Effects of Dry and Wet Sieving of Soil on Identification and Interpretation of Microbial Community Composition

Soil aggregates are microhabitats for microorganisms, and directly influence microorganisms that live within and are influenced by microorganisms in return. Two methods are used to isolate soil aggregates by their size: dry sieving (sieving air-dried soil) and wet sieving (sieving soil in water). Wet-sieving methods are generally considered to represent separation of aggregate classes that are stable to physical disaggregation in water, a condition considered favorable for protecting soil structure over time. However, little is known about the effect of sieving methods on microbial abundance, diversity, and functions, hindering the understanding of the relationship between soil structure and soil aggregates as habitat and soil microorganisms. In this study, the effect of dry and wet sieving on bacterial diversity, and abundance of microorganisms involved in N fixation (nifH gene), nitrification (amoA bacteria and archaea), and denitrification (narG, nirS and nosZ genes), was determined for four sizes of soil aggregates from a cropland and grassland. Quantitative-PCR (Q-PCR) showed little differences in relative gene abundance between size fractions of soil aggregates, but wet-sieving method significantly increased gene abundance for amoA bacteria, nirS and nosZ genes. When the N functional genes were expressed as percentage of the bacterial 16S rRNA genes, the wet sieving resulted in significantly higher genes percentage for all the genes (except for narG gene), and significant differences between soil aggregate size fractions at the grassland site. The different sieving methods resulted in different bacterial community compositions, but only the wet-sieving method was able to reveal significant differences in bacterial community composition between soil fractions in grassland. The results demonstrate significantly different quantitative and qualitative interpretation of soil microbial community depending on whether aggregate samples were obtained from wet or dry sieving, highlighting the importance in the choice of the sieving method

Bacterial biogeography and functional genes abundance in Koiliaris critical zone observatory

The microorganisms drive the nutrient biogeochemical cycles, ecosystems functioning and the delivered by them services. Understanding the environmental variables that drive the spatial distribution patterns of microbial communities is crucial to anticipate ecosystem responses to global changes, to human perturbation and to develop proper tools for predicting their functioning. The objective of this PhD Thesis was to characterize the variations of the distribution of soil microbial community and to investigate the factors that affect that distribution, at the scale of the watershed, by adopting phylogenetic (16S rRNA) and functional gene (N cycle) data.This study investigated the distribution of Archaea, Bacteria and Fungi as well as the dominant bacterial phyla (Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes), and classes of Proteobacteria (Alpha- and Betaproteobacteria) across the Koiliaris watershed by qPCR and linked them with environmental variables. Predictive maps of microorganisms’ distribution at watershed scale were generated by co-kriging, using the most significant predictors. Our findings showed that 31–79% of the spatial variation in microbial taxa abundance could be explained by the parameters measured, with total organic carbon and pH being identified as the most important. Moreover, strong correlations were set between microbial groups. Our findings shed light on the factors shaping microbial communities at a high taxonomic level and provide evidence for ecological coherence and syntrophic interactions at the watershed scale.In the following step, a next-generation, Illumina-based sequencing approach was employed to characterize the bacterial community within a 3-year field sampling campaigns, from 22 sites including two soil depths (0-15 and 15-30 cm). The interannual variation patterns that observed for α- and β-diversity attributed mainly to climatic variations. Land use and soil depth explained 14 and 2%, respectively, of the β-diversity variations, while they had not any effect on α- diversity metrics. Moreover, environmental variations and geographic distance showed a weak correlations with both biodiversity levels. This led as to consider that other processes drive the community assembly. In fact, fitting the Sloan’s neutral community assembly model to our data, provided a very good prediction of R2=0.84. Even, the community found to be assembled mainly by stochastic processes, there were indices for environmental influences at a more local scale. Machine-learning approaches demonstrated that agricultural lands were enriched with Proteobacteria, Acidobacteria, Bacteroidetes, Elusimicrobia, OD1 and WS3, while natural ecosystems with Verrucomicrobia, Actinobacteria, Chlotoflexi, Firmicutes, AD3 and Gemmatimonadetes. Network analysis revealed non-random co-occurrence patterns for discriminant communities of each land use and soil depth. Moreover, keystone OTUs was members of model sub community that occurred more frequently that the model’s prediction.Emphasis was also given on functional microorganism regulating the cycling of N (diazotrophs, ammonia oxidizers, denitrifiers) and on the environmental factors shaping their biogeographic patterns across the Koiliaris watershed. Our findings revealed that a proportion of variance ranging from 40 to 80% of functional genes abundance could be explained by the environmental variables monitored, with pH, soil texture, total organic carbon and potential nitrification rate being identified as the most important drivers. Prediction maps, generated by cokriging, revealed distinct patterns of functional genes. Strong relationships were set between functional groups, which were further mediated by the land use (natural vs agricultural lands). These relationships, in combination with the environmental variables, allow us to provide insights regarding the ecological preferences of N-functional groups and among them the recently identified clade II of nitrous oxide reducers.Οι μικροοργανισμοί του εδάφους κατέχουν κυρίαρχο ρόλο στους βιογεωχημικούς κύκλους των θρεπτικών στοιχείων και κατά συνέπεια στη λειτουργία και παραγωγικότητα των φυσικών αλλά και ανθρωπογενών οικοσυστημάτων. Είναι κρίσιμη λοιπόν η κατανόηση των παραμέτρων, βιοτικών και αβιοτικών, που επηρεάζουν τα μοτίβα της χωρικής κατανομής των μικροβιακών κοινοτήτων ώστε να είναι εφικτή η πρόβλεψη της απόκρισης τους στην κλιματική αλλαγή αλλά και σε ανθρωπογενείς επεμβάσεις, καθώς επίσης για την ανάπτυξη κατάλληλων “εργαλείων” πρόβλεψης των διεργασιών που εμπλέκονται οι μικροοργανισμοί του εδάφους. Στόχος της παρούσας διδακτορικής διατριβής είναι η μελέτη, σε μεγάλη κλίμακα (λεκάνη απορροής), των διακυμάνσεων της κατανομής της μικροβιακής κοινότητας του εδάφους. Για τον στόχο αυτό, η μικροβιακή κοινότητα, μελετήθηκε με την χρήση φυλογενετικών δεικτών και λειτουργικών γονιδίων καθώς επίσης και προσδιορίστηκαν βιογεωχημικές διεργασίες (ρυθμός νιτροποίησης και ανοργανοποίησης C, ενεργότητα ενζύμων) και οι παράγοντες που επιδρούν σε αυτήν (περιβάλλον, κλίμα, χρήσεις γης, ιδιότητες και βάθος εδάφους, χρόνος). Τα αποτελέσματα της διατριβής ερμηνεύτηκαν συνδυάζοντας τα αποτελέσματα των μοριακών αναλύσεων (qPCR, 16S rRNA αλληλούχιση) με μετρήσεις φυσικοχημικών και βιοχημικών παραμέτρων, χρησιμοποιώντας προηγμένες μεθόδους στατιστικής και γεωστατιστικής ανάλυσης. Αρχικά, ο ολικός οργανικός C (TOC) και το pH αναγνωρίστηκαν ως οι σημαντικότερες περιβαλλοντικές παράμετροι που συσχετίζονται με την κατανομή της αφθονίας των μικροβιακών τάξων, καθώς επίσης και παρέχονται ενδείξεις ύπαρξης οικολογικής συνοχής και συντροφικών αλληλεπιδράσεων μεταξύ των μικροβιακών τάξων, σε επίπεδο λεκάνης απορροής. Η συγκρότηση της μικροβιακής κοινότητας βρέθηκε να διέπεται κυρίως από στοχαστικές διαδικασίες και ειδικότερα από ένα συνδυασμό τυχαίων μεταναστεύσεων, γεννήσεων και θανάτων και διασποράς. Στην συνέχεια, η εφαρμογή μηχανικών μεθόδων μάθησης έδειξε πως οι χρήσεις γης και το βάθος του εδάφους μπορεί να κατηγοριοποιηθεί με βάση τη δομή της μικροβιακής κοινότητας σε υψηλό ταξινομικό επίπεδο. Η ανάλυση των δικτύων για τις χρήσεις γης και τα εδαφικά βάθη, ανέδειξαν στοιχεία σχετικά με τις σχέσεις αλληλεπίδρασης των μικροοργανισμών αλλά και τον πιθανό ρόλο τους στους βιογεωχημικούς κύκλους του C και N για την περιοχή μελέτης μας. Πιο συγκεκριμένα, για τον κύκλο του Ν, μελετήθηκαν οι διάφορες διεργασίες του στην περιοχή του ποταμού Κοιλιάρη και οι περιβαλλοντικές παράμετροι που τις επηρεάζουν, μέσω της ποσοτικοποίησης των λειτουργικών γονιδίων που εμπλέκονται σε αυτές. Υπολογίστηκε η αφθονία των αζωτοδεσμευτικών βακτηρίων, των οξειδωτών της αμμωνίας και των απονιτροποιητών, συμπεριλαμβανομένου και του πρόσφατα αναγνωρισμένου clade II του γονιδίου nosZ. Οι περιβαλλοντικές παράμετροι που προσδιορίστηκαν, με σημαντικότερη το pH, εξήγησαν μέχρι και το 80% της διακύμανσης της αφθονίας των λειτουργικών ομάδων και οι χρήσεις γης βρέθηκε να καθορίζουν την ισχύ και το είδος των σχέσεων που παρατηρήθηκαν μεταξύ των λειτουργικών ομάδων

Evaluation de la toxicité de pesticides sur la germination de Rhizophagus intraradices en systèmes miniaturisés

L’UE dispose de l’un des systèmes les plus stricts au monde en matière d’autorisation et de contrôle de l’utilisation des pesticides. Minimiser l’impact de ceux-ci sur la santé humaine et la nature est un objectif prioritaire des instances européennes. Le projet ARISTO a pour but de développer des outils et procédures pour évaluer la toxicité des pesticides sur les organismes du sol, dont les champignons mycorhiziens à arbuscules (CMA). Notre étude a pour but d’optimiser et standardiser un système de culture in vitro pour évaluer la toxicité d'une gamme de pesticides utilisés en production agricole. Dans un premier temps, la dynamique de germination des spores de Rhizophagus intraradices a été évaluée sur microplaques en présence de divers pesticides (6 fongicides, 6 insecticides, 7 herbicides) à concentrations croissantes (0.01, 0.1, 1, 10 et 20 fois la dose recommandée au champ). Dans un second temps, les spores non germées ont été prélevées et déposées sur milieu sans pesticides pour évaluer l’effet fongistatique ou fongitoxique de ces molécules. Nos résultats montrent que la plupart des fongicides, inhibiteurs de la croissance mycélienne, de la peroxydation des lipides et de la synthèse du glycérol, ont davantage d'effet sur la germination des spores que les autres pesticides. Des effets retards ont été observés pour le 3,5,6-Trichloro-2-pyridinol, Chlorpyrifos, Clethodim et Spiromesifen alors que le Fludioxonil et Flutolanil ont totalement inhibé la germination. Les Pyraclostrobin, Flutolanil et Fludioxonil ont montré un effet fongitoxique à toutes les concentrations alors qu’avec l’Iprodione, le Clethodim et le Chlorpyrifos cet effet n’a été observé qu’à concentration 10 et 20 fois celle appliquée au champ. Les Etridiazole et le Glyphosate ont un effet fongistatique. Cette étude, couplée à d’autres études, doit permettre aux instances européennes de juger de la pertinence du retrait ou de l’autorisation de mise sur le marché des produits phytosanitaires

Midlife omega-3 fatty acid intake predicts later life white matter microstructure in an age- and APOE-dependent manner

Omega-3 intake has been positively associated with healthy brain aging, yet it remains unclear whether high omega-3 intake beginning early in life may optimize its protective effects against brain aging. We examined whether omega-3 intake is associated with brain microstructure over 2 decades later among dementia-free older adults. The 128 participants (62% women; age at magnetic resonance imaging: 76.6 ± 7.9) from the Rancho Bernardo Study of Healthy Aging completed at least 1 dietary assessment between 1984 and 1996 and underwent restriction spectrum imaging (RSI) 22.8 ± 3.1 years later. We evaluated associations between prior omega-3 intake and RSI metrics of gray and white matter (WM) microstructure. Higher prior omega-3 intake was associated with greater restricted diffusion in the superior cortico-striatal fasciculus. A correlation between higher prior omega-3 intake and greater cingulum restricted diffusion was stronger among participants >80 years old. Higher omega-3 intake correlated with greater restricted diffusion in the inferior longitudinal and inferior fronto-occipital fasciculus more strongly for apolipoprotein E (APOE) ε4 carriers than noncarriers. Associations were not modified by adjustment for dietary pattern, health, or lifestyle. High omega-3 intake in midlife may help to maintain WM integrity into older age, particularly in the latest decades of life and among APOE ε4 carriers

Multi-species relationships in legume roots: From pairwise legume-symbiont interactions to the plant - Microbiome - soil continuum

Mutualistic relationships of legume plants with, either bacteria (like rhizobia) or fungi (like arbuscular mycorrhizal fungi), have been investigated intensively, usually as bi-partite interactions. However, diverse symbiotic interactions take place simultaneously or sequentially under field conditions. Their collective, but not additive, contribution to plant growth and performance remains hard to predict, and appears to be furthermore affected by crop species and genotype, non-symbiotic microbial interactions and environmental variables. The challenge is: (i) to unravel the complex overlapping mechanisms that operate between the microbial symbionts as well as between them, their hosts and the rhizosphere (ii) to understand the dynamics of the respective mechanisms in evolutionary and ecological terms. The target for agriculture, food security and the environment, is to use this insight as a solid basis for developing new integrated technologies, practices and strategies for the efficient use of beneficial microbes in legumes and other plants. We review recent advances in our understanding of the symbiotic interactions in legumes roots brought about with the aid of molecular and bioinformatics tools. We go through single symbiont-host interactions, proceed to tripartite symbiont-host interactions, appraise interactions of symbiotic and associative microbiomes with plants in the root-rhizoplane-soil continuum of habitats and end up by examining attempts to validate community ecology principles in the legume-microbe-soil biosystem. © 2021 The Author(s) 2020

Unraveling negative biotic interactions determining soil microbial community assembly and functioning

Microbial communities play important roles in all ecosystems and yet a comprehensive understanding of the ecological processes governing the assembly of these communities is missing. To address the role of biotic interactions between microorganisms in assembly and for functioning of the soil microbiota, we used a top-down manipulation approach based on the removal of various populations in a natural soil microbial community. We hypothesized that removal of certain microbial groups will strongly affect the relative fitness of many others, therefore unraveling the contribution of biotic interactions in shaping the soil microbiome. Here we show that 39% of the dominant bacterial taxa across treatments were subjected to competitive interactions during soil recolonization, highlighting the importance of biotic interactions in the assembly of microbial communities in soil. Moreover, our approach allowed the identification of microbial community assembly rule as exemplified by the competitive exclusion between members of Bacillales and Proteobacteriales. Modified biotic interactions resulted in greater changes in activities related to N- than to C-cycling. Our approach can provide a new and promising avenue to study microbial interactions in complex ecosystems as well as the links between microbial community composition and ecosystem function