Dissertationen zum Thema „Rhizosphere microbiota“

Limiting environmentally harmful consequences of crop production while increasing productivity amid climatic modifications is one of the major challenges facing agriculture in the XXIst century. Crucially, over the course of the next 30 years, innovative strategies are required to tackle this challenge and ensure a sustainable and safe access to food resources to a global population of over 9 billion people in 2050. One of these strategies proposes to exploit the microbial communities thriving in association with plant roots, collectively referred to as the rhizosphere microbiota, to uncouple profitable crop yield from the input of synthetic compounds in the agroecosystem. In the last decade technical advances have allowed scientists to gain unprecedented insights into plant-microbiota interactions in the rhizosphere. However, a precise understanding of how plants can shape these communities is still missing. This is information will be crucial to assist breeders in developing crops capable of maximising the mutualistic relationships with soil microbes. To fill this knowledge gap, in this thesis I use Barley (Hordeum vulgare), a global crop and an excellent experimental model, and will capitalise on state-of-the art sequencing and computational approaches to gain fundamentally novel insights into the host genetic control of the rhizosphere microbiota. The overarching hypothesis of my work is that the host genotype has the capacity to shape the rhizosphere microbiota to sustain plant growth in given soil conditions. I further hypothesize that this capacity impacts both the taxonomic and functional compositions of the rhizosphere microbiota and can be ultimately traced to loci in the barley genome. To test these hypotheses, I developed three major experimental lines aimed at a) characterising the microbiota of wild and cultivated barley genotypes grown in agricultural soils and how this impacts on plant growth; b) assessing the modulation of structure and function of the rhizosphere microbiota by the plant host under different nitrogen regimes and c) identifying the barley genetic region(s) responsible for the microbiota recruitment using experimental segregating populations between wild and modern barley genotypes. These experiments will contribute to decipher the genetic relationships between a plant genome and its associated microbiota and, in the long term, they will be key to devise novel strategies to enhance nutrient uptake efficiency in cereals.

The effect of genetically modified crop plants on the microbiology of the rhizosphere was investigated using the single-gene Bt cotton as a case study. The project compared the rhizosphere microbiota of four Ingard® 1cotton plant varieties that were closely matched with their non-GM parental strains. The plants were grown in three different Australian soils, ie, a vertisol from a cotton-growing region, and two soils, a fine sandy loam and a red sand from South Australia that had not been exposed to cotton. At the time of the commencement of the project, the only commercially available genetically modified plants were cotton and carnations. The cotton industry in Australia is worth $1.5b annually, and care of the soil and the dynamics of its living microbial consortia needs to be understood for optimum management to enable agricultural sustainability. The general outline of the thesis incorporated four main sections: 1. Experimental setup and analysis of the soils and plants to be used, quantification of the Cry1A(c) plant-produced Bt protein, and its persistence in the soil environment. 2. Measurement of the selected microbial populations of bacteria, fungi, AMfungi, protozoans and nematodes, by counting and estimation by dilution and most-probable number methods. 3. Assessment of selected metabolic pathways to determine the effects on the soil microbial community by chemical and other biochemical methods 4. An overall analysis between different group ratios of expression of each of the variables tested, and the summary of the risk analysis and conclusion. The outcome of this work was the acquisition of scientific data to produce an environmental impact report. The findings of this study showed that generally the microbial populations and the products of major metabolic pathways correlated more closely within the non-GM and GM plant rhizospheres of the paired trials than those of separate trials, indicating that soil and plant cultivar had a stronger environmental effect. The results obtained from the paired trials did not show that there were consistent effects on the rhizosphere soil microbiota that could be attributed to the presence of the Cry1A(c) Bt plant protein on the selected strains of cotton plants. The results from the tests of the paired trials correlate highly with previously published work that the risk factors of genetically modified cotton plants on the microbiology of the rhizosphere soil were found be negligible and not consistent across trials. 1 ® Monsanto Co. St Louis, MO.

The current population growth will demand a higher productive agriculture to full the food requirement. To supply this need and preserve the environment, many resources are applied to promote sustainable agriculture. Phosphorus depletion is the main factor that limits crops yields in tropical soils, where the pH and clay content rapid fixate this nutrient. Plant breeders aim to solve this issue by changing the plant requirements for phosphorus and adapting them to low P availability. However, with these approaches the demand for phosphorus fertilizers will continue and so the depletion of the natural deposits. In this study is proposed that plants with contrasting phosphorus uptake efficiency, i.e. P-efficient and P-inefficient, recruits distinct rhizosphere microbiome specialized in phosphorus mobilization. This hypothesis was tested growing plants in a gradient of two sources of P, triple superphosphate or rock phosphate Bayovar. Thebean rhizosphere microbiome was assessed with culture dependent and independent approaches, enzymatic assays, predictive metagenomics and networks analysis. A differential enrichment of several OTUs in the rhizosphere of the P-inefficient common bean genotype, and the enrichment of bacterial chemotaxis functions and functions involved in phosphorus mobilization suggest that this genotype has superior communication with the rhizosphere microbiome and is highly dependent on it for phosphorus mobilization. As a proof of concept, the P-efficientefficient genotype was sown in soil previously cultivated with P-inefficientinefficient genotype. The results showed that P-efficientefficient genotype positively responded to the modified rhizosphere in early stages, that is, the microbiome selected and enriched by the P-inefficient genotype improved the P uptake in the genotype cultivated afterwards in the same soil. Taken collectively, these results suggest that plants partly rely on the rhizosphere microbiome for P uptake and that the exploration of these interactions during plant breeding would allow the selection of even more efficient genotypes, leading to a sustainable agriculture by exploring soil residual P.
O atual aumento populacional irá demandar uma maior produção agrícola para completar a necessidade de alimento. Para suprir essa necessidade e preservar o meio ambiente, muitos recursos serão aplicados para promover a agricultura sustentável. A depleção de fósforo é um dos principais fatores que limita a produção agrícola em solos tropicais, onde o pH e o conteúdo de argila fixam rapidamente esse nutriente. Os melhoristas de plantas visam solucionar esse problema alterando a necessidade de fósforo das plantas e adaptando-as as baixas disponibilidade de fósforo. No entanto, com essas estratégias a demanda por fertilizantes fosfatados irá continuar assim como a exploração das reservas naturais de fósforo. Nesse estudo foi proposto que as plantas contrastantes em relação a eficiência na absorção de fósforo, i.e. P-eficiente e P-ineficiente, recrutariam um microbioma rizosférico distinto em relação a mobilização de fósforo. Essa hipótese foi testada cultivando plantas em um gradiente usando duas fontes distintas de P, triplo fosfato ou fosfato de rocha Bayovar. O microbioma da rizosfera de feijão foi então avaliado por técnicas dependentes e independentes de cultivo, análise enzimática, predição metagenômica e análises de network. Um enriquecimento diferencial de várias OTUs observado na rizosfera do genótipo de feijão P-ineficiente, e o enriquecimento de funções de quimiotaxia bacteriana e envolvidas na mobilização de fósforo sugerem que esse genótipo tem uma maior comunicação com o microbioma rizosférico e é altamente dependente deste para a mobilização de fósforo. Como prova de conceito, o genótipo P-eficiente foi plantado em solo previamente cultivadocom o genótipo P-ineficiente. Os resultados mostraram que o genótipo P-eficiente responde positivamente à rizosfera modificada nos estádios iniciais de crescimento, ou seja, o microbioma selecionado e enriquecido pelo genótipo P-ineficiente melhorou a absorção de fósforo no genótipo cultivado posteriormente no mesmo solo. Coletivamente, esses resultados sugerem que as plantas dependem parcialmente do microbioma da rizosfera para a absorção de P e que a exploraçãodestas interações durante o melhoramento vegetal permitiria a seleção de genótipos muito mais eficientes, conduzindo à uma agricultura sustentável explorando o fósforo residual do solo.

Three model plant and three crop plant species were grown for three generations in sand and compost. Pots were inoculated with 10 % soil initially, and with 10% of growth medium from the previous generation in generations 2 and 3, keeping replicates separate for all three generations. The microbiome community structure of the plant rhizosphere in each generation was characterised using ARISA DNA fingerprinting and 454 sequencing. Rhizosphere bacterial and fungal communities are different from those in bulk soil and there are also differences in the microbial community between different plant species. Plants both select and suppress specific bacteria and fungi in the rhizosphere microbiome, presumably via composition of their root exudates. Two out of three most abundant bacteria selected in the rhizosphere were isolated. These isolates proved to possess plant growth promotion properties. Plants are able to “farm” the soil in order to enrich it with plant growth promoting rhizobacteria (PGPR) species. However, in some plant species rhizospheres, invasions of opportunists and pathogens take place, mimicking events in plant monocultures. Other experiments using this multi-replicate system allowed for statistical analysis of the influence of Arabidopsis and Medicago mutants on the rhizosphere microbiome. Three groups of Arabidopsis mutants were tested: plants unable to produce aliphatic glucosinolates, plants impaired in the PAMP-triggered immune response and plants unable and over-expressed in methyl halides production and one group of Medicago mutants which are impaired in the mycorrhization ability. All these plant genotypes, except those for methyl-halide production and one genotype involved in PAMP response, significantly altered the rhizosphere microbiome.

Plant-microbe interactions in the rhizosphere, the region of soil influenced by plant roots, are integral to biogeochemical cycling, and maintenance of plant health and productivity. Interactions between model plants and microbes are well understood, but relatively little is about the plant microbiome. Here, comparative metatranscriptomics was used to determine taxonomic compositions and metabolic responses of microbes in soil and the rhizospheres of wheat, oat and pea. Additionally a wild-type oat was compared to a mutant (sad1) deficient in production of antifungal avenacins. Analyses of taxonomic compositions and functions based on rRNA and protein coding genes agreed that rhizosphere microbiomes differed from soil and between plant species. Pea had a stronger effect than wheat and oat, suggesting distinct cereal and legume microbiomes. Proportions of eukaryotic rRNA in the oat and pea rhizospheres were more than fivefold higher than in the wheat rhizosphere or soil. Nematodes and bacterivorous protozoa were enriched in all rhizospheres, while the pea rhizosphere was highly enriched for fungi. Only the eukaryotic community was distinct from wild-type oat in the sad1 mutant, suggesting avenacins have a broader role than protecting from fungal pathogens. The addition of an internal RNA standard allowed quantitative determination of global transcriptional activity in each environment. This was generally higher in the rhizospheres, particularly pea, than in soil. Taxa known to possess metabolic traits potentially important for rhizosphere colonisation, plant growth promotion and pathogenesis were selected by plants. Such traits included cellulose and other plant polymer degradation, nitrogen fixation, hydrogen oxidation, methylotrophy and antibiotic production. These functions were also more highly expressed in rhizospheres than soil. Microbes also induced functions involved in chemotaxis, motility, attachment, pathogenesis, responses to oxidative stress, cycling of nitrogen and sulphur, acquisition of phosphorous, iron and other metals, as well as metabolism of a variety of sugars, aromatics, organic and amino acids, many plant species specific. Profiling microbial communities with metatranscriptomics allowed comparison of relative and quantitative abundance of microbes and their metabolism, from multiple samples, across all domains of life, without PCR bias. This revealed profound differences in the taxonomic composition and metabolic functions of rhizosphere microbiomes between crop plants and soil.

L'exsudation racinaire est connue pour avoir une influence sur le fonctionnement des communautés microbiennes, en particulier celles impliquées dans le cycle de l'azote (Haichar et al, 2012). Elle est liée à la physiologie de la plante, cette dernière pouvant être évaluée via les traits fonctionnels végétaux, permettant une classification des plantes en fonction de leur performance dans leur environnement. Ainsi, nous pouvons distinguer d'une part les espèces exploitatrices, avec une efficience de la photosynthèse élevée et une acquisition rapide de l'azote dans les sols, et d'autre part les plantes conservatrices, possédant des caractéristiques contraires (Aerts & Chapin, 1999) et des plantes intermédiaires dont les caractéristiques sont intermediaires.L'objectif de ces travaux de thèse est de déterminer l'influence de la stratégie de gestion des ressources de 6 poacées, réparties le long d'un gradient de stratégie de gestion des ressources, allant de stratégies conservatrices (Sesleria caerulea et Festuca paniculata), intermédiaires (Antoxanthum odoratum, Bromus erectus) à des stratégies exploitatrices (Dactylis glomerata et Trisetum flavescens), sur la diversité et le fonctionnement des communautés totales et dénitrifiantes. I) Dans un premier temps nous avons étudié le lien entre la stratégie de gestion de ressources des plantes et la quantité d'exsudats racinaires dans le sol adhérent aux racines (SAR). Nous avons ensuite déterminé l'influence de la quantité d'exsudats racinaire sur les activités microbiennes potentielles des communautés microbiennes du SAR (respiration et dénitrification potentielles), puis par une approche ADN-SIP (Stable Isotope Probing) couplée à du séquençage haut-débit, l'influence de l'exsudation racinaire sur la structure et la diversité des communautés bactérienne colonisant le SAR et le système racinaire. II) Dans un second temps, nous avons étudié le lien entre la stratégie de gestion des ressources des plantes et la nature des exsudats racinaires libérés au niveau du SAR et présents dans les extraits racinaires en analysant les profils des métabolites primaires chez Festuca paniculata, Bromus erectus et Dactylis glomerata, représentant respectivement des stratégies de gestion des ressources conservative, intermédiaire et exploitatrice
Root exudation is known to influence microbial communities functioning, in particular those involve in nitrogen cycle. (Haichar et al, 2012). It’s linked to plant physiology, which can be evaluated with functional traits, allowing a plant distribution in function of their performance in their environment. Thus, we can distinguish competitive species, with higher photosynthetic capacity and rapid rates of N acquisition, conservative species with the opposite characteristics (Aerts & Chapin, 1999) and intermediate plants, with intermediate characteristics.The objective of this work is to determinate the influence of nutrient management strategiy of 6 poaceae, along a strategies gradient from conservative strategy (Sesleria caerulea and Festuca paniculata), intermediate (Antoxanthum odoratum and Bromus erectus) to competitive strategy (Dactylis glomerata and Trisetum flavescens), on diversity and functioning of total and denitrifying communities.I) Firstly, we studied the link between the plant nutrient management strategy and the root exudates quantity in the root adhering soil (RAS). Then, we determined the influence of the rate of root exudation on potential microbial activities (respiration and denitrification), and with a DNA-SIP (Stable Isotope Probing) approach coupled to high-throughput sequencing, the influence of root exudation on the bacterial structure and diversity of communities colonizing the RAS and the root system. II) Secondly, we studied the link between the plant nutrient management strategy and the nature of molecules exuded in RAS and present in root extracts by analyzing primary metabolites profile to Festuca paniculata, Bromus erectus and Dactylis glomerata, respectively a conservative, an intermediate and a competitive plant. Then, we determined the influence of primary metabolites profile of each plant on semi-real denitrification of communities colonizing RAS of plants. III) Finally, an mRNA-SIP approach is in progress to determine the influence of exuded metabolites on active bacterial communities functioning and the expression of genes involved in denitrification process in RAS and root system. Our results show an influence of the nutrient management strategy on the rate of carbon exudation, the competitive plants exuding more than conservatives ones

O atual cenário mundial das mudanças climáticas, somado ao aquecimento global e ao aumento das áreas em processo de desertificação tem impactado diretamente nos padrões de produção agrícola. A Caatinga é um bioma que só ocorre no Brasil e possui um clima semiárido, quente e de baixa pluviosidade, sendo que na estação seca a temperatura do solo pode chegar até 60ºC. A Caatinga apresenta uma grande riqueza de ambientes e espécies, e boa parte dessa diversidade não é encontrada em nenhum outro bioma. Uma característica muito peculiar da Caatinga é a existência de duas estações bem contrastantes durante o ano, o inverno caracterizado por ser a estação da chuva e o verão a época da seca. A vegetação é composta por Euforbiáceas, Bromeliáceas e Cactáceas, dentre as quais destacam-se o Cereus jamacaru (mandacaru), Pilosocereus gounellei (xique-xique) e Melocactus sp. (cabeça-de-frade). O mandacaru planta que sobrevive às altas temperaturas e baixa disponibilidade de água da Caatinga possui adaptações morfológicas estruturais que contribuem para a sobrevivência da mesma. Além dessas adaptações a comunidade microbiana da rizosfera foi estudada para descobrir quais micro-organismos presentes nesse ambiente auxiliam na manutenção do hospedeiro frente a essas condições adversas. Assim como, quais grupos e funções são mais abundantes nessas condições. Nesse estudo foi feito o sequenciamento parcial do gene 16S rRNA e do DNA total da rizosfera de mandacaru. A comunidade bacteriana foi bem representada pelos filos Actinobacteria, Proteobacteria e Acidobacteria, sendo que o filo Actinobacteria foi mais abundante na seca de acordo com o sequenciamento metagenômico e o filo Acidobacteria foi mais abundante no período de chuva. Em geral o sequenciamento do gene 16S rRNA, indicou que Actinobacteria e Proteobacteria são os filos mais abundantes e os genes relacionados às funções de resistência a doenças foram mais abundantes na estação seca, enquanto genes relacionados ao metabolismo do nitrogênio foram mais abundantes durante o período chuvoso, revelando assim, um pouco do potencial que o microbioma da rizosfera de mandacaru possui para auxiliar a planta hospedeira.
The present world scenario of climate change, global warming and the increase in areas undergoing desertification, have directly impacted on current patterns of agricultural crop production. The Caatinga is a specific Brazilian biome because of its semi-arid climate, hot and low rainfall, and the temperature that reaches the 60°C in the dry season. The Caatinga has a huge biodiversity and much of its diversity is not found in any other biome. A peculiar characteristic of the Caatinga biome is the occurrence of two very contrasting seasons during the year, the winter which is characterized by a rainy season and summer the dry season. The vegetation is composed by Euphorbiaceae , Bromeliaceae and Cactaceae, represented by Cereus jamacaru (Mandacaru) Pilosocereus gounellei (xique-xique) and Melocactus sp. (head-to-brother). Mandacaru is the plant that can survive through the specifics climate conditions of the Caatinga biome such as high temperatures and low water availability and this is probably due to some structural and morphological adaptations that contribute to its survival. Therefore, we assessed which microorganisms are associated with the plant rhizosphere, and which microbial groups contribute to the maintenance of the host throughout these adverse conditions. Also, we identified which are the most abundant microbial groups in these conditions and which microbial functions are more abundant in both evaluated seasons. Thus the present study assessed the mandacaru rhizosphere microbiome through a partial 16S rRNA gene sequencing and metagenomic sequencing. The bacterial community was well represented by the phyla Actinobacteria, Proteobacteria and Acidobacteria. The Actinobacteria was the most abundant microbial phyla in the dry season according to shotgun sequencing while the Acidobacteria was the most abundant microbial phyla in the rainy season. Overall, the 16S rRNA sequencing indicated that Actinobacteria and Proteobacteria were the most abundant groups and additionally, and genes related to disease resistance functions were more abundant in the dry season. Genes related to nitrogen metabolism were more abundant during the rainy season revealing some of the potential traits that the mandacaru can explore from its microbiome.

A Mata Atlântica é uma floresta tropical úmida considerada um \"hotspot\" de biodiversidade e endemismo. É uma das florestas mais antigas do mundo e uma das maiores florestas da América, abrangendo aproximadamente 150 milhões de hectares em condições ambientais altamente heterogêneas. Estudos em diferentes ambientes da Mata Atlântica, nos núcleos de Picinguaba e Santa Virginia no Parque Estadual da Serra do Mar (PESM), têm sido realizados para determinar a diversidade de espécies e alterações da estrutura das comunidades de bactérias, tanto na filosfera, quanto na dermosfera e solo rizosférico. No entanto, pouco se sabe sobre as funções ecológicas dessas bactérias, e sobre as interações ecológicas entre as comunidades microbianas e os ambientes onde se desenvolvem. Assim o objetivo desse trabalho foi explorar as interações entre as comunidades microbianas da filosfera, dermosfera e solo coletado sobre a projeção da copa de duas espécies arbóreas da Mata Atlântica ao longo de um gradiente altitudinal, usando análises de co-ocorrência, a partir dos dados obtidos por pirosequenciamento da região V4 do gene rRNA 16S de bactérias, para determinar padrões de associações de bactérias em diferentes níveis taxonômicos em cada microambiente. Para esse estudo, foi proposta a hipótese de que mesmo que as condições ambientais sejam diferentes em cada tipo de floresta (gradiente altitudinal), pode existir grupos de bactérias específicos que co-ocorrem na filosfera, dermosfera ou solo das plantas, funcionando como taxons chaves na estruturação das comunidades bacterianas. Com base do sequenciamento dos genes rRNA 16S, as comunidades bacterianas associadas à filosfera e dermosfera de E. edulis e G. opposita nas diferentes florestas foram mais similares entre si do que as do solo. Actinobacteria, Firmicutes, Bacteroidetes e Proteobacteria foram mais abundantes em todos os microambientes estudados. Diferenças nas estruturas das comunidades bacterianas na filosfera, dermosfera e solo foram observadas ao longo do gradiente altitudinal, independente da espécie de planta. Na floresta de terras baixas, a comunidade bacteriana associada à filosfera foi mais similar entre E. edulis e G. opposita. No solo, a comunidade bacteriana foi mais similar dentro de cada tipo de floresta do que entre florestas, sugerindo um efeito da fisionomia da floresta nas comunidades de bactérias dos solos. Explorando as redes de co-ocorrência das comunidades bacterianas em cada microambiente observou-se que no nível de UTOs, cada microambiente têm diferentes táxons chaves que podem regular as interações ecológicas da comunidade. Embora táxons chaves não representam as UTOs mais abundantes em cada microambiente, eles pertencem, predominantemente às classes Alphaproteobacteria e Gammaproteobacteria, sugerindo que na filosfera, dermosfera e solo o core microbioma não pode ser definido ao nível de UTO, mas possivelmente a níveis taxonômicos mais elevados representando grandes grupos microbianos que apresentam funções redundantes.
The Atlantic Forest is a rainforest considered a hotspot of biodiversity and endemism. It is one of the oldest forests in the world and one of the largest forests of America, covering approximately 150 million hectares in highly heterogeneous environmental conditions. Studies in different environments of the Atlantic forest, in the Picinguaba and Santa Virginia areas in the Serra do Mar State Park (PESM) have been conducted to determine the species diversity and changes in the structure of the bacterial communities in the phyllosphere, dermosphere and rhizosphere. However, little is known on the ecological functions of these bacteria, and on the ecological interactions between microbial communities and the environment in which they develop. The aim of this study was to explore the interactions between the microbial communities of the phyllosphere, dermosphere and rhizosphere of two tree species of the Atlantic Forest along an altitudinal gradient. Co-occurrence analysis based on data obtained by pyrosequencing of the 16S rRNA gene V4 region of bacteria to determine patterns of bacterial associations in different taxonomic levels in each microenvironment. For this study, the hypothesis that even if the environmental conditions are different in each type of forest (altitudinal gradient), there may be specific groups of bacteria that co-occur in the phyllosphere, dermosphere or rhizosphere, functioning as keystone taxa in the bacterial communities. Based on the sequencing of 16S rRNA genes, bacterial communities associated with the E. edulis and G. opposita phyllosphere and dermosphere in different forests were more similar to each other than the rhizosphere. Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes were the more abundant taxa in all studied microenvironments. Differences in the bacterial community structures in the phyllosphere, dermosphere and rhizosphere were observed along the altitudinal gradient, regardless of the plant species. In the lowland forest, the bacterial community associated with the phyllosphere was more similar between E. edulis and G. opposita. The rhizosphere bacterial community was more similar within each forest type than between forests, suggesting an effect of the forest physiognomy on the bacterial communities of the rhizosphere. Exploring the co-occurrence networks in the bacterial communities of each microenvironment it was observed that at the OTU level each microenvironment has different keystoine taxa that may regulate the ecological interactions in the community. Although the keystone taxa do not represent the most abundant OTUs in each microenvironment, they belong predominantly to Alphaproteobacteria and Gammaproteobacteria classes, suggesting that in the phyllosphere, dermosphere and rhizosphere the core microbiome cannot be determined at the OTU level, but possibly at higher taxonomic levels representing microbial groups having redundant functions.

Fe biofortification of potato is a promising strategy to prevent Fe deficiency worldwide either through traditional breeding or biotechnological approaches. These approaches require the identification of candidate genes to uptake, transport and store Fe in potato tubers. We employed multiple approaches including SNP genotyping, QTL analysis, identifying genes orthologous to Arabidopsis ferrome, yeast complementation assay and genetic transformation to avoid the limitation from a single approach. We revealed several candidate genes potentially associated with potato plant Fe acquisition, PGSC0003DMG400024976 (metal transporter), PGSC0003DMG400013297 (oligopeptide transporter), PGSC0003DMG400021155 (IRT1) and IRTunannotated (an ortholog to the IRT gene that is not annotated in the potato genome). The microorganisms in the rhizosphere react intensely with the various metabolites released by plant roots in a variety of ways such as positive, negative, and neutral. These interactions can influence the uptake and transport of micronutrients in the plant roots. Therefore, the contribution of soil microorganisms in the rhizosphere to improve Fe supply of plants may play a key role in Fe biofortification, especially under real world field-based soil scenarios. We thus investigated rhizosphere microbial community diversity in Andean potato landraces to understand the role of plant-microbial interaction in potato Fe nutrient cycling. From the analysis of the high-throughput Illumina sequences of 16S and ITS region of ribosomal RNA gene, we found that both potato landraces with low and high Fe content in tubers and a landrace on which low or high Fe content fertilizer was applied to the leaf surface had large impacts on the rhizosphere fungal community composition. Indicator species analysis (ISA) indicated that Operational Taxonomic Units (OTUs) contributing most to these impacts were closely related to Eurotiomycetes and Leotiomycetes in the phylum Ascomycota, Glomeromycetes in the phylum Glomeromycota and Microbotryomycetes in the phylum Basidiomycota. Lots of species from these groups have been shown to regulate plant mineral nutrient cycling. Our research revealed potential candidate genes and fungal taxa involved in the potato plant Fe nutrient dynamics, which provides new insights into crop management and breeding strategies for sustainable Fe fortification in agricultural production.
Ph. D.

For the last 150 years many studies have shown the importance of earthworms for plant growth, but the exact mechanisms involved in the process are still poorly understood. Many important functions required for plant growth can be performed by soil microbes in the rhizosphere. To investigate earthworm influence on the rhizosphere microbial community, it was performed a macrocosm experiment with and without Pontoscolex corethrurus (EW+ and EW-, respectively) and followed various soil and rhizosphere processes for 217 days with sugarcane. In the second chapter of this thesis it was demonstrate that in EW+ treatments, N2O concentrations belowground (15 cm depth) and relative abundances of nitrous oxide genes (nosZ) were higher in bulk soil and rhizosphere, suggesting that soil microbes were able to consume earthworm-induced N2O. Shotgun sequencing (total DNA) revealed that around 70 microbial functions in bulk soil and rhizosphere differed between EW+ and EW- treatments. Overall, genes indicative of biosynthetic pathways and cell proliferation processes were enriched in EW+ treatments, suggesting a positive influence of worms. In EW+ rhizosphere, functions associated with plant-microbe symbiosis were enriched relative to EW- rhizosphere. Ecological networks inferred from the datasets revealed decreased niche diversification and increased keystone functions as an earthworm-derived effect. Plant biomass was improved in EW+ and worm population proliferated. Considering that earthworms contributed to with extra resources, it was evaluated in chapter three response of the soil resistome of sugarcane macrocosms under the influence of earthworms. Mechanisms of resistance against antimicrobial compounds appear to be an obligatory feature for the ecology and evolution of prokaryotic forms of life. However, most studies on resistance dynamics have been conducted in artificial conditions of anthropogenic inputs of antibiotics into very specific communities such as animal microbiomes. To resolve why and how resistance evolves, it is important to track antibiotics resistance genes (ARGs) (i.e., the resistome) in their natural hosts and understand their ecophysiological role in the environment. The results demonstrated that earthworms influenced changes of ARGs in bulk soil and rhizosphere. Negative correlations between ARGs and taxonomical changes were increased in EW+. Differential betweenness centrality (DBC=nBCEW+ - nBCEW-) values comparing the network models with and without earthworms showed earthworm presence changed the composition and the importance of the keystone members from the models. Redundancy analysis suggested that ARGs may be associated with microbial fitness, as the variance of relative abundance of members of the group Rhizobiales could be significantly explained by the variance of a specific gene responsible for one mechanism of tetracycline detoxification
Ao longo dos últimos 150 anos muitos estudos têm demonstrado a importância das minhocas para o crescimento de plantas. Porém o exato mecanismo envolvido neste processo ainda é muito pouco compreendido. Muitas funções importantes necessárias para o crescimento de plantas podem ser realizadas pela comunidade microbiana da rizosfera. Para investigar a influência das minhocas na comunidade microbiana da rizosfera, foi desenvolvido um experimento de macrocosmo com cana-de-açúcar com e sem Pontoscolex corethrurus (EW+ e EW-, respectivamente) seguindo diversos procedimentos por 217 dias. No Segundo capítulo da tese é demonstrado que no tratamento EW+, as concentrações de N2O dentro do solo (15 cm profundidade) e a abundância relativa dos genes óxido nitroso redutase (nosZ) foram elevadas no solo e na rizosfera, sugerindo que microrganismos do solo foram capazes de consumir a emissão de N2O induzida pelas minhocas. O sequenciamento do DNA total revelou que aproximadamente 70 funções microbianas no solo e na rizosfera apresentaram diferenças entre os tratamentos EW+ e EW-. No geral, genes associados a biossíntese e proliferação de células foram enriquecidos em EW+, sugerindo uma influencia positiva por parte das minhocas. Na rizosfera EW+, funções associadas a simbiose entre planta e microrganismos foram relativamente enriquecidas comparado com rizosfera EW-. Modelos de rede de interação ecológica revelam menor número de diversificação de nichos e aumento de funções importantes como um efeito derivado da influência das minhocas. A biomassa das plantas foi aumentada no tratamento EW+ e a população de minhocas proliferou. Considerando que as minhocas contribuíram com o aumento de nutrientes, foi avaliado no capítulo três a resposta do resistoma presente nas comunidades microbianas dos solos do experimento. Mecanismos de resistência contra compostos antimicrobianos parecem ser características obrigatórias para a ecologia e evolução de procariotos. Entretanto, a maior parte dos estudos sobre genes de resistência tem sido conduzida em condições artificiais utilizando fontes antropogênicas de antibióticos em comunidades microbianas muito específicas como por exemplo o microbioma animal. Para resolver por que e como a resistência evolui, é importante estudar genes de resistência a antibióticos (GRA) (i.e., resistoma) no seu ambiente natural e entender seu papel ecofisiologico no ambiente. Os resultados demonstraram que minhocas influenciaram a mudança na composição de GRA no solo e na rizosfera. Tratamentos EW+ apresentaram maior número de correlações negativas entre ARG e grupos taxonômicos. A medida de centralidade diferencial (DBC=nBCEW+ - nBCEW-) comparando os modelos de rede de interações obtidos mostrou que a composição e o nível de importância dos indivíduos mais influentes é alterado nos tratamentos EW+ comparado com EW-. Além disso, por meio de uma análise de redundância (RDA) foi demonstrado que as alterações na abundancia relativa de GRA podem ser explicadas pelas alterações verificadas em grupos taxonômicos

O fósforo é um elemento-chave para a manutenção da vida, desde os processos de transferência energética (ATP) até a estruturação das cadeias de ácidos nucléicos. Frequentemente é o que mais limita o desenvolvimento vegetativo em função da alta reatividade do PO43- na solução do solo. Mais de 80% do P adicionado ao solo pode ser imobilizado na forma de compostos de baixa labilidade, adsorvendo-se as superfícies das argilas e dos oxi-hidróxidos de Fe3+ e Al3+, processo irreversível em condições naturais. A solubilização do P não lábil às plantas é possível mediante a liberação de ácidos orgânicos sintetizados por micro-organismos nativos do solo, os quais também contribuem efetivamente com a mineralização do P orgânico através da produção de enzimas (fosfatases), entretanto, esses mecanismos ainda são pouco conhecidos. O principal objetivo desse estudo foi correlacionar as possíveis mudanças nas frações orgânicas (Po) e inorgânicas (Pi) de fósforo no solo rizosférico de plantas de cana-de-açúcar com a estrutura e a diversidade do seu microbioma. Para tanto, as plantas foram cultivadas durante 180 dias em diferentes microcosmos com fosfato monoamônico (MAP), superfosfato triplo (SFT) e fosfato natural reativo (NAT), tanto na ausência quanto na presença da torta de filtro. As diferentes frações de fósforo no solo rizosférico foram correlacionadas com os dados moleculares baseados no sequenciamento parcial dos amplicons do gene 16S rRNA obtidos do DNA total extraído desses solos. Aos 60 dias, houve uma maior diversidade geral de bactérias, sendo um período com altos teores de P-lábil na rizosfera, beneficiando principalmente as bactérias de hábitos copiotróficas (r-estrategistas): divisão Halophaga/Acidobacterium, Chloracidobacteria e Bacilli. A diminuição da diversidade aos 120 dias ocorreu como resposta da redução dos teores de P-lábil na rizosfera, beneficiando mais as bactérias oligotróficos (k-estrategistas): Actinobacteria e Proteobacterias. A associação da torta de filtro com o MAP revelou fortes indícios do aumento do processo de mineralização na superfície do solo, uma vez que houve rápida liberação de P lábil para a rizosfera. Isso teria aumentado a atividade de micro-organismos envolvidos na mineralização da torta de filtro, havendo forte correlação com o aumento da população de β-Proteobacteria aos 120 dias. Neste período, as Proteobacterias foram mais abundantes nos solos com NAT+torta. Isso explicaria o incremento de P moderadamente lábil desse tratamento aos 180 dias, pois esse filo possui várias espécies capazes de solubilizar o P ligado ao Ca, presente nos fosfatos de rocha. No geral, a torta de filtro alterou a solubilização e disponibilidade do P nas fontes minerais de modo diferente, contribuindo com os incrementos tanto das frações de Po como as de Pi. Esses resultados demonstraram que as bactérias participam ativamente dos processos de disponibilização do P de fontes minerais no solo. Nessas condições, a torta de filtro pode modular essas transferências, em benefício às plantas, por estimular a atividade de micro-organismos solubilizadores de P e mineralizadores da matéria orgânica do solo.
Phosphorus is a key element for the maintenance of life from the energy transfer processes (ATP) to the structuring of chains of nucleic acids. Often, it is more limited vegetative growth due to the high reactivity of PO43- in the soil solution. Up to 80% of P added to the soil can be immobilized as low-labile compounds adsorbing to the clay surfaces and oxy-hydroxides of Fe3+ and Al3+, irreversible process under natural conditions. Solubilization of non-labile P by plants may be carried out for organic acids synthesized by native soil microorganisms, which also effectively contribute for mineralization of organic P through the production of enzymes (phosphatases) although these mechanisms are still poorly understood. The main objective of this study was to correlate the changes in the organic (Po) and inorganic (Pi) fractions of P in the rhizosphere of sugarcane plants with the structure and diversity in their microbiome. Therefore, the plants were grown for 180 days in different microcosms with monoammonium phosphate (MAP), triple superphosphate (TSP) and rock phosphate (NAT), both in absence and in presence of the filter cake. The different P fractions in the rhizosphere soil were correlated with the molecular data based on the partial sequencing of 16S rRNA gene amplicons obtained from total DNA extracted from soils. At 60 days, there was a greater overall diversity of bacteria, it is a period with high labile-P in the rhizosphere, especially benefiting the copiotrophic bacteria (r-strategists): Halophaga/Acidobacterium division, Chloracidobacteria and Bacilli. The decrease in diversity at 120 days occurred in response to the reduction of labile-P content in the rhizosphere, more benefiting oligotrophic bacteria (k-strategists): Actinobacteria and Proteobacteria. The association filter cake with MAP showed strong evidence of increased mineralization process on the soil surface, since there was rapid release of labile P to the rhizosphere. It would have increased the activity of microorganisms involved in the mineralization of the filter cake with a strong correlation with increase in β-Proteobacteria population at 120 days. Then, Proteobacteria were more abundant in soils with NAT+filtercake at 120 days. That would explain the increases in moderately labile P content this treatment at 180 days, because this phylum has several species capable of solubilizing the P linked to Ca present in rock phosphates. In general, filter cake changed solubilization and availability of P in mineral sources of different ways, contributing to the increments of Po and Pi fractions. These results showed that bacteria participate in the processes that afford phosphorus in the soil. Under these conditions, the filter cake can modulate these transfers in beneficial to plants by stimulating the activity of P solubilizing microorganisms and mineralizing the soil organic matter.

Face aux enjeux sociétaux, environnementaux et d’érosion de la biodiversité, la reconception des systèmes de cultures mobilisant les interactions écologiques en place des intrants est cruciale à développer. L’interface sol-plante présente un intérêt majeur car elle héberge une diversité de processus impliqués dans la nutrition et la santé des plantes. L’effet rhizosphère induit par les plantes est très dynamique, et influencé non seulement par le type de sol ou l’espèce cultivée mais également par les pratiques agricoles. Dans une approche combinant expérimentation et modélisation statistiques par équation structurale, cette thèse a permis de développer des indicateurs de fonctionnement de la rhizosphère du blé (Triticum aestivum var Absalon) au cours du cycle de développement de la plante.La méthodologie appliquée a permis de mettre en avant des effets synergiques de la diversification des cultures au cours de la succession avec le travail réduit du sol afin de promouvoir l’effet rhizosphère. Enfin, ce travail a permis de mettre en évidence un schéma structurel causal commun entre les conditions contrôlées et le terrain grâce à la modélisation par équation structurales. En conclusion, ce travail de thèse menée en collaboration avec les adhérents de trois coopératives permet d’ouvrir des perspectives de conception et d’évaluation des systèmes de cultures agroécologiques à bas intrants basés sur l’effet rhizosphère
Faced with societal and environmental challenges and loss of biodiversity, the redesign of cropping systems mobilizing ecological interactions instead of synthetic inputs is crucial to develop. The soil-plant interface influenced by root activities is of major interest because it harbors a diversity of processes involved in plant nutrition and health. However, the rhizosphere effect induced by plants is very dynamic, and influenced not only by soil type or crop species but also by agricultural practices. In an approach combining experimentation and statistical modeling by structural equations, indicators of rhizosphere functioning in wheat (Triticum aestivum var Absalon) were developped.The applied methodology allowed to put forward synergistic effects of crop diversification during the succession with reduced tillage in order to promote the rhizosphere effect. Finally, we highlighted a common scheme of causal structure between controlled conditions and the field by structural equation modeling that pave the way for further development of rhizosphere indicators. In conclusion, this thesis work carried out in collaboration with the members of three cooperatives opens perspectives for the design and evaluation of low-input agroecological cropping systems

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior
Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
Os microrganismos sÃo essenciais para manter as funÃÃes do solo em Ãreas naturais e agrÃcolas, por causa de seu envolvimento em processos-chave na estrutura do solo, envolvendo a decomposiÃÃo de matÃria orgÃnica, a ciclagem de nutrientes e obtenÃÃo de energia, a fixaÃÃo de nitrogÃnio atmosfÃrico, a decomposiÃÃo de xenobiÃticos e o controle biolÃgico de pragas e doenÃas. Partindo do pressuposto de que ambientes de sistemas agrÃcolas tÃm mais indivÃduos r-estrategistas, enquanto Ãreas de floresta tÃm mais indivÃduos K-estrategistas, objetivou-se neste estudo avaliar a dinÃmica populacional e atividade dos microrganismos na rizosfera de fruteiras tropicais e comparar com uma Ãrea de vegetaÃÃo natural do semiÃrido cearense. Foram coletadas amostras de solo sob influÃncia de abacaxizeiro, cultivares MD2 e Imperial, de sapotÃcea, clone BRS 228, em Ãreas sem a influÃncia das fruteiras e numa faixa de vegetaÃÃo natural adjacente ao pomar, no municÃpio de Trairi, estado do CearÃ. As amostragens de solo superficial (camada de 0 a 10 cm) foram realizadas em junho, setembro e dezembro de 2010, para posterior anÃlise de atributos microbiolÃgicos e bioquÃmicos do solo. A biomassa microbiana e o quociente metabÃlico (qCO2) variaram entre as Ãreas e as Ãpocas avaliadas, enquanto o teor de carbono orgÃnico e o quociente microbiano (qMIC) variaram apenas com as Ãreas e a respiraÃÃo edÃfica mudou ao longo do ano. Houve pouca variaÃÃo na estimativa da quantidade glomalina entre as Ãreas e foi observada uma tendÃncia de reduÃÃo desta proteÃna ao longo dos perÃodos do ano. A fosfatase Ãcida e a urease apresentaram maior atividade no solo sob vegetaÃÃo natural, enquanto a enzima desidrogenase foi mais ativa em Ãreas do pomar. A densidade populacional de bactÃrias cultivÃveis foi maior no solo do pomar, enquanto a de fungos cultivÃveis foi semelhante para as Ãreas avaliadas e variando entre os perÃodos do ano. A densidade de esporos de fungos micorrÃzicos arbusculares (FMA) no solo foi influenciada negativamente pelo estabelecimento de fruteiras, em comparaÃÃo com a Ãrea de vegetaÃÃo natural. A prevalÃncia de Glomus foi comum nas diferentes Ãreas, e a abundÃncia relativa das comunidades foi maior no solo com vegetaÃÃo natural. Para as condiÃÃes do estudo pode-se inferir: os parÃmetros microbiolÃgicos sÃo sensÃveis à cobertura vegetal e podem ser usados para avaliar a qualidade do solo; os teores de carbono orgÃnico e da biomassa microbiana, a respiraÃÃo basal e os quocientes metabÃlico e microbiano do solo sÃo influenciados pelo manejo do solo no pomar e as prÃticas agrÃcolas influenciam na diversidade de FMA, selecionando provavelmente os fungos r-estrategistas.
Microorganisms are essential to keep the normal soil functioning in both natural and agricultural areas, mainly due to their implication in key processes occurring in soil structure, such as organic matter decomposition, nutrients cycling and energy captation, atmospheric nitrogen fixation, xenobiotics degradation and biological pests and diseases control. Assuming the existence of a higher number of r-strategists individuals in agricultural systems, while forest areas have more K-strategists, this study aimed to evaluate the populational dynamics and microbial activity in the rhizosphere of fruit plants, comparing them to a natural vegetation from the semiarid region of CearÃ. Soil samples under the influence of pineapple plants, cultivars MD2 and imperial, and of sapodilla plants, clone BRS 228, and also without the influence of fruit plants, but from a natural vegetation adjacent to the orchard, were collected. This collection was made in Trairi municipality, Cearà state. Superficial soil sampling (layer from 0 to 10 cm) were made in June, September and December 2010, for microbiological and biochemical analysis. Microbial biomass and metabolic quotient (qCO2) varied between the areas and periods evaluated, whereas the organic carbon content and the microbial quotient (qMIC) varied only between areas. Soil respiration varied along the year. There was a little variation in the glomalin concentration estimation between the areas and there was a tendency of reduction of this protein along the year. Acid phosphatase and urease showed greater activity in soil under natural vegetation, while the dehydrogenase enzyme was most active in orchard areas. The population density of culturable bacteria was higher in the orchard soil, while the populations of culturable fungi were similar between areas and ranged between periods of the year. The spores density of arbuscular mycorrhizal fungi (AMF) in soil was negatively influenced by the establishment of fruit plants, when compared to the natural vegetation area. The prevalence of Glomus species was common in different areas; relative abundance of those fungi communities was higher in soil under natural vegetation. Under conditions of this study we conclude: microbiological parameters are sensitive to the type of vegetation cover and can be used to evaluate the soil quality, the organic carbon and microbial biomass, basal respiration and metabolic and microbial quotients are influenced by the soil and orchard management. Agricultural practices can influence the diversity of AMF species, presumably selecting the r-strategists fungi.

La rhizosphère est la zone étroite de sol soumise à l’influence des racines des plantes qui libèrent un mélange moléculaire complexe : les exsudats racinaires. Ils permettent à la plante de recruter son microbiote rhizosphérique qui joue un rôle clé dans sa croissance et sa résistance aux stress biotiques et abiotiques. Dans le cadre de l’agroécologie, la compréhension du dialogue moléculaire racines-microbiote pourrait permettre de promouvoir l’installation de rhizobactéries bénéfiques pour les plantes (PGPR) dans la rhizosphère. Lors de cette thèse, la capacité des exsudats racinaires de colza (Brassica napus), de pois (Pisum sativum) et de ray-grass (Lolium perenne) à attirer et nourrir trois PGPR (Bacillus subtilis ATCC 6633, Pseudomonas fluorescens ATCC 17400 et Azospirillum brasilense Sp245) a été mesurée et comparée grâce à la définition d’un nouvel indicateur, le score de « love match ». Pour toutes ces bactéries, les exsudats de colza sont les plus attractifs et induisent la croissance la plus rapide, ceux de pois permettent la production de biomasse la plus élevée, tandis que ceux de ray-grass sont les moins efficaces. Si l’on compare les PGPR, P. fluorescens et A. brasilense semblent répondre plus efficacement aux exsudats racinaires que B. subtilis. L’analyse transcriptomique révèle quant à elle que B. subtilis régule l’expression de nombreux gènes en réponse aux exsudats racinaires, tandis que P. fluorescens semble déjà exprimer la plupart des gènes nécessaires à cette réponse. Ces résultats mettent en évidence la sélection spécifique des PGPR par la plante à travers ses exsudats racinaires, et pourraient aider à sélectionner les exsudats les plus efficaces pour promouvoir l'établissement de bioinoculants dans la rhizosphère
The rhizosphere is the narrow zone of soil under the influence of plant roots that release a complex molecular mixture: root exudates. They allow the plant to recruit its rhizosphere microbiota, which plays a key role in its growth and resistance to biotic and abiotic stresses. In the context of sustainable agriculture, understanding the molecular root-microbiota dialogue could help to promote the establishment of Plant Growth-Promoting Rhizobacteria (PGPR) in the rhizosphere. In this thesis, the ability of root exudates from rapeseed (Brassica napus), pea (Pisum sativum) and ryegrass (Lolium perenne) to attract and feed three PGPR (Bacillus subtilis ATCC 6633, Pseudomonas fluorescens ATCC 17400 and Azospirillum brasilense Sp245) was measured and compared by defining a new indicator, the « love match » score. For all bacteria, rapeseed exudates are the most attractive and induce the fastest growth, pea exudates allow the highest biomass production, while ryegrass exudates are the least effective. When comparing PGPR, P. fluorescens and A. brasilense seem to respond more efficiently to root exudates than B. subtilis. Transcriptomic analysis reveals that B. subtilis regulates the expression of many genes in response to root exudates, whereas P. fluorescens appears to already express most of the genes required for this response. These results highlight the specific selection of PGPR by the plant through its root exudates, and could help to select the most efficient exudates in order to promote the establishment of bioinoculants in the rhizosphere

A interação entre bactérias e plantas resulta na ocorrência de vários processos biológicos no ambiente e pode ser regulada por diferentes fatores. Considerando que um recíproco reconhecimento ocorre entre as espécies de bactérias e plantas, alterações nos fatores bióticos e abióticos interferem diretamente nesta interação levando a modificações na composição das comunidades bacterianas associadas às plantas. No presente trabalho foram avaliados diferentes fatores que estão diretamente relacionados a este assunto. Atualmente, a aplicação de técnicas de microbiologia molecular permite acessar alterações causadas nestas comunidades de maneira independente do cultivo bacteriano. Desta maneira, foi demonstrado que na rizosfera de plantas de tabaco, os diferentes estágios de desenvolvimento são os principais determinantes da composição da comunidade bacteriana em detrimento do genótipo das plantas, sejam estas transgênicas ou convencionais. Utilizando plantas transgênicas e convencionais de eucalipto de diferentes genótipos, foi verificado que diferentes plantas não transgênicas podem apresentar comunidades bacterianas mais distintas do que quando os clones não transgênicos são comparados com os transgênicos. No entanto, efeitos específicos podem ocorrer na interação bactéria-planta, como o observado pela inibição da população de Methylobacterium spp. em plantas transgênicas de eucalipto TR-15. Avaliando o efeito da inoculação de bactérias endofíticas na composição de comunidades bacterianas associadas à plantas de batata de diferentes cultivares, os resultados mostram que a inoculação de Pseudomonas putida altera a comunidade bacteriana de maneira similar a alteração da variedade da planta. Os demais isolados avaliados, classificados como Paenibacillus sp. e M. mesophilicum, causam menores alterações na composição das comunidades bacterianas. Adicionalmente foi demonstrado que a colonização das plantas de batata pelo endófito P. putida resulta em pequena alteração no perfil metabólico da planta hospedeira. Por fim, buscando melhores métodos de isolamento da comunidade bacteriana da rizosfera de batata, os resultados mostraram que a mimetização do ambiente pode resultar em melhor acesso da diversidade bacteriana por meio de isolamento e cultivo das espécies componentes desta comunidade.
The plant-bacteria interactions result in the occurrence of biological process in the environment and might be regulated by different factors. Considering that a reciprocal recognition occur amongst bacteria and plants species, shifts in biotic and abiotic factors interfere directly on these interactions, leading to modifications in the composition of bacterial communities to plants associated. In the present work different factors related to this subject were evaluated. Nowadays, the applications of techniques of molecular microbiology allow assessing the shifts caused on these communities by a culture independent approach. On this way, it was demonstrated that in rhizosphere of tobacco plants, different stages of plants development are main determinants of bacterial community composition rather than the plants genotypes, transgenic or not. Using plants transgenic or not, carrying different genotypes, it was verified that different non-transgenic plants could harbor bacterial communities more distinct than those observed in association with transgenic plants. However, specific effects could be observed like the inhibition of Methylobacterium spp. population in eucalyptus transgenic plants TR-15. Considering the effect of endophytic bacteria inoculation in the composition of bacterial communities associated to different cultivars of potato plants, the results show that inoculation of Pseudomonas putida causes similar shifts in the bacterial community similarly to those observed when different cultivars are considered. Other evaluated strains, classified as Paenibacillus sp. and M. mesophilicum, caused minor alterations in the composition of bacterial communities. In addition, it was demonstrated that plant colonization by endophytic P. putida results in small effects on the metabolic profile of host plant. At least, aiming better methodologies for bacteria isolation from potato rhizosphere, the results show that mimicking the natural environment could result in a better assessment of bacterial diversity by isolation of species present in this community.

La vigne est confrontée à de nombreuses maladies parmi lesquelles le mildiou, causé par l’oomycète Plasmopara viticola, est l’une des plus préoccupantes dont le contrôle repose essentiellement sur l’utilisation de fongicides de synthèse. L’un des défis majeurs pour la viticulture est donc de développer des stratégies de protection durables basées sur l'exploitation des fonctions bénéfiques du microbiome rhizosphérique comme levier actionnable du biocontrôle. Le potentiel fonctionnel du microbiome rhizosphérique de la vigne est encore inexploré et les relations entre l'assemblage microbien et la résistance aux maladies sont loin d’être établies. Dans cette étude, des approches métagénomiques et culturomique ont été mises en œuvre pour la caractérisation des communautés bactériennes de la rhizosphère, de l’endosphère et du rhizoplan de deux cépages de sensibilité différente à P. viticola, Chardonnay (sensible) et Voltis (hybride résistant). Les analyses de métabarcoding 16S et métagénomique shotgun ont révélé des différences taxonomiques et fonctionnelles entre les rhizocompartiments des deux cépages. Elles ont également mis en évidence des traits fonctionnels communs entre les bactériomes de la rhizosphère et du rhizoplan chez les deux cépages. Les analyses ont mis en évidence des différences fonctionnelles entre les plantes saines et infectées, avec une perte des taxons bénéfiques dans la rhizosphère du cépage sensible après infection. Cependant, le cépage résistant a montré un microbiote rhizosphérique enrichi en fonctions pour la colonisation racinaire et l’antagonisme direct. De plus, ce microbiote bactérien s’est maintenu chez le Voltis même après attaque de P. viticola. L’approche culturomique, reposant sur des milieux synthétiques (LB, R2A) et à base d’extrait de sol supplémenté ou non avec un extrait de racines de vigne, combinée à une identification taxonomique a révélé des taxons bactériens communs comme des Pseudomonas, Phyllobacterium, Bacillus, et des taxons peu étudiés (i.e. Mycetocola, Isoptericola). Le séquençage du génome des taxons cultivés les plus abondants de la rhizosphère de Chardonnay sain (Pseudomonas et Phyllobacterium) et de Voltis (Sphingomonas, Streptomyces et Agromyces) a révélé davantage de fonctions pour l'induction de l'immunité de la plante chez les genres du Chardonnay, et pour la dégradation des composés végétaux et la colonisation des racines chez ceux du Voltis. La comparaison des souches Pseudomonas spp. et Phyllobacterium spp. issues de chaque cépage a également mis en évidence des fonctions pour la production d'acétoïne, de 2,3-butanediol, de sidérophores, de lipopeptides et de GABA chez celles provenant du Chardonnay. La réalisation de communautés microbiennes synthétiques (SynComs) combinée à des analyses de métabarcoding 16S ont permis également de mettre en évidence la présence de souches individuelles et cooccurrentes dans des communautés naturelles (NatComs) stabilisées, avec des Enterobacter et Serratia spp. communs chez Chardonnay, et des Achromobacter, Pseudomonas et Serratia chez Voltis. Le criblage in planta de différentes bactéries cultivées a permis d’identifier des souches (Pseudomonas et Chryseobacterium) capables d’induite une résistance systémique de la vigne contre P. viticola. L’analyse de leurs génomes, la caractérisation de certains traits bénéfiques pour la plante et de leur capacité à induire l’immunité systémique de la vigne ont également été appréhendées
Grapevines are faced to numerous diseases, including downy mildew, caused by the oomycete Plasmopara viticola, which is one of the most threatening and controlled mainly through the use of synthetic fungicides. One of the major challenges for viticulture is therefore to develop sustainable protection strategies based on harnessing the beneficial functions of the rhizosphere microbiome as a lever for biocontrol. The functional potential of the grapevine rhizosphere microbiome is still unexplored, and the relationships between microbial assembly and disease resistance are not yet well established. In this study, metagenomic and culturomic approaches were used to characterize the rhizosphere, endosphere and rhizoplane bacterial communities of two grape cultivars with contrasting susceptibilities to P. viticola, Chardonnay (susceptible) and Voltis (resistant hybrid). 16S metabarcoding and shotgun metagenomic analyses revealed taxonomic and functional differences between the rhizocompartments of the two grape cultivars. They also revealed common functional traits between the rhizosphere and rhizoplan bacteriomes of the two grape cultivars. Analyses revealed functional differences between healthy and infected plants, with a loss of beneficial taxa in the rhizosphere of the susceptible cultivar after infection. However, the resistant cultivar showed a rhizosphere microbiota enriched with functions for root colonization and direct antagonism. Moreover, this bacterial microbiota was maintained in the resistant cultivar even after P. viticola attack. The culturomic approach, based on synthetic media (LB, R2A) and soil extract supplemented or not with grapevine root extract, combined with taxonomic identification revealed common bacterial taxa as Pseudomonas, Phyllobacterium, Bacillus, and poorly studied taxa (i.e. Mycetocola, Isoptericola). Genome sequencing of the most abundant cultivated taxa from the rhizosphere of healthy Chardonnay (Pseudomonas and Phyllobacterium) and Voltis (Sphingomonas, Streptomyces and Agromyces) revealed more functions for the induction of plant immunity in the Chardonnay genera, and for the degradation of plant compounds and root colonization in the Voltis genera. Comparison of the Pseudomonas spp. and Phyllobacterium spp. strains from each grape cultivar also revealed functions for the production of acetoin, 2,3-butanediol, siderophores, lipopeptides and GABA in those from Chardonnay. Synthetic microbial communities (SynComs) combined to 16S metabarcoding approach also revealed the presence of individual and co-occurring strains in stabilized natural communities (NatComs), with Enterobacter and Serratia spp. common in Chardonnay, and Achromobacter, Pseudomonas and Serratia in Voltis. In planta screening of various cultivated bacteria allowed us to identify strains belonging to Pseudomonas and Chryseobacterium able to induce systemic resistance in grapevines against P. viticola. Analysis of their genomes, characterization of some of their beneficial traits for the plant, and their ability to induce systemic immunity in grapevines were also carried out

Diazotróficos de vida-livre podem ser encontradas associadas à filosfera, dermosfera e rizosfera das espécies vegetais. Alguns dados sugerem que a fixação biológica de N2 (FBN) por bactérias assimbióticas representa uma entrada importante de nitrogênio nos ecossistemas tropicais, variando com as espécies vegetais e nas diferentes partes da planta. O presente trabalho teve como objetivos estimar a quantidade de N2 fixado de forma assimbiótica na filosfera, dermosfera e rizosfera sobre a copa das espécies vegetais Guapira oposita e Euterpe edulis, e avaliar a diversidade das bactérias assimbióticas, através da análise do gene rRNA 16S, em uma Restinga ,em Ubatuba, SP. O estudo foi realizado no Parque Estadual da Serra do Mar, Núcleo Picinguaba, em épocas de baixa e alta pluviosidade. A atividade da nitrogenase foi determinada pela técnica de redução do acetileno e as concentrações de etileno foram determinadas por cromatografia gasosa. A diversidade de bactérias que habitam filosfera, dermosfera e solo foi acessada por pirosequenciamento da região V4 do gene rRNA 16S. A maior fixação de N foi observada na dermosfera de E. edulis nas duas épocas de coleta (175,1± 53,4 ng cm-2 h-1; 97,2 ± 21 ng cm-2 h-1), as taxas de fixação de N mais baixas foram observadas no solo. Na época de alta pluviosidade, a FBN na filosfera de G. oposita (52,0 ± 12 ng cm-2 h-1) foi significativamente maior do que a filosfera de E. edulis (3,6 ± 06 ng cm-2. h-1) e do que no mesmo compartimento mas em diferentes épocas de coleta (7,5 ± 1,3 ng cm-2 h-1). O valor do 15N foi maior no solo onde a fixação de N foi mais baixa. Na filosfera e na dermosfera, a relação C/N foi mais baixa quando a FBN foi mais alta. A FBN no solo e serrapilheira de restinga apresentou grande variação espacial, com locais de alta atividade. As 188629 sequências obtidas foram agrupadas em 16727 Unidades Taxonômicas Operacionais (UTOs), distribuídos em 35 filos. Os principais filos detectados foram Proteobacteria (38%) e Acidobacteria (12%). As classes Alphaproteobacteria e Gammaproteobacteria foram as mais abundantes nos três compartimentos. Potenciais fixadores de N foram detectados nas classes Alpha Beta e Gammaproteobacteria. A abundância de cianobacterias fixadoras de N na filosfera e na dermosfera foi baixa, indicando que outros diazotróficos também colonizam esses ambientes e contribuem com a FBN.
Free-living N2 fixing bacteria can be found associated with the phyllosphere, bark and rizosphere of the diferent plant species. Some data suggest that biological N2 fixation (BNF) by free-living bacteria represents an important input of nitrogen in tropical ecosystem, varying with the plant species and in different parts of the plant. This study aimed to estimate the amount of N2 fixed in the phyllosphere, bark and soil under the canopy of Guapira opposite and Euterpe edullis, and evaluate the diversity of bacteria through the sequencing of the 16S rRNA gene analysis, the phyllosphere, bark and soil in a Restinga area, Ubatuba, SP. The study was conducted in the Parque Estadual da Serra do Mar, Núcleo Picinguaba in seasons of low and high rainfall. Nitrogenase activity was determined by the acetylene reduction assay (ARA) and ethylene concentrations were determined by gas chromatography. The diversity of bacteria in the phyllosphere, bark and soil was accesed using pyrosequencing of the 16S rRNA V4 region. The bark of Euterpe edullis was higher at both sampling times (175,1±53,4 ng. cm-2. h-1, 97,2±21 ng. cm-2. h-1). The BNF rates were lower in soil. In high rainfall conditions, the BNF in the phyllosphere of Guapira opposite increased significantly (52,0±12 ng. cm-2. h-1) when compared with Euterpe edullis (3,6 ± 06 ng. cm-2. h-1) and Guapira opposite (7,5 ± 1,3 ng. cm-2. h-1) phyllosphere. The value of 15N was higher in the soil where the rates of FBN was lower. In the phyllosphere and bark, C/N was lower when BNF was higher. BNF in soil great spatial variation with areas of high activity. The 18.629 sequences obtained were grouped into 16.727 Operational Taxonomic Units (OTUs) distributed in 35 phyla. The main phyla Proteobacteria represented 38% of the OTUs and Acidobacteria 12% of the UTOs. The classes Alphaproteobacteria and Gammaproteobacteria were the most abundant in the three compartmens. Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria were the main potential N-fixers. The abundance of nitrogen-fixing Cyanobacteria in the phyllosphere and bark was low, indicating that others diazotrophics also colonize these environments and contribute with BNF.

Le phytomanagement compte parmi les méthodes innovantes de gestion des sites et sols pollués en raison de son adéquation avec le développement durable. Pour répondre à la double exigence de performances environnementales et économiques, les phytotechnologies sont désormais combinées à la valorisation de la biomasse produite sur sols pollués. Parmi les filières éco-innovantes et non-alimentaires de valorisation de cette biomasse, la culture de plantes à parfums, aromatiques et médicinales (PPAM) pour la production d’huiles essentielles (HE), substances biosourcées à haute valeur ajoutée, a été proposée dans le cadre du projet PhytEO, financé par l’ADEME. Ainsi, ma contribution à ce projet a permis d‘évaluer la pertinence d’un mode de gestion reposant sur la culture de la sauge sclarée et de la coriandre, en présence ou non d’un amendement mycorhizien, en s’appuyant sur des démonstrateurs in situ à l’échelle de l’hectare, parcelles historiquement polluées ou non par les éléments traces (ET). La coriandre ainsi que la sauge sclarée ont toutes deux montré une bonne capacité à s’installer, à se développer et à produire une quantité importante de biomasse sur des sols présentant une forte pollution historique par les ET. Bien que l’ajout d’un amendement biologique à base de champignons mycorhiziens à arbuscules ait permis d’augmenter de façon significative les taux de mycorhization des racines de coriandre et de sauge sclarée, aucun gain n’a été observé ni au niveau de la croissance de ces deux PPAM, ni au niveau de la quantité et de la qualité des HE. Un effet positif en termes d’immobilisation des ET dans le sol, en particulier du Pb, et une réduction des transferts des ET (Cd et Pb) dans les parties aériennes a toutefois été montré chez la sauge sclarée. D’autre part, les HE distillées à partir de la biomasse de sauge sclarée (inflorescences) ou de coriandre (parties aériennes ou graines) cultivées sur sols pollués présentent une qualité très satisfaisante, caractérisée par leur non contamination en ET ou en résidus de pesticides (traces). Leurs compositions chimiques, non altérées par la pollution en ET et l’inoculation mycorhizienne, affichent divers principes actifs dont le linalol, l’acétate de linalyle, le germacrène, l’α-pinène, le γ-terpinène, le 2-décénal, le décanal et le 2-dodécénal. Ainsi, plusieurs propriétés biologiques potentiellement valorisables dans des domaines à vocation non-alimentaire, tels que la protection des cultures (effets fongistatiques, anti-germinatifs et herbicides) ou la santé humaine (effets anti-inflammatoires et antioxydants) ont été mises en évidence. En revanche, en dépit de son fort pouvoir complexant des HE, la β-cyclodextrine n’a montré aucun effet positif sur les propriétés biologiques testées. Par ailleurs, la végétalisation du sol pollué par la sauge sclarée permet une modulation de la structure des communautés bactériennes et fongiques, telluriques et racinaires. Enfin, l’ensemble des résultats de cette étude et de l’analyse technico-économique de la filière PPAM-HE soulignent le haut potentiel de la sauge sclarée dans une démarche de phytomanagement de sols pollués par les ET
To cope with the soil trace element (TE) pollution, phytomanagement was brought forward as an eco-friendly and feasible approach. To meet the dual requirements of environmental and economic performances, phytotechnologies are now combined with the valorisarion of biomass produced on polluted soils. Among the eco-innovative channels intended for the non-food valorisation of the produced biomass, the cultivation of aromatic and medicinal plants producing essential oils (EO), high-added value biosourced products, has been suggested within the framework of PhytEO project, funded by ADEME. Thus, this thesis contributed to evaluate the relevance of a management approach based on clary sage and coriander cultivation, combined or not with a mycorrhizal inoculum, on in situ experimental plots polluted or not with TE. Both clary sage and coriander have shown a good ability to settle in, to grow and to produce high amounts of biomass, despite the presence of high TE concentrations in the soil. The addition of an amendment based on arbuscular mycorrhizal fungi enhanced the mycorrhizal colonisation rates of both coriander and clary sage roots. However, no significant improvement was observed in terms of plants’ growth, EO amounts and quality. Moreover, mycorrhizal inoculation allowed TE immobilisation in soils, in particular Pb, and reduced TE (Cd and Pb) transfers in aerial parts of clary sage. Besides, the EO distilled from clary sage inflorescences and coriander (aerial parts orseeds) grown on TE polluted soils, displayed a highly satisfactory quality, regarding the absence of contamination by TE or pesticide residues (trace amounts). Their chemical compositions were not altered by both TE pollution and mycorrhizal inoculation and were characterized by several active principles, such as linalool, linalyl acetate, germacrene, α-pinene, γ-terpinene, 2-decenal, decanal and 2-dodecenal. Furthermore, several biological properties with potential applications in non-food fields such as crop protection (antifungal, antigerminative and herbicidal) or human healthcare (antioxidant and anti-inflammatory) have been brought forward. However, despite its ability to form inclusion complexes with EO components, β-cyclodextrin has shown no significant improvement of the tested EO biological activities. On another note, the soil revegetation with clary sage displayed a clear shaping of the bacterial and fungal communities, in both the rhizospheric soil and the roots of the aromatic plant species. Altogether, our results combined with the techno-economic feasibility assessment of the channel “aromatic and medicinal plants/EO” have emphasised clary sage as a good candidate for the phytomanagement of TE-polluted soils

Endophytes are organisms that live inside plants without causing disease and include microbes that benefit their hosts by aiding in nutrient acquisition and pathogen control. This thesis concerns the endophytes of the genus Zea which includes modern maize (Zea mays L.). Beginning 9,000 years ago, maize was domesticated from wild grasses in Mexico (teosintes), bred into diverse varieties and moved to new soils throughout the Americas. The impact of these long-term changes on the associated endophytic communities has not been examined. Furthermore, today, maize is routinely transplanted around the world to facilitate breeding, but the short-term impact of switching soils on endophyte composition is not known. I attempted to answer the first question by surveying the bacterial endophytes that inhabit 14 diverse ancestral, ancient and modern Zea genotypes. To answer the second question, three extreme Zea genotypes, ancestral, intermediate and modern, were grown side by side on two extreme soils that span the tropical-to-temperate migration route of maize. Endophyte populations from seeds, roots and shoots were DNA fingerprinted using terminal restriction length polymorphism (TRFLP) of 16S rDNA. To understand microbial functions, bacteria were cultured and tested for >13 in vitro traits including nitrogen fixation, phosphate solubilization, plant hormone production and antibiosis. Relationships between endophyte communities were analyzed using principle component analysis (PCA) and Sᴓrensen’s similarity index. The results show that different Zea tissues and genotypes have diverse endophytic communities. The community composition of seed endophytes correlates with host phylogeny suggesting that as humans bred maize, they inadvertently impacted its microbial inhabitants, though the change was gradual. Soil swapping and growth on sterile sand confirm that shoot and root endophyte communities in juvenile plants are primarily inherited. However, a given maize genotype can also select and take up the same microbes (based on TRFLP) from geographically diverse soils. These latter results show that the endophyte communities of Zea plants are significantly buffered from the short-term effects of migration. A few microbes and microbial traits are conserved across all Zea genotypes and soil treatments, suggestive of a core taxonomic and functional microbiota for this agriculturally important genus.
OMAFRA New Directions, Ontario Research Fund, Canadian Foundation for Innovation

Tese de mestrado, Biologia Evolutiva e do Desenvolvimento, Universidade de Lisboa, Faculdade de Ciências, 2020
Interactions between microorganisms and plants have occurred for millions of years. Combining the capacity of light and CO2 usage by the plants with the capacity of efficient substrate usage by their microbiota, the water-to-land-transition was possible. Since then, plants and plant microbiota have coevolved, and today, the microbiome is considered as an extension of the plant’s genetic assembly. However, the agricultural revolution led to progressive alterations in habitat, crop managing practices, and breeding to promote crop production changing their evolutionary trajectory. Moreover, the trajectory of the co-evolution between crops and their microbiome is also changed. The soil, the plant, and the microorganisms are connected and impact each other. The rhizosphere is considered to be the most dynamic interface on Earth, and the microorganisms that exist there might promote plant growth and resilience. These microorganisms are referred to as Plant Growth-Promoting Microbes (PGPM), including Plant Growth-Promoting Rhizobacteria (PGPR) and Arbuscular Mycorrhizal Fungi (AMF). The experimental work is divided into three chapters according to the plant-model. In the first one, microorganisms (AMF and bacteria) were collected from a wild relative of chrysanthemum (Dendranthema grandiflora). Plant Growth Promoting Rhizobacteria (PGPR) was submitted for molecular analysis. Three of them (previously selected by plant growth-promoting traits) were inoculated on five chrysanthemum commercial cultivars to test their impact on plant performance and root microbiome assembly. AMF was also inoculated with the same propose. PGPR impacted the number of nodes and root biomass of commercial chrysanthemum cultivars. AMF affected the root biomass of cultivars of chrysanthemum cultivated in autoclaved and non-autoclaved soil. AMF root colonization was not found. All the treatments impacted the microbiome assembly in the tested commercial cultivars. Concluding, PGPR and AMF obtained from wild chrysanthemum impacted growth performance and microbiome assembly in five commercial cultivars. In the second one, AMF collected from wild chrysanthemum were proliferated using millet (Panicum miliaceum) as a host in order to have an inoculum of two morphotypes. The two morphotypes of AMF spores were successfully multiplied in millet roots and sorrowing soil. Millet appeared as a good host for the propagation of AMF spores. Lastly, in the third chapter, the same AMF were inoculated in sorghum (Sorghum bicolor) to test their impact on plant performance and resilience against Striga hermonthica, a parasitic weed. Sorghum growth performance and resilience were impacted by the presence of S. hermonthica and/or AMF in the soil. AMF root colonization on sorghum was observed and AMF treatments reduced S. hermonthica germination by 59%. However, the reduction of the germination of S. hermonthica seeds did not increase the sorghum growth performance, so more studies are needed to understand these mechanisms. Promising results were found, but additional work is needed to understand how these inocula are best applied in the field and the mechanisms behind it. It is also necessary to comprehend how the entire microbiome is affected by the inocula and how these changes impact growth performance and resilience in both, chrysanthemum, and sorghum species.

Eastern black walnut (Juglans nigra L.) ranks among the most highly valued timber species in the central hardwood forest and across the world. This valuable tree fills a critical role in native ecosystems as a mast bearing pioneer on mesic sites. Along with other Juglans spp. (Juglandaceae), J. nigra is threatened by thousand cankers disease (TCD), an insect-vectored disease first described in 2009. TCD is caused by the bark beetle Pityophthorus juglandis Blackman (Corthylini) and the phytopathogenic fungus Geosmithia morbida Kol. Free. Ut. & Tiss. (Bionectriaceae). Together, the P. juglandis-G. morbida complex has expanded from its historical range in southwest North America throughout the western United States (U.S.) and Europe. This range expansion has led to widespread mortality among naïve hosts J. nigra and J. regia planted outside their native distributions.

The severity of TCD was previously observed to be highest in urban and plantation environments and outside of the host native range. Therefore, the objective of this work was to provide information on biotic and abiotic environmental factors that influence the severity and impact of TCD across the native and non-native range of J. nigra and across different climatic and management regimes. This knowledge would enable a better assessment of the risk posed by TCD and a basis for developing management activities that impart resilience to natural systems. Through a series of greenhouse-, laboratory- and field-based experiments, environmental factors that affect the pathogenicity and/or survival of G. morbida in J. nigra were identified, with a focus on the microbiome, climate, and opportunistic pathogens. A number of potentially important interactions among host, vector, pathogen and the rest of the holobiont of TCD were characterized. The holobiont is defined as the whole multitrophic community of organisms—including J. nigra, microinvertebrates, fungi and bacteria—that interact with one another and with the host.

Our findings indicate that interactions among host, vector, pathogen, secondary pathogens, novel microbial communities, and novel abiotic environments modulate the severity of TCD in native, non-native, and managed and unmanaged contexts. Prevailing climatic conditions favor reproduction and spread of G. morbida in the western United States due to the effect of wood moisture content on fungal competition. The microbiome of soils, roots, and stems of trees and seedlings grown outside the host native range harbor distinct, lower-diversity communities of bacteria and fungi compared to the native range, including different communities of beneficial or pathogenic functional groups of fungi. The pathogen G. morbida was also associated with a distinct community of microbes in stems compared to G. morbida-negative trees. The soil microbiome from intensively-managed plantations facilitated positive feedback between G. morbida and a disease-promomting endophytic Fusarium solani species complex sp. in roots of J. nigra seedlings. Finally, the nematode species Bursaphelenchus juglandis associated with P. juglandis synergizes with G. morbida to cause foliar symptoms in seedlings in a shadehouse; conversely, experiments and observations indicated that the nematode species Panagrolaimus sp. and cf. Ektaphelenchus sp. could suppress WTB populations and/or TCD outbreaks.

In conclusion, the composition, function, and interactions within the P. juglandis and J. nigra holobiont play important roles in the TCD pathosystem. Managers and conservationists should be aware that novel associations outside the host native range, or in monocultures, intensive nursery production, and urban and low-humidity environments may favor progression of the disease through the effects of associated phytobiomes, nematodes, and climatic conditions on disease etiology. Trees in higher diversity, less intensively managed growing environments within their native range may be more resilient to disease. Moreover, expatriated, susceptible host species (i.e., J. nigra) growing in environments that are favorable to novel pests or pest complexes (i.e., the western U.S.) may provide connectivity between emergent forest health threats (i.e., TCD) and native host populations (i.e., J. nigra in its native range).