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In agricoltura, i pesticidi sono stati usati molto frequentemente per salvaguardare le colture dagli attacchi di parassiti e dalle malattie. Questi pesticidi, oltre a uccidere gli organismi target, molte volte colpiscono anche gli organismi non-target. Tra gli organismi non-target, possiamo individuare molti microrganismi utili a determinare la fertilità e la qualità del terreno. La presenza di questi xenobiotici nel terreno può influenzare i principali cicli biogeochimici (N, C, S, P) e altre vie metaboliche (es. β-ketoadipate). In questo studio abbiamo analizzato gli effetti di isoproturon, tebuconazole e chlorpyrifos sull’abbondanza, sulla struttura e sulla diversità della comunità microbica. Inoltre, abbiamo anche studiato gli effetti di questi pesticidi sui geni coinvolti nel ciclo dell’azoto. Si è potuto notare che l’abbondanza della comunità batterica è molto influenzata dall’applicazione del fungicida tebuconazole . Per quanto riguarda gli studi sulla funzionalità e diversità della popolazione microbica, l’applicazione di questi pesticidi sembra non indurre una chiara dose-dipendente e un effetto tempo. Diversamente, in relazione all’analisi sulla diversità microbica, possiamo affermare che l’applicazione di questi tre pesticidi ha influenzato il numero di OTU rilevate; tuttavia, l’indice di diversità (H’) ci dice che l’uso di questi pesticidi porta ad un incremento della diversità all’interno dei campioni trattati. In conclusione, è possibile affermare che l’applicazione di questi pesticidi influenza l’abbondanza e la funzionalità della popolazione microbica, ma non induce una diminuzione della diversità all’interno della medesima comunità. / In agriculture, pesticides have been frequently used to protect crops from pest and disease attacks. Many times such pesticides, besides killing the target organisms, hit non-target organisms. Among the non-target organisms, we can find many useful microorganisms that determine fertility and soil quality. The presence of these xenobiotics in soil can influence the main biogeochemical cycles (N, C, S, P) and other metabolic pathways (eg. Β-ketoadipate). In this study, we investigated the effects of isoproturon, tebuconazole and chlorpyrifos on the abundance, the structure and the diversity of the microbial community. We have also studied the effects of these pesticides on the genes involved in the nitrogen cycle. It was observed that the abundance of the bacterial community is significantly affected by the application of the fungicide tebuconazole. As for the studies on the functionality and the diversity of the bacterial population, the application of these pesticides does not seem to induce a clear dose-dependent nor a time effect. On the contrary, with respect to the analysis on microbial diversity, we observed that the application of these three pesticides did influence the number of detected OTU, whereas the diversity index (H') tells us that the use of such pesticides leads to an increase of diversity within the treated samples. Finally, we can conclude that the application of these pesticides affects the abundance and function of the microbial population, but does not lead to lower diversity within the same community.

The effects of managing soil with organic amendments were examined with respect to soil microbial community dynamics, macroaggregate formation, and plant physio-genetic responses. The objective was to examine the possibility of managing soil microbial communities via soil management, such that the microbial community would provide agronomic benefits. In part one of this research, effects of three amendments (hairy vetch residue, manure, compost) on soil chemical and microbial properties were examined relative to formation of large macroaggregates in three different soils. Vetch and manure promoted fungal proliferation (measured via two biomarkers: fatty acid methyl ester 18:2ω6c and ergosterol) and also stimulated the greatest macroaggregate formation. In part two of this research, effects of soil management (same amendments as above, inorganic N fertilization, organic production) on soil chemical and microbial properties were examined relative to the expression of nitrogen assimilation and defense response genes in tomato (Solanum lycopersicum L.). Soil management affected expression of a nitrogen assimilation gene (GS1, glutamine synthetase) and several defense-related genes. The GS1 gene was downregulated with inorganic N fertilization, expression of the pathogenesis-related PR1b gene (which codes for the pathogenesis-related PR1b protein) was increased in plants grown in soil amended with compost, vetch, and N fertilizer, and expression of three other defense-related genes coding for chitinase (ChiB), osmotin (Osm), and β-1,3-glucanase (GluA) were decreased in plants from soil amended with manure and in plants from the organically managed soil. Differential expression of defense-related genes was inversely related to the relative abundance of Gram-negative bacteria. The relative abundance of the 18:1ω7c Gram‑negative bacterial biomarker was greatest in manure treated soil and in organically managed soil (which recieves seasonal manure applications). These treatments also had the lowest expression of ChiB, Osm, and GluA, leading to speculation that manure, through increases in Gram-negative bacteria, may have suppressed populations of soil organisms that induce a defense response in plants, possibly allowing for less-stressed plants. Outcomes of this research may be useful for those interested in developing management strategies for maintaining or improving soil structure as well as those interested in understanding management effects plant physio-genetic responses.

The behaviour and impact of pharmaceuticals in the environment are still poorly understood. Pharmaceuticals are widely used and continually released into the environment causing increasing concerns about their impact on the environment beyond the intended human or veterinary use. Prescribed pharmaceuticals, typically, enter the environment either through excretion after human use or disposal of surplus medication. Sewage treatment plants do not completely remove pharmaceuticals and their metabolites and these have been detected in sewage treatment plant effluent and receiving waters. Land application of treated sewage effluent is widely practiced in New Zealand as an alternative to surface water discharge. Methods were developed to determine selected pharmaceuticals in environmental matrices such as sewage effluent, sewage solids, soil, and soil water. From these, pharmaceutical removal efficiencies were determined for three common sewage treatment processes; activated sludge, composting and land application of sewage effluent. The impacts of some common pharmaceuticals on soil microbial communities, together with the effect of prolonged exposure to sewage effluent on these communities were examined. Additionally, toxicity of sewage effluent, and toxicity mechanisms of specific pharmaceuticals were investigated using luminescent micro-organisms and lettuce seedlings. Pharmaceuticals were successfully detected in sewage effluent, sewage solids, compost, soil and soil water. The sewage treatment processes investigated, including land application, were able to remove or reduce pharmaceutical concentrations in sewage. In case of land application, volcanic soils were more efficient than sandy soils in pharmaceutical removal while irrigation rate and level of sewage pre-treatment also showed some effect on removal efficiency. Pharmaceuticals were not acutely toxic at environmental levels currently detected. Exposure of microbial communities to unnaturally high levels of pharmaceuticals did demonstrate that most pharmaceuticals were potentially able to induce stress in the microbial community although microbes were able to metabolise some of these pharmaceuticals. Twelve years of effluent irrigation resulted in microbial adaptation to aspirin, acetaminophen and tetracycline, indicating that these pharmaceuticals had an effect on microbial community. Presently, land application of treated sewage waste may be a suitable treatment for additional pharmaceutical removal provided that land application schemes are designed appropriately, and pharmaceutical accumulation in the soil is occasionally monitored. It would be prudent to recognise the potential risk that could be caused by chronic exposure to pharmaceuticals such that continued vigilance may lead to future indications of chronic effects at an early stage.

I investigated the effects ofhabitat loss and subsequent restoration on invertebrate community structure and ecosystem functioning in a mined peat bog in the North Island, New Zealand. In an experimental trial, the impact of peat bog habitat loss and isolation on the invertebrate community associated with Sporadanthus ferrugineus (Restionaceae) was investigated. Potted S. ferrugineus plants were exposed to invertebrates at various distances up to 800 m from an intact habitat (the presumed source population) over 18 weeks. Invertebrates rapidly colonised the experimental plants, with all major Orders and trophic groups present on Sc ferrugineus within 6 weeks. However. with increasing distance away from the undisturbed habitat, there was a significant decrease in total richness and abundance of invertebrates associated with the potted plants. Additional tests showed that even a moderate degree of isolation (i.e. greater than 400 m) from the intact habitat caused an almost complete failure of 'Batrachedra' sp. to colonise its host plant, at least in the short-term, The density of eggs and larvae, and the average larval size of 'Batrachedra' sp. (Lepidoptera: Coleophoridae) colonising Si ferrugineus plants, as well as the proportion of Si ferrugineus stems damaged by 'Batrachedra' sp. herbivory, all decreased logarithmically with increasing distance from the intact habitat. Surprisingly, though, the rate of recovery of the insect-plant interaction following experimental habitat restoration was remarkably rapid (i.e. between 3Y2 and 6 years). After just 6 years there was no significant difference in insect-plant interactions between the intact peat bog sites and any of the experimentally restored sites up to 800 m away. These results suggest that the degree of isolation from undisturbed habitat has a major impact on the rate and patterns of restoration recovery in the invertebrate community and that some insect-plant interactions can recover rapidly from habitat loss with restoration management. Restoration of mined peat bogs in northern New Zealand is initiated by establishing a native vegetation cover to minimize further peat degradation. The effects of various restoration techniques on litter decomposition, microbial community activity and beetle community composition were investigated within an experimental trial, These treatments included translocation ofpeat bog habitat (direct transfer of islands), milled peat islands with no seed and milled peat islands with seed, and were compared with an unrestored mined site and an undisturbed peat bog. In all the response variables measured, the undisturbed peat bog sites had significantly higher decomposition rates and microbial respiration rates, and significantly higher abundance and species richness of beetles than any of the restoration treatments. Inaddition, the technique used to restore mined peatlands had a significant effect on the beetle community composition and litter decomposition processes. Despite a rapid initial change in the beetle community following habitat translocation, the direct transfer islands were still the most similar in beetle species composition to the undisturbed peat bog. Microbial activity and decomposition rates were higher in the direct transfer and mined peat surface after 6 months. However, even after 12 months, decomposition rates in the restored habitats were still far from reaching the levels recorded in the undisturbed peat bog. The results suggest that beetle community structure and ecosystem processes such as decomposition and microbial activity rates may be able to recover faster with certain restoration techniques, such as direct transfer of intact habitat islands. Subsequently, I examined long-term beetle community reassembly on islands that had been restored by creating raised areas ofprocessed peat with the addition of Leptospermum scoparium seed. Monitoring of different-aged restored islands representing the full range of restoration ages (up to 6 years) available at the peat mine, indicated that as the peat islands became older and the vegetation structure became more complex, the abundance, species richness and composition of the beetle community became increasingly similar to the community in the undisturbed peat bog. Despite this, distinct differences between the intact peat bog and older restored peat islands still persisted, even after 6 years, particularly at an individual species level. However, it is predicted that within 12 years the restored peat islands will share 100% ofbeetle species in common with the undisturbed peat bog. Taken together, these results indicate that restoration is effective in initiating the recovery of beetle assemblages and ecosystem processes (such as litter decomposition and microbial community activity) in cut-over peat bogs. However, it is estimated to take at least 12 years before pre-mining communities and functions are attained, and ongoing monitoring to develop an understanding of the longer-term dynamics of such ecosystems and processes is clearly required.

The field of microbial ecology has just entered a new era of rapid technological development and generation of big data. The high-throughput sequencing techniques presently available provide an opportunity to extensively inventorize the blueprints of life. Now, millions of microbes of natural microbial communities can be studied simultaneously without prior cultivation. New species and new functions (genes) can be discovered just by mining sequencing data. However, there is still a tremendous number of microorganisms not yet examined, nor are the ecosystem functions these carry out. The modern genomic technologies can contribute to solve environmental problems and help us understand ecosystems, but to most efficiently do so, methods need to be continuously optimised.   During my Ph. D. studies, I developed a method to survey eukaryotic microbial diversity with a higher accuracy, and applied various sequencing-based approaches in an attempt to answer questions of importance in environmental research and ecology. In PAPER-I, we developed a set of 18S rRNA gene PCR primers with high taxonomic coverage, meeting the requirements of currently popular sequencing technologies and matching the richness of 18S rRNA reference sequences accumulated so far. In PAPER-II, we conducted the first sequencing-based spatial survey on the combined eukaryotic and bacterial planktonic community in the Baltic Sea to uncover the relationship of microbial diversity and environmental conditions. Here, the 18S primers designed in PAPER-I and a pair of broad-coverage 16S primers were employed to target the rRNA genes of protists and bacterioplankton for amplicon sequencing. In PAPER-III, we integrated metagenomic, metabarcoding, and metatranscriptomic data in an effort to scrutinise the protein synthesis potential (i.e., activity) of microbes in the sediment at a depth of 460 m in the Baltic Sea and, thus, disclosing microbial diversity and their possible ecological functions within such an extreme environment. Lastly, in PAPER-IV, we compared the performance of E. coli culturing, high-throughput sequencing, and portable real-time sequencing in tracking wastewater contamination in an urban stormwater system. From the aspects of cost, mobility and accuracy, we evaluated the usage of sequencing-based approaches in civil engineering, and for the first time, validated the real-time sequencing device in use within water quality monitoring.   In summary, these studies demonstrate how DNA sequencing of microbial communities can be applied in environmental monitoring and ecological research. / <p>Yue Hu was supported by a scholarship from the China Scholarship Council (CSC #201206950024)</p><p>Yue Hu has been publishing papers under the name "Yue O. O. Hu".</p><p>QC 20170403</p>

Os microorganismos estão entre os maiores responsáveis pela transformação dos compostos orgânicos em uma lagoa de estabilização e são a chave do sucesso deste tratamento. A utilização de técnicas de biologia molecular em conjunto é uma das melhores formas para obter resultados mais confiáveis. A estrutura da comunidade microbiana na lagoa de estabilização facultativa da estação de tratamento de esgoto doméstico do Município de Cajati foi descrita baseada nos padrões dos fragmentos do gene RNAr 16S por PCR-DGGE, CARD-FISH, biblioteca de clones de gene RNAr 16S e sequenciamento. Os padrões obtidos com o DGGE foram correlacionados com as variáveis ambientais coletadas por análise de redundância (RDA). As amostras foram coletadas em três períodos para o estudo da variação temporal, coletou-se em três profundidades (superfície, 0,7 m e 1,5 m) para o estudo espacial vertical e em nove pontos a 0,7 m de profundidade para o estudo espacial horizontal da comunidade. / Microorganisms are amongst the most responsible for the conversion of sewage organic compounds and they are the key of the treatment success. The combination of two or more molecular techniques in this kind of assessment allows getting more accurate results concerning a microbial community. The facultative stabilization pond microbial community structure of domestic sewage treatment from Cajati city was characterized based on 16S RNAr gene fragments patterns from PCR-DGGE, FISH, 16S RNAr gene clone library and sequencing. The patterns obtained by DGGE were co-related to environmental variables by redundancy analysis (RDA). The samples were collected in three intervals to study the seasonal variation, it was collected in three depth (surface, 0,7 m and 1,5 m) to the vertical assessment and in nine spots at 0,7 m depth for the horizontal assessment of the microbial community.

O Brasil é considerado o maior produtor mundial de cana-de-açúcar, voltada para a produção de açúcar, etanol e derivados. O aproveitamento de resíduos da usina, como torta de filtro e vinhaça no condicionamento do solo pode contribuir com a manutenção da sua fertilidade. Por outro lado, a cada operação agrícola necessária para o cultivo da cana-de-açúcar está associada uma emissão de GEE que deve ser contabilizada. O objetivo desta pesquisa foi estimar a emissão dos principais GEE (CO2, N2O e CH4) pela torta de filtro, e avaliar as alterações de alguns atributos das comunidades microbianas durante o processo de decomposição. Após avaliação de uma enquete sobre o modo de utilização da torta de filtro por diversas usinas, um estudo foi desenvolvido na Usina Costa Pinto localizada em Piracicaba (SP). A torta aplicada no sulco de plantio da cana foi monitorada quanto à emissão dos gases, sendo que as concentrações dos mesmos nas amostras foram determinadas por cromatografia gasosa. Os atributos microbiológicos examinados foram a biomassa, a atividade enzimática (fosfatase ácida, alcalina e ?-glicosidase) e a estrutura da comunidade através do polimorfismo de fragmento de restrição terminal (T-RFLP). Foi observada emissão significativa dos principais GEE predominantemente da torta quando aplicada nos sulcos de plantio, com destaque para o N2O, com uma proporção doze vezes maior em massa do que o CH4, em 56 dias de experimento. Quanto aos aspectos microbiológicos, o maior valor encontrado de carbono e nitrogênio da biomassa microbiana para a dose usualmente aplicada (25 Mg ha-1), foi com dois meses de experimento, com respectivamente 484,89 ?g C g solo seco-1 e 62,95 ?g N g solo seco-1, e correlacionado pelo coeficiente de Pearson com a atividade enzimática no material. Pela técnica de T-RFLP foi possível avaliar a estrutura dos Domínios de Archaea, Bacteria e Fungi na comunidade microbiana da torta de filtro. Não houve modificação dessa estrutura ao longo do tempo analisado. Os resultados obtidos reforçam a importância dos atributos microbiológicos aliados a fatores químicos e físicos e a influência dos mesmos sobre as emissões de GEE. / Brazil is the greatest worldwide producer of sugarcane with production of sugar, ethanol and derived. Usually applied to soil as fertirrigation, filter cake and vinasse on soil conditioning have contributed to the maintenance of fertility. On the contrary, each agricultural operation is associated to GHG emissions that must be accounted for the balance of products. This work aims evaluate GHG emissions (CO2, N2O and CH4) from the filter cake, as well as evaluate the main differences in the microbiological community available within the decomposition process. After evaluation of a survey about to use the filter cake by industries, the study has been developed at Usina Costa Pinto located in Piracicaba (SP). The filter cake applied to the row of sugarcane planting have been monitored by taking regular samples of the emissions. The concentration of the three gases in the samples has been determined by gas chromatography. The microbiological aspects has been evaluated by biomass, enzymatic activity (acid phosphatase, alkaline phosphatase, and beta-glycosidase) and the community structure through terminal restriction fragment length polymorphism (T-RFLP). Significant GHG emission has been observed; mainly from the filter cake applied to the row of sugarcane planting especially N2O, with ratio twelve times greater than CH4 in 56 days of experiment. For microbiological aspects, the maximum of carbon and nitrogen from the microbial biomass for the treatment usually applied (25 Mg ha-1), within two months of experimentation, with respectively 484,89 ?g C g dry soil-1 e 62,95 ?g N g dry soil-1, and correlated by the coefficient of Pearson with the enzymatic activity existent in the material. The T-RFLP analysis has allowed evaluate the community structures of Archaea, Bacteria e Fungi in the microbiological community of the filter cake. Modification in the community structures was not observed over this time examined. The results obtained reinforce the importance of microbiological aspects combined with chemical and physical factors and their influence on GHG emissions.

Plants regulate responses to their environment through complex hormone signaling; these hormones can be categorized broadly into two categories: growth and defense, though many have roles in both. Much remains to be understood about the complexity of hormone signaling in relation to environmental responses, especially species- and genotype-specific differences. Unraveling this complexity of hormone signaling will lead to the development of resilient crops that are able to respond appropriately to their environment. In this dissertation, I hypothesize novel roles for growth and defense hormones in <i>Solanum </i>spp. responses to 1) biochar, a black carbon soil amendment (Chapter 2), 2) infection with<i> Ralstonia solanacearum</i>, an economically important soilborne pathogen causing bacterial wilt (Chapter 3), and 3) endophytic colonization by the soil bacterial community (Chapter 4). In Chapter 2, I showed that biochar upregulates GA signaling and affects GA-related traits in a species- and cultivar-specific manner. Biochar amendment also downregulates defense signaling. In Chapter 3, I demonstrated a novel role for auxin in resistance against <i>R. solanacearum, </i>including differential expression of auxin signaling genes in resistant genotype H7996 compared to susceptible WV in response to <i>R. solanacearum</i> infection. In addition, I observed stronger and faster upregulation of defense hormone marker genes for SA and ET in H7996 compared to WV. In Chapter 4, I showed that SA and ET are required for normal tomato root microbial community assembly, affecting the colonization of a few key taxa in order to promote alpha diversity. H7996 and WV root communities differ in alpha diversity, and a panel of H7996 x WV RILs showed quantitative variation in alpha diversity that correlated negatively with the abundance of these key taxa. In conclusion, I elucidated novel roles for hormones in responses to the soil environment, pathogen infection, and root community colonization. These findings are important for developing resilient, sustainable crops.

En Europe, le bois est la première source d’énergie renouvelable. La transition énergétique se traduit par une intensification de l’exploitation des forêts. L’effet de ces pratiques sylvicoles sur les communautés microbiennes du sol est encore peu étudié. Au cours de ma thèse, j’ai évalué les conséquences d’une manipulation artificielle de matière organique en forêt tempérée sur la diversité fonctionnelle et taxonomique des communautés bactériennes et fongiques telluriques dans six sites expérimentaux (réseau expérimental MOS). Parallèlement, une caractérisation fonctionnelle des communautés microbiennes a également été réalisée dans un contexte proche des réalités de l’intensification des pratiques sylvicoles sous climat tropical en plantation d’Eucalyptus. Si certains descripteurs fonctionnels de la dégradation de la matière organique sont particulièrement informatifs, les activités microbiennes de dégradation de la chitine, polymère azoté des arthropodes et champignons, sont apparues très sensibles au retrait de matière organique. C’est pourquoi, par des approches de génomiques comparatives, nous avons cherché à estimer le potentiel chitinolytique des différentes guildes fongiques des sols. En conditions contrôlées, nous avons ensuite quantifié les capacités potentielles de mobilisation et de transfert du carbone et de l’azote, à partir d’une matière organique microbienne riche en chitine, par un champignon ectomycorhizien en symbiose avec son hôte. Enfin, la généricité des fonctions chitinolytiques d’un plus large spectre d’espèces fongiques ectomycorhiziennes a été évaluée par le couplage d’approches enzymatiques et isotopiques. L’ensemble de nos résultats met en lumière le rôle significatif des champignons ectomycorhiziens dans la mobilisation du carbone et de l’azote à partir de certaines formes de matière organique, et la nécessité de prendre en compte le compartiment microbien dans les études d’impact des pratiques sylvicoles / One of the main usages of wood in Europe is renewable energy supply that implies intensification of forest management to respond to this increasing demand. However, the impact of intense forestry practices on soil microbial communities remains poorly investigated. In the frame of my PhD thesis, I evaluated effects of artificial organic matter removal on functional and taxonomical diversity of soil bacterial and fungal communities in temperate forest, using six experimental sites across France (INRA MOS experimental network). In parallel, I also characterised impact of intensified forest management practices on functional microbial communities in tropical plantation of Eucalyptus trees. This work permitted to identify several sensitive functional indicators of organic matter degradation. Notably, the degradation of chitin – a nitrogen polymer main component of arthropods and fungal cell walls – was revealed to be particularly sensitive to organic matter removal. Genomics and enzymatic approaches were then used to estimate chitinolytic potentials of the different genera of soil fungi. In controlled conditions, we were able to quantify ectomycorrhizal fungus carbon and nitrogen mobilisation and transfer capacities from chitin enriched organic matter to its host during symbiotic interaction. Finally, we evaluated chitinolytic functions of ectomycorrhizal fungi at large scale by combining enzymatic and isotopic approaches. Taken together, the results acquired in the frame of my PhD thesis, illustrate the significant role of ectomycorrhizal fungi in carbon and nitrogen mobilisation from organic matter. We particularly highlight that microbial compartment in soil must be considered in studies of forest management practices