Energy sorrel (Rumex patientia L. x Rumex tianschanicus A. Los.) is a plant with a high yield of above-ground biomass. Because of this, it is grown as energy crop, but in Ukraine it is also important forage crop. Mycobiota has not been examined, even though energy sorrel is grown in the Czech Republic experimentally since 1992 (in agricultural operations since 2001). The aims of this thesis were: to determine the composition of mycobiota of healthy plants and of mycobiota associated with leaf and stem spots, to verify whether fungi isolated from spots can cause these spots and if previously isolated fungus Phomopsis sp. can cause spots. Fungi were cultivated on 2% malt extract agar and then morphologically determined. From representative strains were isolated DNA and sequences of the ITS region were compared with the GenBank databases, this verified the morphological determination and the sterile species were determined. In total 24 morphotypes of endophytic fungi were cultivated from healthy plants. The most common species were: Clonostachys rosea f. rosea, Cladosporium cladosporioides, Rhizopus arrhizus var. arrhizus, Alternaria alternata and Acremonium strictum. From the spots were isolated in total 29 morphotypes, the dominant species were: A. strictum, Aureobasidium pullulans, C....

The fungal microbiome, or “mycobiome” of plants is diverse and important to host health, but the fluxes of fungi among plant hosts and with the surrounding environment are poorly understood. In chapter two, we employed sterile culture techniques and spatial sampling to examine leaves as possible vectors for transfer of their endophytic fungi from the canopy to substrate on the forest floor, as predicted by the Foraging Ascomycete hypothesis. Some foliar endophytic fungal species are also present as wood-decomposing fungi on the forest floor, that transfer of mycelium across these two life history stages can occur, that endophytic life history stages are buffered from environmental conditions in comparison to wood-decomposing fungi, and that spatial linkages between the two life history stages can be observed. In another study, described in chapter 3, wood and leaf wood endophytes were sampled across a 25 ha plot, to explore landscape patterns of mycobiomes, and to explore the concept of a core microbiome in aerial plant tissues. We found that core microbiomes may be observed in a real ecological setting, but that the concept of core must be carefully defined and that some level of buffering from disturbance may be necessary to allow core microbiomes to assemble. In chapter four, we return to examine some of the assumptions and implications of the Foraging Ascomycete hypothesis, with an agent-based model. We model the conditions under which dispersal through falling leaves may represent a fitness-enhancing dispersal strategy for fungi, and that deforestation as is currently underway throughout the world may have impacts on fungi that rely upon a canopy- inhabiting life stage for dispersal. In chapter five, some challenges associated with environmental sampling of microbes using illumina© MiSeq sequences are critically examined. We find that biases introduced by random sampling at various stages of IVenvironmental DNA extraction and illumina© MiSeq sequencing are not well corrected by currently accepted bioinformatic algorithms. In addition, information loss from differential extraction, PCR amplification, and sequencing success, requires that users of MiSeq read libraries to interpret read abundances carefully. This dissertation includes previously published, co-authored material.

Diversos estudos evidenciam dezenas de espécies de fungos de diferentes gêneros nas colônias de formigas-cortadeiras. No entanto, a procedência desses microrganismos ainda é incerta. Alguns desses podem ser endofíticos oriundos do material vegetal transportado para o ninho. Assim sendo, determinados fungos endofíticos possuem a capacidade de contaminar a colônia das cortadeiras ou influenciar no seu microbioma simbiótico. Com isso, os endófitos fúngicos têm potencial no controle de formigas-cortadeiras como parceiros mutualistas das plantas. Esse sistema simbiótico pode se tornar uma estratégia no manejo dessa importante praga dentro do ecossistema florestal. O objetivo deste trabalho foi avaliar o potencial de colonização endofítica de mudas de Eucalyptus urophylla pelos fungos Escovopsis sp., Metarhizium anisopliae e Trichoderma strigosellum, e analisar, por meio da avaliação de características biométricas, a influência da inoculação desses microrganismos sobre o desenvolvimento das plantas. O delineamento adotado foi o inteiramente casualizado, usandose o esquema fatorial 4 x 3, com 15 repetições, sendo cada parcela constituída por uma muda. Os fatores foram: três espécies de fungos (Escovopsis sp., M. anisopliae, T. strigosellum) mais um controle e três métodos de inoculação (inoculação via foliar, inoculação via solo e inoculação via plântula). Para avaliação da colonização endofítica, utilizaram-se dez mudas, sendo cinco destinadas para avaliação das características biométricas. No método de inoculação via plântula, o fungo T. strigosellum foi isolado apenas nas raízes. Contudo, no método de inoculação via solo, além das raízes, esse fungo colonizou também o caule. As plantas de E. urophylla não foram colonizadas endofiticamente quando inoculadas pelo método de inoculação via foliar. As plantas inoculadas com o fungo T. strigosellum, pelo método de inoculação via plântula, apresentaram maiores valores nas características altura de plantas, número de folhas, massa seca da parte aérea e massa seca total quando comparado com os outros métodos de inoculação. Houve incremento também, por esse método, para a variável altura de plantas, quando comparadas às plantas controle e às inoculadas pelos fungos Escovopsis sp. e M. anisopliae. Dentre os fungos estudados, o isolado T. strigosellum colonizou endofiticamente o E. urophylla e influenciou positivamente no seu desenvolvimento, quando inoculado via plântula. / Several studies evidenced dozens of fungus species of different genera in the colonies of leafcutting ants. However, the origin of these microorganisms is still uncertain. Some of these may be endophytes from plant material transported to the nest. Thus, certain endophytic fungi have the ability to contaminate the leaf-cutting ant colony or influence its symbiotic microbiome. Thus, fungal endophytes have the potential to control leaf-cutting ants as mutual plant partners. This symbiotic system could become a strategy in the management of this important forest pest. The objective of this work was to evaluate the potential of endophytic colonization of Eucalyptus urophylla plants by fungi Escovopsis sp., Metarhizium anisopliae e Trichoderma strigosellum, and to analyze, by means of the evaluation of biometric characteristics, the influence of the inoculation of these microorganisms on the plant development. Experimental design was completely randomized, using the factorial scheme 4 x 3, with 15 replicates, each plot consisting of one plant. The factors were: three fungi species (Escovopsis sp., M. anisopliae, T. strigosellum) plus one control and three methods of inoculation (foliar inoculation, inoculation via soil and seedling inoculation). For the evaluation of endophytic colonization, ten plants were used; five of them were destined to evaluate the biometric characteristics. In the seedling inoculation method, the T. strigosellum fungus was isolated only in the roots. However, in the soil inoculation method, besides the roots, this fungus also colonized the stem. E. urophylla plants were not colonized endophiatically when inoculated by foliar inoculation method. The plants inoculated with T. strigosellum by the seedling inoculation method had higher values in the characteristics of plant height, number of leaves, root dry mass and total dry mass when compared to the other inoculation methods. There was also an increase, by this method, for the plant height variable, when compared to the control plants and those inoculated by the fungi Escovopsis sp. and M. anisopliae. Among the fungi studied, the isolate T. strigosellum endofitically colonized E. urophylla and positively influenced its development when inoculated via seedling.

Two hundred and forty three fungal isolates were obtained from roots of four Woollsia pungens (Cav.) F. Muell plants collected from a field site in New South Wales, Australia. 175 sterile isolates were slow growing and dark-coloured on 2 percentage malt agar and were selected for further analysis. Microsatellite-primed PCR using the primers (GACA)4 and (GTG)5 separated these isolates into 50 genets. Isolates representative of 43 genets (including 168 isolates in total) formed typical ericoid mycorrhizal structures when inoculated onto roots of Vaccinium macrocarpon Ait (Ericaceae), confirming their status as mycorrhizal endophytes. It was estimated that a minimum of 43 genetically-distinct mycorrhizal mycelial genets were present in the root systems of the sampled W. pungens population with 7 to 15 distinct endophytic genets identified in each host plant, indicating that considerable genetic diversity exists within the endophyte population. While most genets were represented by less than 8 isolates, 3 genets contained up to 41 isolates, suggesting that root system colonisation by some mycelia may be extensive. While most fungal genets were shown to be confined to individual plants, 2 genets (genets 32 and 33), however, were present within the root systems of 2 adjacent plants (plants C and D), suggesting that the two root systems were interconnected by the endophyte mycelia. The ITS region of 13 mycorrhizal endophytes and a non-mycorrhizal isolate selected from the endophyte population were sequenced and compared to the sequence of Hymenoscyphus ericae as well as sequences from the GenBank database. A phylogenetic tree was generated from the nucleotide sequence data. This analysis revealed that 6 putative taxa were present in the root systems of 4 host plants. No isolates were positively identified to genus or species level. Closest matches with fungal sequences in the database indicated that most isolates probably belonged to the order Leotiales. Cluster analysis on the basis of the ITS sequences indicated that H. ericae was not clustered together with any endophytes from W. pungens, suggesting that endophytes of W. pungens are not identical to the known ericoid mycorrhizal fungus H. ericae. H. ericae had a low degree of sequence similarity with isolates from W. pungens, with similarities ranging from 68.3-80.6%. Cluster analysis based on DNA sequences of the ITS region did not fully support the groupings inferred from microsatellite-based fingerprinting. / Master of Science (Hons)

Echinacea purpurea is a native herbaceous perennial with substantial economic value for its medicinal and ornamental qualities. Arbuscular mycorrhizae are symbiotic fungi that form relationships with plant roots and are known to enhance growth in the host. Mycorrhizae and other fungal endophytes often affect stress resistance and secondary metabolism in the host, as well as the ecology of other endophytes in the plant. A newly emerging paradigm in sustainable biotechnique is the targeted use of fungal endophytes to enhance growth and secondary metabolism in crops. Many of the therapeutic compounds in E. purpurea could be affected by fungal colonization. In this research the effects of inoculation of Echinacea purpurea with two classes of fungal endophytes: the arbuscular mycorrhizal fungi Glomus intraradices and Gigaspora margarita and the entomopathogenic endophyte Beauveria bassiana were evaluated . Endophyte colonization and impacts on plant growth and phytochemistry were tested in multiple greenhouse experiments. Arbuscular mycorrhizae and B. bassiana effectively colonized E. purpurea with some significant interactive effects. Consistent, substantial, and significant increases in all growth parameters were observed in mycorrhizal plants; mycorrhizal plants produced up to four times the biomass of controls in 12 weeks. Broad spectrum changes in fertilization were necessary to produce mycorrhizal and nonmycorrhizal samples of equal size, and severely nutrient-limited mycorrhizal E. purpurea seedlings maintained growth rates comparable to well fertilized samples. Treatment with B. bassiana had minor and inconsistent effects on some plant growth parameters, and there were significant interactive effects with arbuscular mycorrhizae. Phytochemical concentrations in all metabolite classes tested responded significantly to inoculation with both classes of fungal endophytes. Changes were observed in various pigments, caffeic acid derivatives, alkylamides, and terpenes. Many of the affected compounds have important roles in metabolism or have bioactive value as natural products. When considered from a net production perspective (concentration X dry weight), compared to controls, plants inoculated with endophytes produced as much as 30 times the content of some compounds in 12 weeks. This work effectively demonstrates that fungal endophytes can enhance the bioactivity of plant tissues and the production of natural products in E. purpurea.

Phytoremediation systems for petroleum hydrocarbons rely on a synergistic relationship between plants and their root-associated microbial communities. Plants exude organic compounds through their roots, which increase the density, diversity and activity of plant-associated microorganisms, which in turn degrade hydrocarbons. Understanding the mechanisms driving this relationship poses one of the more intriguing challenges in phytoremediation research. This study was designed to address that challenge. Plant-microbe interactions in a weathered-hydrocarbon contaminated soil were examined under controlled growth chamber, and field conditions. In both environments single-species grass treatments initially facilitated greater total petroleum hydrocarbon (TPH) degradation than <i> Medicago sativa </i> (alfalfa), mixed species, or control treatments. In growth chamber studies increased degradation was linked to increased aliphatic-hydrocarbon degrader populations within the rhizosphere. Under field conditions, specific recruitment of endophytic aliphatic-hydrocarbon degraders in response to high TPH levels may have facilitated increased degradation by the grass <i> Elymus angustus</i>(Altai wild rye, AWR). AWR stably maintained these communities during times of local drought, enabling them to act as subsequent source populations for rhizosphere communities. The broad phylogenetic diversity of AWR endophytes, compared to the <i> Pseudomonas</i>-dominated communities of other plants, contributed to the observed stability. The relative composition of exudates released by plants also impacted both degradation activity and potential. Alfalfa released higher concentrations of malonate, which hindered degradation by decreasing metabolic activity and concomitantly inhibiting catabolic plasmid transfer. In contrast, AWR exudates contained high levels of succinate, which was linked to increased catabolic gene expression and plasmid transfer. A reciprocal relationship between exudation patterns and endophytic community structure likely exists, and both parameters have a specific influence on rhizosphere degradation capacity. In this study, grasses were more successful in maintaining the specific balance of all parameters required for the transfer, preservation, and stimulation of hydrocarbon catabolic competency.

Phytoremediation systems for petroleum hydrocarbons rely on a synergistic relationship between plants and their root-associated microbial communities. Plants exude organic compounds through their roots, which increase the density, diversity and activity of plant-associated microorganisms, which in turn degrade hydrocarbons. Understanding the mechanisms driving this relationship poses one of the more intriguing challenges in phytoremediation research. This study was designed to address that challenge. Plant-microbe interactions in a weathered-hydrocarbon contaminated soil were examined under controlled growth chamber, and field conditions. In both environments single-species grass treatments initially facilitated greater total petroleum hydrocarbon (TPH) degradation than <i> Medicago sativa </i> (alfalfa), mixed species, or control treatments. In growth chamber studies increased degradation was linked to increased aliphatic-hydrocarbon degrader populations within the rhizosphere. Under field conditions, specific recruitment of endophytic aliphatic-hydrocarbon degraders in response to high TPH levels may have facilitated increased degradation by the grass <i> Elymus angustus</i>(Altai wild rye, AWR). AWR stably maintained these communities during times of local drought, enabling them to act as subsequent source populations for rhizosphere communities. The broad phylogenetic diversity of AWR endophytes, compared to the <i> Pseudomonas</i>-dominated communities of other plants, contributed to the observed stability. The relative composition of exudates released by plants also impacted both degradation activity and potential. Alfalfa released higher concentrations of malonate, which hindered degradation by decreasing metabolic activity and concomitantly inhibiting catabolic plasmid transfer. In contrast, AWR exudates contained high levels of succinate, which was linked to increased catabolic gene expression and plasmid transfer. A reciprocal relationship between exudation patterns and endophytic community structure likely exists, and both parameters have a specific influence on rhizosphere degradation capacity. In this study, grasses were more successful in maintaining the specific balance of all parameters required for the transfer, preservation, and stimulation of hydrocarbon catabolic competency.

Echinacea purpurea is a native herbaceous perennial with substantial economic value for its medicinal and ornamental qualities. Arbuscular mycorrhizae are symbiotic fungi that form relationships with plant roots and are known to enhance growth in the host. Mycorrhizae and other fungal endophytes often affect stress resistance and secondary metabolism in the host, as well as the ecology of other endophytes in the plant. A newly emerging paradigm in sustainable biotechnique is the targeted use of fungal endophytes to enhance growth and secondary metabolism in crops. Many of the therapeutic compounds in E. purpurea could be affected by fungal colonization. In this research the effects of inoculation of Echinacea purpurea with two classes of fungal endophytes: the arbuscular mycorrhizal fungi Glomus intraradices and Gigaspora margarita and the entomopathogenic endophyte Beauveria bassiana were evaluated . Endophyte colonization and impacts on plant growth and phytochemistry were tested in multiple greenhouse experiments. Arbuscular mycorrhizae and B. bassiana effectively colonized E. purpurea with some significant interactive effects. Consistent, substantial, and significant increases in all growth parameters were observed in mycorrhizal plants; mycorrhizal plants produced up to four times the biomass of controls in 12 weeks. Broad spectrum changes in fertilization were necessary to produce mycorrhizal and nonmycorrhizal samples of equal size, and severely nutrient-limited mycorrhizal E. purpurea seedlings maintained growth rates comparable to well fertilized samples. Treatment with B. bassiana had minor and inconsistent effects on some plant growth parameters, and there were significant interactive effects with arbuscular mycorrhizae. Phytochemical concentrations in all metabolite classes tested responded significantly to inoculation with both classes of fungal endophytes. Changes were observed in various pigments, caffeic acid derivatives, alkylamides, and terpenes. Many of the affected compounds have important roles in metabolism or have bioactive value as natural products. When considered from a net production perspective (concentration X dry weight), compared to controls, plants inoculated with endophytes produced as much as 30 times the content of some compounds in 12 weeks. This work effectively demonstrates that fungal endophytes can enhance the bioactivity of plant tissues and the production of natural products in E. purpurea.

Bacterial endophytes, bacteria residing within plants, play an important role in the growth and development of plants and their ability to thrive under adverse conditions. The endophytes of Acer negundo, Ulmus pumila and Ulmus parvifolia trees sampled from a hydrocarbon-contaminated site were analyzed for variation between seasons and plant species. Branches from the same trees over a span of 3 seasons were collected and analyzed via culture dependent and culture independent methods. Numerous culture independent approaches were tested, culminating in the development of a new method for the amplification of endophytic bacterial ribosomal DNA that excludes plastid DNA. Community analyses using this new method in combination with T-RFLP showed significant differences between the endophytic communities of different plants species and of the same species growing in different seasons. The proposed technique can be used for the future study of endophytic communities of plants.

Bacterial endophytes, bacteria residing within plants, play an important role in the growth and development of plants and their ability to thrive under adverse conditions. The endophytes of Acer negundo, Ulmus pumila and Ulmus parvifolia trees sampled from a hydrocarbon-contaminated site were analyzed for variation between seasons and plant species. Branches from the same trees over a span of 3 seasons were collected and analyzed via culture dependent and culture independent methods. Numerous culture independent approaches were tested, culminating in the development of a new method for the amplification of endophytic bacterial ribosomal DNA that excludes plastid DNA. Community analyses using this new method in combination with T-RFLP showed significant differences between the endophytic communities of different plants species and of the same species growing in different seasons. The proposed technique can be used for the future study of endophytic communities of plants.