drought and protect them against pathogens and insect pests. Given the roles that endophytes play in host stress responses, it is possible that without studying endophytes we may not fully understand a plant’s response to increased temperatures and climate-induced disease.Using Illumina-sequencing of the 16S rRNA-gene the bacterial endophyte community in 15 trees from three of the remaining native populations were studied. By investigating trees from several sites geographical community differences were discovered. The three overall most dominating bacterial taxa can all be connected with genera known to contain members withanti-fungal properties.

Fungal endophytes of the genus Neotyphodium form stable symbiotic associations, with grasses, that are symptomless and generally considered to be mutualistic. The benefits that these fungi confer to their grass hosts are exploited in pastoral agriculture systems. The production of a range of secondary metabolites, specifically alkaloids including peramine and ergovaline can give their host plants an ecological advantage in certain environments. Neotyphodium endophytes are asexual and have lost the ability to transfer horizontally between hosts making seed transmission a vital feature of the association. This thesis reports the occurrence of phenotypically different perennial ryegrass plants (Lolium perenne) in a population infected with Neotyphodium lolii. Here we show that the change in the plants is directly attributable to a variant endophyte that they host. Isolation of the variant endophyte reveals a change in colony growth compared to the wild-type resident endophyte in the population, which has a white and cottony phenotype. Colonies of the variant endophyte are smaller than wild-type colonies and mucoid, with hyphal filaments forming aggregates. Evidence shows that the switch between colony morphologies occurs at a very high frequency, is reversible, and appears to be environmentally induced. This suggests that the switching phenomenon involves gene regulation rather than mutation. When endophyte-free plants are infected, with either white and cottony (wild-type) or mucoid (variant) fungal colonies, they assume a morphology consistent with the state of the fungus at the time of inoculation, that is normal or dwarfed, respectively. In addition, re-isolation of endophyte from either normal or dwarfed plants always yields white and cottony or mucoid colonies, respectively, suggesting that the host environment stabilizes the state of the fungus. Proteomic profiling revealed differences in protein expression between plants infected with either the wild-type or mucoid fungus. Furthermore, host plants containing the mucoid fungus have never flowered or produced seed. Thus, if this change in the fungal symbiont occurs in a competitive natural environment the mucoid fungus and its host plant may not persist beyond the first generation. This thesis provides insights into the plastic nature of fungal endophyte/grass symbiota and discusses possible mechanisms for the observed morphological switching in culture and host dwarfing.

Thesis (Ph. D.)--University of Kentucky, 2008. / Title from document title page (viewed on May 12, 2008). Document formatted into pages; contains: xvi, 221 p. : ill. (some col.). Includes abstract and vita. Includes bibliographical references (p. 214-219).