Bacterial viruses, also known as phages, are intrinsic components of the Earth’s Critical Zone (CZ). Together with diverse communities of bacteria and fungi, they occupy habitats of the CZ extending from the vegetation canopy, through the soils and into the aquifers. In this thesis, I aimed to study the transport of phages in the upper CZ and their interactions with non-host soil bacteria and fungi to reveal their role in regulating the CZ microbial life. To reflect transport processes of CZ-inhabiting phages in soil tracers are highly useful. Thus, in the first study, I evaluated the transport efficiency and particle intactness of marine tracer phages passing through soil. Marine phages were selected as tracer, because they are non-pathogenic, non-toxic, naturally absent and thus non-multiplying in the terrestrial subsurface. I found that the marine phages PSA-HM1 and PSA-HS2 retained high phage particle intactness in contrast to commonly used Escherichia virus T4. This suggests their potential as particle tracers to mimic the transport of (bio-) colloids of similar traits in soil. Soil in the CZ is often unsaturated and restricts mobility of microorganisms. Fungi bridge unsaturated zones in soil and hence provide network for microbial transport. In the second study, I developed a hyphosphere model system mimicking unsaturated soil environment, and reported on the ability of hyphal-riding bacteria to co-transport lytic phages and to utilize phages as “weapons” for improved colonization of water-unsaturated habitats. As the findings emphasize the importance of hyphal transport of bacteria and associated phages, in the third study, I developed a hyphae-assisted approach and isolated five soil bacteria able to co-transport phages. In analogy to invasion frameworks in macroecology, the hyphosphere model system with bacteria and co-transported phages can be useful models to simulate processes of biological invasion at micro-scale. In the fourth study, I investigated dormant phages (i.e. prophages) that are widespread in the CZ and can be induced under environmental stress. I found that volatile fungal metabolites can act as triggers for prophage induction and may exert long-distance manipulation of prophage activity thereby affecting microbial community and nutrient cycling in soil. Altogether, the findings may help to elucidate transport processes of phages in the CZ and to reveal the role of phages in the CZ microbial ecosystem. Approaches (e.g. phage as tracers) and findings (e.g. phage-bacterial co-transport) may also serve as useful tools for testing hypothesis in other disciplines, such as hydrogeology, invasion ecology and chemical ecology.

Fungi and bacteria co-inhabit a wide variety of habitats, and their interactions are significant drivers of many ecosystem services and functions. Creating unique microenvironments, fungal mycelia and their surroundings (defined here as the mycosphere) allow for spatially distinct fungal bacterial activities and interactions at the microscale. Fungi in particular modulate the mycosphere pH and oxygen as the drivers and/or the results of various fungal processes. However, due to the microscopic diameters of hyphae (typically 2-10 μm), it is experimentally difficult to non-invasively access themycosphere to thereby analyze the local pH and oxygen on hyphae or around mycelia. Hence, in this thesis, I aimed to develop and deploy microscale techniques to analyzethe mycosphere pH and oxygen in vitroand thereby to further resolve their influences on the local microbial life for a better understanding of mycosphere habitat properties and functioning.

A imensa diversidade de micro-organismos no solo leva a uma inevitável riqueza de interações entre espécies. Neste intuito, esse projeto é inovador na identificação dos cogumelos da Mata Atlântica, e na descrição da comunidade bacteriana associada às suas micosferas. Usando corpos de frutificação dos fungos (cogumelos) como indicadores para sistemas ricos em nutrientes, as amostras foram coletadas para investigar as interações entre os fungos (maioria do domínio Basidiomycota) e as bactérias presentes no solo em volta das micélios fúngicos (ambiente micosférico). As análises foram feitas com técnicas independentes de cultivo (análise PCR-DGGE, sequenciamento Sanger de fragmentos de ITS/18S e pirosequenciamento de tags da região V4 de 16S DNAr). As famílias fúngicas Marasmiaceae e Lepiotaceae, do domínio Basidiomycota foram as mais abundantes entre os cogumelos amostrados (13 e 5 cogumelos, respectivamente), e estavam presentes entre todas as três parcelas de estudo. As demais amostras foram alocadas dentro das famílias Marasmiaceae, Lepiotaceae, Inocybaceae, Lachnocladiaceae, Bolbitiaceae, Entolomataceae, Hygrophoraceae, Hymenogastraceae, Mycenaceae e Strophariaceae, além de dois do domínio Ascomycota. Baseado na análise de DGGE, é bastante claro que existe uma grande diferença na comunidade bacteriana (de toda a comunidade bacteriana, de ?- proteobacteria e de ?-proteobacteria) entre os solos associados ou não com os corpos de frutificação. Os dados de pirosequenciamento indicaram que dentro dos tratamentos as amostras se agruparam baseado nas famílias fúngicas ou no substrato onde os cocumeglos ocorrem (solo ou serrapilheira), sendo clara em algumas micosferas as alterações na ocorrência de grupos microbianos. O grupo de UTOs mais induzidas na região da micosfera foi composto dos grupos Burkholderia, Acidobacteria Gp1, Comamonadaceae, Sphingobacteriaceae, Burkholderiaceae, Chitinophagaceae), Schlesneria, Acidobacteria Gp3 e Spartobacteria gênero incertae sedis. Isto indica que existe um processo de seleção para bactérias específicas dependendo das diversas variáveis e fatores ambientais presentes no microhabitat micosfera. / The immense microbial diversity in the soil leads to inevitable richness in inter-species interactions. For this reason, this is a novel project that focuses on identifying mushrooms and the associated bacterial community with their mycospheres in the Brazilian Atlantic Rainforest. Using fungal fruiting bodies (mushrooms) as indicators of nutrient-rich systems, samples were taken to investigate the interactions between fungi (mostly of the domain Basidiomycota) and bacteria present in the soil surrounding fungal mycelia (mycosphere environment). The analyses were conducted using culture-independent techniques, where isolating DNA of bacteria and/or mushrooms attempts to provide information on microbial functionality. These culture-independent analyses (PCR-DGGE, Sanger sequencing of ITS/18S fragments, and pyrosequencing of tags from the V4 region of 16S rDNA) generated extensive data on the bacterial diversity selected for in the presence of fungal structures in the soil. The fungal families Maramiaceae and Lepiotaceae, of the domain Basidiomycota were the most abundant among the mushrooms sampled (13 and 5 mushrooms, respectively) and were present in all of the three sampling sites. The rest of the mushrooms were found to be within the families Marasmiaceae, Lepiotaceae, Inocybaceae, Lachnocladiaceae, Bolbitiaceae, Entolomataceae, Hygrophoraceae, Hymenogastraceae, Mycenaceae, and Strophariaceae, in addition to two of the domain Ascomycota. Based on DGGE analysis, it is clear that there is a great difference in the bacterial community (entire bacterial community, of ?- proteobacteria and of ?-proteobacteria) between all fungal fruiting body-associated and non-associated soils. The pyrosequencing data indicated that within each treatment group, the samples did not separate according to fungal families or substrate where the mushrooms were found (in the soil or amoung the leaf litter). However, some mycospheres exhibited clearly altered bacterial communities. The group of OTUs most induced in the mycosphere region consisted of Burkholderia, Acidobacteria Gp1, Comamonadaceae, Sphingobacteriaceae, Burkholderiaceae, Chitinophagaceae), Schlesneria, Acidobacteria Gp3, and Spartobacteria genus incertae sedis. This indicates that there is a selection process for specific bacteria depending on a wide range of variable and environmental factors acting in the mycosphere microhabitat.

A imensa diversidade de micro-organismos no solo leva a uma inevitável riqueza de interações entre espécies. Neste intuito, esse projeto é inovador na identificação dos cogumelos da Mata Atlântica, e na descrição da comunidade bacteriana associada às suas micosferas. Usando corpos de frutificação dos fungos (cogumelos) como indicadores para sistemas ricos em nutrientes, as amostras foram coletadas para investigar as interações entre os fungos (maioria do domínio Basidiomycota) e as bactérias presentes no solo em volta das micélios fúngicos (ambiente micosférico). As análises foram feitas com técnicas independentes de cultivo (análise PCR-DGGE, sequenciamento Sanger de fragmentos de ITS/18S e pirosequenciamento de tags da região V4 de 16S DNAr). As famílias fúngicas Marasmiaceae e Lepiotaceae, do domínio Basidiomycota foram as mais abundantes entre os cogumelos amostrados (13 e 5 cogumelos, respectivamente), e estavam presentes entre todas as três parcelas de estudo. As demais amostras foram alocadas dentro das famílias Marasmiaceae, Lepiotaceae, Inocybaceae, Lachnocladiaceae, Bolbitiaceae, Entolomataceae, Hygrophoraceae, Hymenogastraceae, Mycenaceae e Strophariaceae, além de dois do domínio Ascomycota. Baseado na análise de DGGE, é bastante claro que existe uma grande diferença na comunidade bacteriana (de toda a comunidade bacteriana, de ?- proteobacteria e de ?-proteobacteria) entre os solos associados ou não com os corpos de frutificação. Os dados de pirosequenciamento indicaram que dentro dos tratamentos as amostras se agruparam baseado nas famílias fúngicas ou no substrato onde os cocumeglos ocorrem (solo ou serrapilheira), sendo clara em algumas micosferas as alterações na ocorrência de grupos microbianos. O grupo de UTOs mais induzidas na região da micosfera foi composto dos grupos Burkholderia, Acidobacteria Gp1, Comamonadaceae, Sphingobacteriaceae, Burkholderiaceae, Chitinophagaceae), Schlesneria, Acidobacteria Gp3 e Spartobacteria gênero incertae sedis. Isto indica que existe um processo de seleção para bactérias específicas dependendo das diversas variáveis e fatores ambientais presentes no microhabitat micosfera. / The immense microbial diversity in the soil leads to inevitable richness in inter-species interactions. For this reason, this is a novel project that focuses on identifying mushrooms and the associated bacterial community with their mycospheres in the Brazilian Atlantic Rainforest. Using fungal fruiting bodies (mushrooms) as indicators of nutrient-rich systems, samples were taken to investigate the interactions between fungi (mostly of the domain Basidiomycota) and bacteria present in the soil surrounding fungal mycelia (mycosphere environment). The analyses were conducted using culture-independent techniques, where isolating DNA of bacteria and/or mushrooms attempts to provide information on microbial functionality. These culture-independent analyses (PCR-DGGE, Sanger sequencing of ITS/18S fragments, and pyrosequencing of tags from the V4 region of 16S rDNA) generated extensive data on the bacterial diversity selected for in the presence of fungal structures in the soil. The fungal families Maramiaceae and Lepiotaceae, of the domain Basidiomycota were the most abundant among the mushrooms sampled (13 and 5 mushrooms, respectively) and were present in all of the three sampling sites. The rest of the mushrooms were found to be within the families Marasmiaceae, Lepiotaceae, Inocybaceae, Lachnocladiaceae, Bolbitiaceae, Entolomataceae, Hygrophoraceae, Hymenogastraceae, Mycenaceae, and Strophariaceae, in addition to two of the domain Ascomycota. Based on DGGE analysis, it is clear that there is a great difference in the bacterial community (entire bacterial community, of ?- proteobacteria and of ?-proteobacteria) between all fungal fruiting body-associated and non-associated soils. The pyrosequencing data indicated that within each treatment group, the samples did not separate according to fungal families or substrate where the mushrooms were found (in the soil or amoung the leaf litter). However, some mycospheres exhibited clearly altered bacterial communities. The group of OTUs most induced in the mycosphere region consisted of Burkholderia, Acidobacteria Gp1, Comamonadaceae, Sphingobacteriaceae, Burkholderiaceae, Chitinophagaceae), Schlesneria, Acidobacteria Gp3, and Spartobacteria genus incertae sedis. This indicates that there is a selection process for specific bacteria depending on a wide range of variable and environmental factors acting in the mycosphere microhabitat.

Dans les écosystèmes forestiers, la décomposition du bois est un processus majeur, notamment impliqué dans le cycle du carbone et des nutriments. Les champignons basidiomycètes saprotrophes, incluant les pourritures blanches, sont les principaux agents de cette décomposition dans les forêts tempérées. Bien que peu étudiées, des communautés bactériennes sont également présentes dans le bois en décomposition et cohabitent avec ces communautés fongiques. L'impact des interactions bactéries-champignons sur le fonctionnement d'une niche écologique a été décrit dans de nombreux environnements. Cependant, leur rôle dans le processus de décomposition du bois n'a été que très peu investigué. A partir d'expériences en microcosme et en utilisant une approche non cultivable, il a été démontré que la présence du champignon Phanerochaete chrysosporium influençait significativement la structure et la diversité des communautés bactériennes associées au processus de décomposition du hêtre (Fagus sylvatica). Par une approche cultivable, cet effet mycosphère a été confirmé, se traduisant par une augmentation de la densité des communautés bactériennes en présence du champignon ainsi que par une modification de la diversité fonctionnelle de ces communautés. Enfin, une approche polyphasique a été développée, combinant l'analyse des propriétés physico-chimiques du bois et des activités enzymatiques extracellulaires. Les résultats de cette expérience ont révélé que l'association de P. chrysosporium avec une communauté bactérienne issue de la mycosphère de ce dernier aboutissait à une dégradation plus importante du matériau bois par rapport à la dégradation par le champignon seul, démontrant pour la première fois des interactions bactéries-champignons synergiques dans le bois en décomposition / Wood decomposition is an important process in forest ecosystems in terms of their carbon and nutrient cycles. In temperate forests, saprotrophic basidiomycetes such as white-rot fungi are the main wood decomposers. While they have been less studied, bacterial communities also colonise decaying wood and coexist with these fungal communities. Although the impact of bacterial-fungal interactions on niche functioning has been highlighted in a wide range of environments, little is known about their role in wood decay. Based on microcosm experiments and using a culture-independent approach, we showed that the presence of the white-rot fungus Phanerochaete chrysosporium significantly modified the structure and diversity of the bacterial communities associated with the degradation of beech wood (Fagus sylvatica). Using a culture-dependent approach, it was confirmed that in the presence of the fungus the mycosphere effect resulted in increased bacterial abundance and modified the functional diversity of the fungal-associated bacterial communities. Lastly, a polyphasic approach simultaneously analysing wood physicochemical properties and extracellular enzyme activities was developed. This approach revealed that P. chrysosporium associated with a bacterial community isolated from its mycosphere was more efficient in degrading wood compared to the fungus on its own, highlighting for the first time synergistic bacterial-fungal interactions in decaying wood