Potential of acetic acid to restore methane production in anaerobic reactors critically intoxicated by ammonia as evidenced by metabolic and microbial monitoring.

BACKGROUND: Biogas and biomethane production from the on-farm anaerobic digestion (AD) of animal manure and agri-food wastes could play a key role in transforming Europe's energy system by mitigating its dependence on fossil fuels and tackling the climate crisis. Although ammonia is essential for microbial growth, it inhibits the AD process if present in high concentrations, especially under its free form, thus leading to economic losses. In this study, which includes both metabolic and microbial monitoring, we tested a strategy to restore substrate conversion to methane in AD reactors facing critical free ammonia intoxication. RESULTS: The AD process of three mesophilic semi-continuous 100L reactors critically intoxicated by free ammonia (> 3.5 g_N L-1; inhibited hydrolysis and heterotrophic acetogenesis; interrupted methanogenesis) was restored by applying a strategy that included reducing pH using acetic acid, washing out total ammonia with water, re-inoculation with active microbial flora and progressively re-introducing sugar beet pulp as a feed substrate. After 5 weeks, two reactors restarted to hydrolyse the pulp and produced CH4 from the methylotrophic methanogenesis pathway. The acetoclastic pathway remained inhibited due to the transient dominance of a strictly methylotrophic methanogen (Candidatus Methanoplasma genus) to the detriment of Methanosarcina. Concomitantly, the third reactor, in which Methanosarcina remained dominant, produced CH4 from the acetoclastic pathway but faced hydrolysis inhibition. After 11 weeks, the hydrolysis, the acetoclastic pathway and possibly the hydrogenotrophic pathway were functional in all reactors. The methylotrophic pathway was no longer favoured. Although syntrophic propionate oxidation remained suboptimal, the final pulp to CH4 conversion ratio (0.41 ± 0.10 LN_CH4 g_VS-1) was analogous to the pulp biochemical methane potential (0.38 ± 0.03 LN_CH4 g_VS-1). CONCLUSIONS: Despite an extreme free ammonia intoxication, the proposed process recovery strategy allowed CH4 production to be restored in three intoxicated reactors within 8 weeks, a period during which re-inoculation appeared to be crucial to sustain the process. Introducing acetic acid allowed substantial CH4 production during the recovery period. Furthermore, the initial pH reduction promoted ammonium capture in the slurry, which could allow the field application of the effluents produced by full-scale digesters recovering from ammonia intoxication.

A holobiont approach towards polysaccharide degradation by the highly compartmentalised gut system of the soil-feeding higher termite Labiotermes labralis.

BACKGROUND: Termites are among the most successful insects on Earth and can feed on a broad range of organic matter at various stages of decomposition. The termite gut system is often referred to as a micro-reactor and is a complex structure consisting of several components. It includes the host, its gut microbiome and fungal gardens, in the case of fungi-growing higher termites. The digestive tract of soil-feeding higher termites is characterised by radial and axial gradients of physicochemical parameters (e.g. pH, O2 and H2 partial pressure), and also differs in the density and structure of residing microbial communities. Although soil-feeding termites account for 60% of the known termite species, their biomass degradation strategies are far less known compared to their wood-feeding counterparts. RESULTS: In this work, we applied an integrative multi-omics approach for the first time at the holobiont level to study the highly compartmentalised gut system of the soil-feeding higher termite Labiotermes labralis. We relied on 16S rRNA gene community profiling, metagenomics and (meta)transcriptomics to uncover the distribution of functional roles, in particular those related to carbohydrate hydrolysis, across different gut compartments and among the members of the bacterial community and the host itself. We showed that the Labiotermes gut was dominated by members of the Firmicutes phylum, whose abundance gradually decreased towards the posterior segments of the hindgut, in favour of Bacteroidetes, Proteobacteria and Verrucomicrobia. Contrary to expectations, we observed that L. labralis gut microbes expressed a high diversity of carbohydrate active enzymes involved in cellulose and hemicelluloses degradation, making the soil-feeding termite gut a unique reservoir of lignocellulolytic enzymes with considerable biotechnological potential. We also evidenced that the host cellulases have different phylogenetic origins and structures, which is possibly translated into their different specificities towards cellulose. From an ecological perspective, we could speculate that the capacity to feed on distinct polymorphs of cellulose retained in soil might have enabled this termite species to widely colonise the different habitats of the Amazon basin. CONCLUSIONS: Our study provides interesting insights into the distribution of the hydrolytic potential of the highly compartmentalised higher termite gut. The large number of expressed enzymes targeting the different lignocellulose components make the Labiotermes worker gut a relevant lignocellulose-valorising model to mimic by biomass conversion industries.

Assessing the Interplay between Weather and Septoria Leaf Blotch Severity on Lower Leaves on the Disease Risk on Upper Leaves in Winter Wheat.

Septoria leaf blotch (SLB) is among the most damaging foliar diseases of wheat worldwide. In this study, data for seven cropping seasons (2003−2009) at four representative wheat-growing sites in the Grand-Duchy of Luxembourg (GDL) were used to assess SLB risk on the three upper leaves (L3 to L1, L1 being the flag leaf) based on the combination of conducive weather conditions, simulated potential daily infection events by Zymoseptoria tritici, and SLB severity on lower leaves between stem elongation and mid-flowering. Results indicated that the variability in SLB severity on L3 to L1 at soft dough was significantly (p < 0.05) influenced by the disease severity on the lower leaf L5 at L3 emergence and the sum of daily mean air temperature between stem elongation and mid-flowering. Moreover, analyzing the predictive power of these variables through multiple linear regression indicated that the disease severity on L5 at L3 emergence and mild weather conditions between stem elongation and mid-flowering critically influenced the progress of SLB later in the season. Such results can help fine tune weather-based SLB risk models to guide optimal timing of fungicide application in winter wheat fields and ensure economic and ecological benefits.

Biogas residues in the battle for terrestrial carbon sequestration: A comparative decomposition study in the grassland soils of the Greater Region.

The recycling of biogas residues resulting from the anaerobic digestion of organic waste on agricultural land is among the means to reduce chemical fertilizer use and combat climate change. This in sacco decomposition study investigates (1) the potential of the granulated biogas residue fraction to provide nutrients and enhance soil carbon sequestration when utilized as exogenous organic matter in grassland soils, and (2) the impact of different nitrogen fertilizers on the organic matter decomposition and nutrient release processes. The experiment was conducted in two permanent grasslands of the Greater Region over one management period using rooibos tea as a comparator material. The decomposition and chemical changes of the two materials after incubation in the soil were assessed by measuring the mass loss, total carbon and nitrogen status, and fibre composition in cellulose, hemicellulose and lignin. Overall, after the incubation period, granulated biogas residue maintained up to 68% of its total mass, organic matter and total carbon; increased its content in recalcitrant organic matter by up to 45% and released 45% of its total nitrogen. Granulated biogas residue demonstrated resilience and a higher response uniformity when exposed to different nitrogen fertilizers, as opposed to the comparator material of rooibos tea. However, the magnitude of fertilizer-type effect varied, with ammonium nitrate and the combinatorial treatment of raw biogas residue mixed with urea leading to the highest organic matter loss from the bags. Our findings suggest that granulated biogas residue is a biofertilizer with the potential to supply nutrients to soil biota over time, and promote carbon sequestration in grassland soils, and thereby advance agricultural sustainability while contributing to climate change mitigation.

Compositional and functional characterisation of biomass-degrading microbial communities in guts of plant fibre- and soil-feeding higher termites.

BACKGROUND: Termites are among the most successful insect lineages on the globe and are responsible for providing numerous ecosystem services. They mainly feed on wood and other plant material at different stages of humification. Lignocellulose is often a principal component of such plant diet, and termites largely rely on their symbiotic microbiota and associated enzymes to decompose their food efficiently. While lower termites and their gut flagellates were given larger scientific attention in the past, the gut lignocellulolytic bacteria of higher termites remain less explored. Therefore, in this study, we investigated the structure and function of gut prokaryotic microbiomes from 11 higher termite genera representative of Syntermitinae, Apicotermitinae, Termitidae and Nasutitermitinae subfamilies, broadly grouped into plant fibre- and soil-feeding termite categories. RESULTS: Despite the different compositional structures of the studied termite gut microbiomes, reflecting well the diet and host lineage, we observed a surprisingly high functional congruency between gut metatranscriptomes from both feeding groups. The abundance of transcripts encoding for carbohydrate active enzymes as well as expression and diversity profiles of assigned glycoside hydrolase families were also similar between plant fibre- and soil-feeding termites. Yet, dietary imprints highlighted subtle metabolic differences specific to each feeding category. Roughly, 0.18% of de novo re-constructed gene transcripts were shared between the different termite gut microbiomes, making each termite gut a unique reservoir of genes encoding for potentially industrially applicable enzymes, e.g. relevant to biomass degradation. Taken together, we demonstrated the functional equivalence in microbial populations across different termite hosts. CONCLUSIONS: Our results provide valuable insight into the bacterial component of the termite gut system and significantly expand the inventory of termite prokaryotic genes participating in the deconstruction of plant biomass. Video Abstract.

Integrative omics analysis of the termite gut system adaptation to Miscanthus diet identifies lignocellulose degradation enzymes.

Miscanthus sp. biomass could satisfy future biorefinery value chains. However, its use is largely untapped due to high recalcitrance. The termite and its gut microbiome are considered the most efficient lignocellulose degrading system in nature. Here, we investigate at holobiont level the dynamic adaptation of Cortaritermes sp. to imposed Miscanthus diet, with a long-term objective of overcoming lignocellulose recalcitrance. We use an integrative omics approach combined with enzymatic characterisation of carbohydrate active enzymes from termite gut Fibrobacteres and Spirochaetae. Modified gene expression profiles of gut bacteria suggest a shift towards utilisation of cellulose and arabinoxylan, two main components of Miscanthus lignocellulose. Low identity of reconstructed microbial genomes to closely related species supports the hypothesis of a strong phylogenetic relationship between host and its gut microbiome. This study provides a framework for better understanding the complex lignocellulose degradation by the higher termite gut system and paves a road towards its future bioprospecting.

Carbohydrate Hydrolytic Potential and Redundancy of an Anaerobic Digestion Microbiome Exposed to Acidosis, as Uncovered by Metagenomics.

Increased hydrolysis of easily digestible biomass may lead to acidosis of anaerobic reactors and decreased methane production. Previously, it was shown that the structure of microbial communities changed during acidosis; however, once the conditions are back to optimal, biogas (initially CO2) production quickly restarts. This suggests the retention of the community functional redundancy during the process failure. In this study, with the use of metagenomics and downstream bioinformatics analyses, we characterize the carbohydrate hydrolytic potential of the microbial community, with a special focus on acidosis. To that purpose, carbohydrate-active enzymes were identified, and to further link the community hydrolytic potential with key microbes, bacterial genomes were reconstructed. In addition, we characterized biochemically the specificity and activity of selected enzymes, thus verifying the accuracy of the in silico predictions. The results confirm the retention of the community hydrolytic potential during acidosis and indicate Bacteroidetes to be largely involved in biomass degradation. Bacteroidetes showed higher diversity and genomic content of carbohydrate hydrolytic enzymes that might favor the dominance of this phylum over other bacteria in some anaerobic reactors. The combination of bioinformatic analyses and activity tests enabled us to propose a model of acetylated glucomannan degradation by BacteroidetesIMPORTANCE The enzymatic hydrolysis of lignocellulosic biomass is mainly driven by the action of carbohydrate-active enzymes. By characterizing the gene profiles at the different stages of the anaerobic digestion experiment, we showed that the microbiome retains its hydrolytic functional redundancy even during severe acidosis, despite significant changes in taxonomic composition. By analyzing reconstructed bacterial genomes, we demonstrate that Bacteroidetes hydrolytic gene diversity likely favors the abundance of this phylum in some anaerobic digestion systems. Further, we observe genetic redundancy within the Bacteroidetes group, which accounts for the preserved hydrolytic potential during acidosis. This work also uncovers new polysaccharide utilization loci involved in the deconstruction of various biomasses and proposes the model of acetylated glucomannan degradation by Bacteroidetes Acetylated glucomannan-enriched biomass is a common substrate for many industries, including pulp and paper production. Using naturally evolved cocktails of enzymes for biomass pretreatment could be an interesting alternative to the commonly used chemical pretreatments.

Biogas residues in substitution for chemical fertilizers: A comparative study on a grassland in the Walloon Region.

To provide sufficient quantities of food and feed, farming systems have to overcome limiting factors such as the nutrient depletion of arable soils. Nitrogen being the main mineral element required for plant growth, has led to the extensive use of chemical fertilizers causing nitrogen pollution of the ecosystems. This field study investigates the use of biogas residues (BRs) as biofertilizers and their contribution to the mitigation of nitrate leaching in agricultural soils, while also demonstrating the polluting nature of chemical fertilizers. Nine different fertilization treatments classified in three schemes and two nitrogen doses were tested for three consecutive years on a grassland in the Walloon Region of Belgium. Residual soil mineral nitrogen, percentage contribution of treatments in residual nitrate and agronomic performance were assessed for each fertilization treatment. The results obtained showed significant differences on treatment and scheme level regarding nitrate accumulation in the soil, with chemical fertilizers posing the highest nitrate leaching risk. BRs did not cause nitrate accumulation in the soil, and were N rate and rainfall independent, while the chemical treatments indicated a cumulative tendency under high N rate and low precipitation. Forage yield did not demonstrate statistical differences on treatment and scheme level but varied with changing precipitation, while the maximum application rate suggested a plateau. Aboveground nitrogen content was significantly higher after the application of chemical fertilizers only in the first year, while all the chemical treatments indicated a dilution effect under elevated annual rainfall. Finally, the partial substitution of chemical fertilizers by raw digestate reduced the concentration of NO3- in the soil without having a negative impact on the yield and N content of the biomass. These results strongly advocate for the environmental benefits of BRs over chemical fertilizers and underline their suitability as biofertilizers and substitutes for chemical fertilizers in similar agricultural systems.

First Draft Genome Sequence of a Polymyxa Genus Member, Polymyxa betae, the Protist Vector of Rhizomania.

Polymyxa betae belongs to the Plasmodiophorida (Phytomyxea, Rhizaria). Here, we report the first draft genome sequence of a member of the Polymyxa genus, which includes two obligate root endoparasite species, vectors of important soilborne plant viruses. The genome assembly was represented by 1,001 contigs, with a cumulated length of 27,085,946 bp.

A year of monitoring 20 mesophilic full-scale bioreactors reveals the existence of stable but different core microbiomes in bio-waste and wastewater anaerobic digestion systems.

BACKGROUND: Anaerobic digestion (AD) is a microbe-driven process of biomass decomposition to CH4 and CO2. In addition to renewable and cost-effective energy production, AD has emerged in the European Union as an environmentally friendly model of bio-waste valorisation and nutrient recycling. Nevertheless, due to the high diversity of uncharacterised microbes, a typical AD microbiome is still considered as "dark matter". RESULTS: Using the high-throughput sequencing of small rRNA gene, and a monthly monitoring of the physicochemical parameters for 20 different mesophilic full-scale bioreactors over 1 year, we generated a detailed view of AD microbial ecology towards a better understanding of factors that influence and shape these communities. By studying the broadly distributed OTUs present in over 80% of analysed samples, we identified putatively important core bacteria and archaea to the AD process that accounted for over 70% of the whole microbial community relative abundances. AD reactors localised at the wastewater treatment plants were shown to operate with distinct core microbiomes than the agricultural and bio-waste treating biogas units. We also showed that both the core microbiomes were composed of low (with average community abundance ≤ 1%) and highly abundant microbial populations; the vast majority of which remains yet uncharacterised, e.g. abundant candidate Cloacimonetes. Using non-metric multidimensional scaling, we observed microorganisms grouping into clusters that well reflected the origin of the samples, e.g. wastewater versus agricultural and bio-waste treating biogas units. The calculated diversity patterns differed markedly between the different community clusters, mainly due to the presence of highly diverse and dynamic transient species. Core microbial communities appeared relatively stable over the monitoring period. CONCLUSIONS: In this study, we characterised microbial communities in different AD systems that were monitored over a 1-year period. Evidences were shown to support the concept of a core community driving the AD process, whereas the vast majority of dominant microorganisms remain yet to be characterised.

Complete Genome Sequence of Streptomyces lunaelactis MM109T, Isolated from Cave Moonmilk Deposits.

Streptomyces lunaelactis MM109T is a ferroverdin A (anticholesterol) producer isolated from cave moonmilk deposits. The complete genome sequence of MM109T was obtained by combining Oxford Nanopore MinION and Illumina HiSeq and MiSeq technologies, revealing an 8.4-Mb linear chromosome and two plasmids, pSLUN1 (127,264 bp, linear) and pSLUN2 (46,827 bp, circular).

High-Throughput Sequencing Analysis of the Actinobacterial Spatial Diversity in Moonmilk Deposits.

Moonmilk are cave carbonate deposits that host a rich microbiome, including antibiotic-producing Actinobacteria, making these speleothems appealing for bioprospecting. Here, we investigated the taxonomic profile of the actinobacterial community of three moonmilk deposits of the cave "Grotte des Collemboles" via high-throughput sequencing of 16S rRNA amplicons. Actinobacteria was the most common phylum after Proteobacteria, ranging from 9% to 23% of the total bacterial population. Next to actinobacterial operational taxonomic units (OTUs) attributed to uncultured organisms at the genus level (~44%), we identified 47 actinobacterial genera with Rhodoccocus (4 OTUs, 17%) and Pseudonocardia (9 OTUs, ~16%) as the most abundant in terms of the absolute number of sequences. Streptomycetes presented the highest diversity (19 OTUs, 3%), with most of the OTUs unlinked to the culturable Streptomyces strains that were previously isolated from the same deposits. Furthermore, 43% of the OTUs were shared between the three studied collection points, while 34% were exclusive to one deposit, indicating that distinct speleothems host their own population, despite their nearby localization. This important spatial diversity suggests that prospecting within different moonmilk deposits should result in the isolation of unique and novel Actinobacteria. These speleothems also host a wide range of non-streptomycetes antibiotic-producing genera, and should therefore be subjected to methodologies for isolating rare Actinobacteria.

Optimization of a metatranscriptomic approach to study the lignocellulolytic potential of the higher termite gut microbiome.

BACKGROUND: Thanks to specific adaptations developed over millions of years, the efficiency of lignin, cellulose and hemicellulose decomposition of higher termite symbiotic system exceeds that of many other lignocellulose utilizing environments. Especially, the examination of its symbiotic microbes should reveal interesting carbohydrate-active enzymes, which are of primary interest for the industry. Previous metatranscriptomic reports (high-throughput mRNA sequencing) highlight the high representation and overexpression of cellulose and hemicelluloses degrading genes in the termite hindgut digestomes, indicating the potential of this technology in search for new enzymes. Nevertheless, several factors associated with the material sampling and library preparation steps make the metatranscriptomic studies of termite gut prokaryotic symbionts challenging. METHODS: In this study, we first examined the influence of the sampling strategy, including the whole termite gut and luminal fluid, on the diversity and the metatranscriptomic profiles of the higher termite gut symbiotic bacteria. Secondly, we evaluated different commercially available kits combined in two library preparative pipelines for the best bacterial mRNA enrichment strategy. RESULTS: We showed that the sampling strategy did not significantly impact the generated results, both in terms of the representation of the microbes and their transcriptomic profiles. Nevertheless collecting luminal fluid reduces the co-amplification of unwanted RNA species of host origin. Furthermore, for the four studied higher termite species, the library preparative pipeline employing Ribo-Zero Gold rRNA Removal Kit "Epidemiology" in combination with Poly(A) Purist MAG kit resulted in a more efficient rRNA and poly-A-mRNAdepletion (up to 98.44% rRNA removed) than the pipeline utilizing MICROBExpress and MICROBEnrich kits. High correlation of both Ribo-Zero and MICROBExpresse depleted gene expression profiles with total non-depleted RNA-seq data has been shown for all studied samples, indicating no systematic skewing of the studied pipelines. CONCLUSIONS: We have extensively evaluated the impact of the sampling strategy and library preparation steps on the metatranscriptomic profiles of the higher termite gut symbiotic bacteria. The presented methodological approach has great potential to enhance metatranscriptomic studies of the higher termite intestinal flora and to unravel novel carbohydrate-active enzymes.

Assessment of the Potential Role of Streptomyces in Cave Moonmilk Formation.

Moonmilk is a karstic speleothem mainly composed of fine calcium carbonate crystals (CaCO3) with different textures ranging from pasty to hard, in which the contribution of biotic rock-building processes is presumed to involve indigenous microorganisms. The real microbial input in the genesis of moonmilk is difficult to assess leading to controversial hypotheses explaining the origins and the mechanisms (biotic vs. abiotic) involved. In this work, we undertook a comprehensive approach in order to assess the potential role of filamentous bacteria, particularly a collection of moonmilk-originating Streptomyces, in the genesis of this speleothem. Scanning electron microscopy (SEM) confirmed that indigenous filamentous bacteria could indeed participate in moonmilk development by serving as nucleation sites for CaCO3 deposition. The metabolic activities involved in CaCO3 transformation were furthermore assessed in vitro among the collection of moonmilk Streptomyces, which revealed that peptides/amino acids ammonification, and to a lesser extend ureolysis, could be privileged metabolic pathways participating in carbonate precipitation by increasing the pH of the bacterial environment. Additionally, in silico search for the genes involved in biomineralization processes including ureolysis, dissimilatory nitrate reduction to ammonia, active calcium ion transport, and reversible hydration of CO2 allowed to identify genetic predispositions for carbonate precipitation in Streptomyces. Finally, their biomineralization abilities were confirmed by environmental SEM, which allowed to visualize the formation of abundant mineral deposits under laboratory conditions. Overall, our study provides novel evidences that filamentous Actinobacteria could be key protagonists in the genesis of moonmilk through a wide spectrum of biomineralization processes.

ICoVeR - an interactive visualization tool for verification and refinement of metagenomic bins.

BACKGROUND: Recent advances in high-throughput sequencing allow for much deeper exploitation of natural and engineered microbial communities, and to unravel so-called "microbial dark matter" (microbes that until now have evaded cultivation). Metagenomic analyses result in a large number of genomic fragments (contigs) that need to be grouped (binned) in order to reconstruct draft microbial genomes. While several contig binning algorithms have been developed in the past 2 years, they often lack consensus. Furthermore, these software tools typically lack a provision for the visualization of data and bin characteristics. RESULTS: We present ICoVeR, the Interactive Contig-bin Verification and Refinement tool, which allows the visualization of genome bins. More specifically, ICoVeR allows curation of bin assignments based on multiple binning algorithms. Its visualization window is composed of two connected and interactive main views, including a parallel coordinates view and a dimensionality reduction plot. To demonstrate ICoVeR's utility, we used it to refine disparate genome bins automatically generated using MetaBAT, CONCOCT and MyCC for an anaerobic digestion metagenomic (AD microbiome) dataset. Out of 31 refined genome bins, 23 were characterized with higher completeness and lower contamination in comparison to their respective, automatically generated, genome bins. Additionally, to benchmark ICoVeR against a previously validated dataset, we used Sharon's dataset representing an infant gut metagenome. CONCLUSIONS: ICoVeR is an open source software package that allows curation of disparate genome bins generated with automatic binning algorithms. It is freely available under the GPLv3 license at https://git.list.lu/eScience/ICoVeR . The data management and analytical functions of ICoVeR are implemented in R, therefore the software can be easily installed on any system for which R is available. Installation and usage guide together with the example files ready to be visualized are also provided via the project wiki. ICoVeR running instance preloaded with AD microbiome and Sharon's datasets can be accessed via the website.

A Threshold-Based Weather Model for Predicting Stripe Rust Infection in Winter Wheat.

Wheat stripe rust (caused by Puccinia striiformis f. sp. tritici) is a major threat in most wheat growing regions worldwide, which potentially causes substantial yield losses when environmental conditions are favorable. Data from 1999 to 2015 for three representative wheat-growing sites in Luxembourg were used to develop a threshold-based weather model for predicting wheat stripe rust. First, the range of favorable weather conditions using a Monte Carlo simulation method based on the Dennis model were characterized. Then, the optimum combined favorable weather variables (air temperature, relative humidity, and rainfall) during the most critical infection period (May-June) was identified and was used to develop the model. Uninterrupted hours with such favorable weather conditions over each dekad (i.e., 10-day period) during May-June were also considered when building the model. Results showed that a combination of relative humidity >92% and 4°C < temperature < 16°C for a minimum of 4 continuous hours, associated with rainfall ≤0.1 mm (with the dekad having these conditions for 5 to 20% of the time), were optimum to the development of a wheat stripe rust epidemic. The model accurately predicted infection events: probabilities of detection were ≥0.90 and false alarm ratios were ≤0.38 on average, and critical success indexes ranged from 0.63 to 1. The method is potentially applicable to studies of other economically important fungal diseases of other crops or in different geographical locations. If weather forecasts are available, the threshold-based weather model can be integrated into an operational warning system to guide fungicide applications.

A Phenotypic and Genotypic Analysis of the Antimicrobial Potential of Cultivable Streptomyces Isolated from Cave Moonmilk Deposits.

Moonmilk speleothems of limestone caves host a rich microbiome, among which Actinobacteria represent one of the most abundant phyla. Ancient medical texts reported that moonmilk had therapeutical properties, thereby suggesting that its filamentous endemic actinobacterial population might be a source of natural products useful in human treatment. In this work, a screening approach was undertaken in order to isolate cultivable Actinobacteria from moonmilk of the Grotte des Collemboles in Belgium, to evaluate their taxonomic profile, and to assess their potential in biosynthesis of antimicrobials. Phylogenetic analysis revealed that all 78 isolates were exclusively affiliated to the genus Streptomyces and clustered into 31 distinct phylotypes displaying various pigmentation patterns and morphological features. Phylotype representatives were tested for antibacterial and antifungal activities and their genomes were mined for secondary metabolite biosynthetic genes coding for non-ribosomal peptide synthetases (NRPSs), and polyketide synthases (PKS). The moonmilk Streptomyces collection was found to display strong inhibitory activities against a wide range of reference organisms, as 94, 71, and 94% of the isolates inhibited or impaired the growth of Gram-positive, Gram-negative bacteria, and fungi, respectively. Interestingly, 90% of the cave strains induced strong growth suppression against the multi-drug resistant Rasamsonia argillacea, a causative agent of invasive mycosis in cystic fibrosis and chronic granulomatous diseases. No correlation was observed between the global antimicrobial activity of an individual strain and the number of NRPS and PKS genes predicted in its genome, suggesting that approaches for awakening cryptic metabolites biosynthesis should be applied to isolates with no antimicrobial phenotype. Overall, our work supports the common belief that moonmilk might effectively treat various infectious diseases thanks to the presence of a highly diverse population of prolific antimicrobial producing Streptomyces, and thus may indeed constitute a promising reservoir of potentially novel active natural compounds.

Plant Disease Severity Assessment-How Rater Bias, Assessment Method, and Experimental Design Affect Hypothesis Testing and Resource Use Efficiency.

The effect of rater bias and assessment method on hypothesis testing was studied for representative experimental designs for plant disease assessment using balanced and unbalanced data sets. Data sets with the same number of replicate estimates for each of two treatments are termed "balanced" and those with unequal numbers of replicate estimates are termed "unbalanced". The three assessment methods considered were nearest percent estimates (NPEs), an amended 10% incremental scale, and the Horsfall-Barratt (H-B) scale. Estimates of severity of Septoria leaf blotch on leaves of winter wheat were used to develop distributions for a simulation model. The experimental designs are presented here in the context of simulation experiments which consider the optimal design for the number of specimens (individual units sampled) and the number of replicate estimates per specimen for a fixed total number of observations (total sample size for the treatments being compared). The criterion used to gauge each method was the power of the hypothesis test. As expected, at a given fixed number of observations, the balanced experimental designs invariably resulted in a higher power compared with the unbalanced designs at different disease severity means, mean differences, and variances. Based on these results, with unbiased estimates using NPE, the recommended number of replicate estimates taken per specimen is 2 (from a sample of specimens of at least 30), because this conserves resources. Furthermore, for biased estimates, an apparent difference in the power of the hypothesis test was observed between assessment methods and between experimental designs. Results indicated that, regardless of experimental design or rater bias, an amended 10% incremental scale has slightly less power compared with NPEs, and that the H-B scale is more likely than the others to cause a type II error. These results suggest that choice of assessment method, optimizing sample number and number of replicate estimates, and using a balanced experimental design are important criteria to consider to maximize the power of hypothesis tests for comparing treatments using disease severity estimates.

Start-up phase of an anaerobic full-scale farm reactor - Appearance of mesophilic anaerobic conditions and establishment of the methanogenic microbial community.

The goal of this study was to investigate how the microbial community structure establishes during the start-up phase of a full-scale farm anaerobic reactor inoculated with stale and cold cattle slurry. The 16S/18S high-throughput amplicon sequencing results showed an increase of the bacterial, archaeal and eukaryotic diversity, evenness and richness during the settlement of the mesophilic anaerobic conditions. When a steady performing digestion process was reached, the microbial diversity, evenness and richness decreased, indicating the establishment of a few dominant microbial populations, best adapted to biogas production. Interestingly, among the environmental parameters, the temperature, alkalinity, free-NH3, total solids and O2 content were found to be the main drivers of microbial dynamics. Interactions between eukaryotes, characterized by a high number of unknown organisms, and the bacterial and archaeal communities were also evidenced, suggesting that eukaryotes might play important roles in the anaerobic digestion process.

Analysis of dsDNA and RNA viromes in methanogenic digesters reveals novel viral genetic diversity.

Although viruses are not the key players of the anaerobic digestion process, they may affect the dynamics of bacterial and archaeal populations involved in biogas production. Until now viruses have received very little attention in this specific habitat; therefore, as a first step towards their characterization, we optimized a virus filtration protocol from anaerobic sludge. Afterwards, to assess dsDNA and RNA viral diversity in sludge samples from nine different reactors fed either with waste water, agricultural residues or solid municipal waste plus agro-food residues, we performed metagenomic analyses. As a result we showed that, while the dsDNA viromes (21 assigned families in total) were dominated by dsDNA phages of the order Caudovirales, RNA viruses (14 assigned families in total) were less diverse and were for the main part plant-infecting viruses. Interestingly, less than 2% of annotated contigs were assigned as putative human and animal pathogens. Our study greatly extends the existing view of viral genetic diversity in methanogenic reactors and shows that these viral assemblages are distinct not only among the reactor types but also from nearly 30 other environments already studied, including the human gut, fermented food, deep sea sediments and other aquatic habitats.