RAPiD: a rapid and accurate plant pathogen identification pipeline for on-site nanopore sequencing.

Nanopore sequencing technology has enabled the rapid, on-site taxonomic identification of samples from anything and anywhere. However, sequencing errors, inadequate databases, as well as the need for bioinformatic expertise and powerful computing resources, have hampered the widespread use of the technology for pathogen identification in the agricultural sector. Here we present RAPiD, a lightweight and accurate real-time taxonomic profiling pipeline. Compared to other metagenomic profilers, RAPiD had a higher classification precision achieved through the use of a curated, non-redundant database of common agricultural pathogens and extensive quality filtering of alignments. On a fungal, bacterial and mixed mock community RAPiD was the only pipeline to detect all members of the communities. We also present a protocol for in-field sample processing enabling pathogen identification from plant sample to sequence within 3 h using low-cost equipment. With sequencing costs continuing to decrease and more high-quality reference genomes becoming available, nanopore sequencing provides a viable method for rapid and accurate pathogen identification in the field. A web implementation of the RAPiD pipeline for real-time analysis is available at https://agrifuture.senckenberg.de.

Unexplored microbial diversity from 2,500 food metagenomes and links with the human microbiome.

Complex microbiomes are part of the food we eat and influence our own microbiome, but their diversity remains largely unexplored. Here, we generated the open access curatedFoodMetagenomicData (cFMD) resource by integrating 1,950 newly sequenced and 583 public food metagenomes. We produced 10,899 metagenome-assembled genomes spanning 1,036 prokaryotic and 108 eukaryotic species-level genome bins (SGBs), including 320 previously undescribed taxa. Food SGBs displayed significant microbial diversity within and between food categories. Extension to >20,000 human metagenomes revealed that food SGBs accounted on average for 3% of the adult gut microbiome. Strain-level analysis highlighted potential instances of food-to-gut transmission and intestinal colonization (e.g., Lacticaseibacillus paracasei) as well as SGBs with divergent genomic structures in food and humans (e.g., Streptococcus gallolyticus and Limosilactobabillus mucosae). The cFMD expands our knowledge on food microbiomes, their role in shaping the human microbiome, and supports future uses of metagenomics for food quality, safety, and authentication.

Inter-facility characterization of bacteria in seafood processing plants: Exploring potential reservoirs of spoilage organisms and the resistome.

A study was conducted in fish processing facilities to investigate the microbial composition, microbial metabolic potential, and distribution of antibiotic resistance genes. Whole metagenomic sequencing was used to analyze microbial communities from different processing rooms, operators and fish products. Taxonomic analyses identified the genera Pseudomonas and Psychrobacter as the most prevalent bacteria. A Principal Component Analysis revealed a distinct separation between fish product and environmental samples, as well as differences between fish product samples from companies processing either Gadidae or Salmonidae fish. Some particular bacterial genera and species were associated with specific processing rooms and operators. Metabolic analysis of metagenome assembled genomes demonstrated variations in microbiota metabolic profiles of microbiota across rooms and fish products. The study also examined the presence of antibiotic-resistance genes in fish processing environments, contributing to the understanding of microbial dynamics, metabolic potential, and implications for fish spoilage.

The gut microbiome of farmed Arctic char (Salvelinus alpinus) is shaped by feeding stage and nutrient presence.

The gut microbiome plays an important role in maintaining health and productivity of farmed fish. However, the functional role of most gut microorganisms remains unknown. Identifying the stable members of the gut microbiota and understanding their functional roles could aid in the selection of positive traits or act as a proxy for fish health in aquaculture. Here, we analyse the gut microbial community of farmed juvenile Arctic char (Salvelinus alpinus) and reconstruct the metabolic potential of its main symbionts. The gut microbiota of Arctic char undergoes a succession in community composition during the first weeks post-hatch, with a decrease in Shannon diversity and the establishment of three dominant bacterial taxa. The genome of the most abundant bacterium, a Mycoplasma sp., shows adaptation to rapid growth in the nutrient-rich gut environment. The second most abundant taxon, a Brevinema sp., has versatile metabolic potential, including genes involved in host mucin degradation and utilization. However, during periods of absent gut content, a Ruminococcaceae bacterium becomes dominant, possibly outgrowing all other bacteria through the production of secondary metabolites involved in quorum sensing and cross-inhibition while benefiting the host through short-chain fatty acid production. Whereas Mycoplasma is often present as a symbiont in farmed salmonids, we show that the Ruminococcaceae species is also detected in wild Arctic char, suggesting a close evolutionary relationship between the host and this symbiotic bacterium.

Metagenomic insight to apprehend the fungal communities associated with leaf blight of Welsh onion in Taiwan.

Plants are associated with a large diversity of microbes, and these complex plant-associated microbial communities are critical for plant health. Welsh onion (Allium fistulosum L.) is one of the key and oldest vegetable crops cultivated in Taiwan. The leaf of the Welsh onion is one of the famous spices in Taiwanese cuisine, thus, it is crucial to control foliar diseases. In recent years, Welsh onion cultivation in Taiwan has been severely threatened by the occurrence of leaf blight disease, greatly affecting their yield and quality. However, the overall picture of microbiota associated with the Welsh onion plant is still not clear as most of the recent etiological investigations were heavily based on the isolation of microorganisms from diseased plants. Therefore, studying the diversity of fungal communities associated with the leaf blight symptoms of Welsh onion may provide information regarding key taxa possibly involved in the disease. Therefore, this investigation was mainly designed to understand the major fungal communities associated with leaf blight to identify key taxa potentially involved in the disease and further evaluate any shifts in both phyllosphere and rhizosphere mycobiome assembly due to foliar pathogen infection by amplicon sequencing targeting the Internal Transcribed Spacer (ITS) 1 region of the rRNA. The alpha and beta-diversity analyses were used to compare the fungal communities and significant fungal groups were recognized based on linear discriminant analyses. Based on the results of relative abundance data and co-occurrence networks in symptomatic plants we revealed that the leaf blight of Welsh onion in Sanxing, is a disease complex mainly involving Stemphylium and Colletotrichum taxa. In addition, genera such as Aspergillus, Athelia and Colletotrichum were abundantly found associated with the symptomatic rhizosphere. Alpha-diversity in some fields indicated a significant increase in species richness in the symptomatic phyllosphere compared to the asymptomatic phyllosphere. These results will broaden our knowledge of pathogens of Welsh onion associated with leaf blight symptoms and will assist in developing effective disease management strategies to control the progress of the disease.

Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments.

Deep investigation of the microbiome of food-production and food-processing environments through whole-metagenome sequencing (WMS) can provide detailed information on the taxonomic composition and functional potential of the microbial communities that inhabit them, with huge potential benefits for environmental monitoring programs. However, certain technical challenges jeopardize the application of WMS technologies with this aim, with the most relevant one being the recovery of a sufficient amount of DNA from the frequently low-biomass samples collected from the equipment, tools and surfaces of food-processing plants. Here, we present the first complete workflow, with optimized DNA-purification methodology, to obtain high-quality WMS sequencing results from samples taken from food-production and food-processing environments and reconstruct metagenome assembled genomes (MAGs). The protocol can yield DNA loads >10 ng in >98% of samples and >500 ng in 57.1% of samples and allows the collection of, on average, 12.2 MAGs per sample (with up to 62 MAGs in a single sample) in ~1 week, including both laboratory and computational work. This markedly improves on results previously obtained in studies performing WMS of processing environments and using other protocols not specifically developed to sequence these types of sample, in which <2 MAGs per sample were obtained. The full protocol has been developed and applied in the framework of the European Union project MASTER (Microbiome applications for sustainable food systems through technologies and enterprise) in 114 food-processing facilities from different production sectors.

Enhancement of Soybean Meal Alters Gut Microbiome and Influences Behavior of Farmed Atlantic Salmon (Salmo salar).

Atlantic salmon (Salmo salar) is one of the worlds most domesticated fish. As production volumes increase, access to high quality and sustainable protein sources for formulated feeds of this carnivorous fish is required. Soybean meal (SBM) and soy-derived proteins are the dominant protein sources in commercial aquafeeds due to their low-cost, availability and favorable amino acid profile. However, for Atlantic salmon, the inclusion of soybean meal (SBM), and soy protein concentrate (SPC) in certain combinations can impact gut health, which has consequences for immunity and welfare, limiting the use of soy products in salmonid feeds. This study sought to address this challenge by evaluating two gut health-targeted enhancements of SBM for inclusion in freshwater phase salmon diets: enzyme pre-treatment (ETS), and addition of fructose oligosaccharide (USP). These were compared with untreated soybean meal (US) and fish meal (FM). This study took a multi-disciplinary approach, investigating the effect on growth performance, gut microbiome, and behaviors relevant to welfare in aquaculture. This study suggests that both enhancements of SBM provide benefits for growth performance compared with conventional SBM. Both SBM treatments altered fish gut microbiomes and in the case of ETS, increased the presence of the lactic acid bacteria Enterococcus. For the first time, the effects of marine protein sources and plant protein sources on the coping style of salmon were demonstrated. Fish fed SBM showed a tendency for more reactive behavior compared with those fed the FM-based control. All fish had a similar low response to elicited stress, although ETS-fed fish responded more actively than US-fed fish for a single swimming measure. Furthermore, SBM-fed fish displayed lower repeatability of behavior, which may indicate diminished welfare for intensively farmed fish. The implications of these findings for commercial salmonid aquaculture are discussed.

Impact of Putative Probiotics on Growth, Behavior, and the Gut Microbiome of Farmed Arctic Char (Salvelinus alpinus).

Beneficial bacteria promise to promote the health and productivity of farmed fish species. However, the impact on host physiology is largely strain-dependent, and studies on Arctic char (Salvelinus alpinus), a commercially farmed salmonid species, are lacking. In this study, 10 candidate probiotic strains were subjected to in vitro assays, small-scale growth trials, and behavioral analysis with juvenile Arctic char to examine the impact of probiotic supplementation on fish growth, behavior and the gut microbiome. Most strains showed high tolerance to gastric juice and fish bile acid, as well as high auto-aggregation activity, which are important probiotic characteristics. However, they neither markedly altered the core gut microbiome, which was dominated by three bacterial species, nor detectably colonized the gut environment after the 4-week probiotic treatment. Despite a lack of long-term colonization, the presence of the bacterial strains showed either beneficial or detrimental effects on the host through growth rate enhancement or reduction, as well as changes in fish motility under confinement. This study offers insights into the effect of bacterial strains on a salmonid host and highlights three strains, Carnobacterium divergens V41, Pediococcus acidilactici ASG16, and Lactiplantibacillus plantarum ISCAR-07436, for future research into growth promotion of salmonid fish through probiotic supplementation.

Torula yeast in the diet of Atlantic salmon Salmo salar and the impact on growth performance and gut microbiome.

Atlantic salmon aquaculture is expanding, and with it, the need to find suitable replacements for conventional protein sources used in formulated feeds. Torula yeast (Cyberlindnera jadinii), has been identified as a promising alternative protein for feed and can be sustainably cultivated on lignocellulosic biomasses. The present study investigated the impact of torula yeast on the growth performance and gut microbiome of freshwater Atlantic salmon. A marine protein base diet and a mixed marine and plant protein base diet were tested, where conventional proteins were replaced with increasing inclusion levels of torula yeast, (0%, 10%, 20%). This study demonstrated that 20% torula yeast can replace fish meal without alteration to growth performance while leading to potential benefits for the gut microbiome by increasing the presence of bacteria positively associated with the host. However, when torula yeast replaced plant meal in a mixed protein diet, results suggested that 10% inclusion of yeast produced the best growth performance results but at the 20% inclusion level of yeast, potentially negative changes were observed in the gut microbial community, such as a decrease in lactic acid bacteria. This study supports the continued investigation of torula yeast for Atlantic salmon as a partial replacement for conventional proteins.

Basalt-Hosted Microbial Communities in the Subsurface of the Young Volcanic Island of Surtsey, Iceland.

The island of Surtsey was formed in 1963-1967 on the offshore Icelandic volcanic rift zone. It offers a unique opportunity to study the subsurface biosphere in newly formed oceanic crust and an associated hydrothermal-seawater system, whose maximum temperature is currently above 120°C at about 100m below surface. Here, we present new insights into the diversity, distribution, and abundance of microorganisms in the subsurface of the island, 50years after its creation. Samples, including basaltic tuff drill cores and associated fluids acquired at successive depths as well as surface fumes from fumaroles, were collected during expedition 5059 of the International Continental Scientific Drilling Program specifically designed to collect microbiological samples. Results of this microbial survey are investigated with 16S rRNA gene amplicon sequencing and scanning electron microscopy. To distinguish endemic microbial taxa of subsurface rocks from potential contaminants present in the drilling fluid, we use both methodological and computational strategies. Our 16S rRNA gene analysis results expose diverse and distinct microbial communities in the drill cores and the borehole fluid samples, which harbor thermophiles in high abundance. Whereas some taxonomic lineages detected across these habitats remain uncharacterized (e.g., Acetothermiia, Ammonifexales), our results highlight potential residents of the subsurface that could be identified at lower taxonomic rank such as Thermaerobacter, BRH-c8a (Desulfallas-Sporotomaculum), Thioalkalimicrobium, and Sulfurospirillum. Microscopy images reveal possible biotic structures attached to the basaltic substrate. Finally, microbial colonization of the newly formed basaltic crust and the metabolic potential are discussed on the basis of the data.

Impact of onboard chitosan treatment of whole cod (Gadus morhua) on the shelf life and spoilage bacteria of loins stored superchilled under different atmospheres.

The initial handling of marine fish on board fishing vessels is crucial to retain freshness and ensure an extended shelf life of the resulting fresh products. Here the effect of onboard chitosan treatment of whole, gutted Atlantic cod (Gadus morhua) was studied by evaluating the quality and shelf life of loins processed six days post-catch and packaged in air or modified atmosphere (% CO2/O2/N2: 55/5/40) and stored superchilled for 11 and 16 days, respectively. Sensory evaluation did not reveal a clear effect of chitosan treatment on sensory characteristics, length of freshness period or shelf life of loins under either packaging conditions throughout the storage period. However, directly after loin processing, microbiological analysis of loins showed that onboard chitosan treatment led to significantly lower total viable counts as well as lower counts of specific spoilage organisms (SSO), such as H2S-producers and Pseudomonas spp., compared to the untreated group. In addition, the culture-independent approach revealed a lower bacterial diversity in the chitosan-treated groups compared to the untreated groups, independently of packaging method. Partial 16S rRNA gene sequences belonging to Photobacterium dominated all sample groups, indicating that this genus was likely the main contributor to the spoilage process.

The effect of ultrasonic antifouling control on the growth and microbiota of farmed European sea bass (Dicentrarchus labrax).

Biofouling is a serious threat to marine renewable energy structures and marine aquaculture operations alike. As an alternative to toxic surface coatings, ultrasonic antifouling control has been proposed as an environmentally friendly means to reduce biofouling. However, the impact of ultrasound on fish farmed in offshore structures or in marine multi-purpose platforms, combining renewable energy production and aquaculture, has not yet been assessed. Here we study the impact of ultrasound on the growth and microbiota of farmed European sea bass (Dicentrarchus labrax) under laboratory conditions. Whereas growth and survival were not reduced by ultrasound exposure, microbiological analysis using plate counts and 16S rRNA gene based metataxonomics showed a perturbation of the gill and skin microbiota, including an increase in putative pathogenic bacteria. This warrants further research into the long-term effects of ultrasonic antifouling control on the health and wellbeing of farmed fish.

Microbial intrusion and seasonal dynamics in the groundwater microbiome of a porous basaltic rock aquifer used as municipal water reservoir.

Groundwater is a key resource for safe drinking water supply. Yet unconfined aquifers can be vulnerable to microbial contamination during extreme weather events that lead to surface runoff. The current study characterises the groundwater microbiome of a porous basaltic rock aquifer in South-West Iceland used for drinking water extraction and analyses the microbial community dynamics during surface runoff. The groundwater microbial community sampled from 12 wells across the extraction area contained over 745 prokaryotic genera and was phylogenetically similar between wells and most seasons, representing a diverse but homogenous ecosystem. The largest seasonal variation in the microbial community composition was detected during a period of concurrent snow melt and high precipitation leading to surface runoff. This period was characterised by an increased abundance of soil-associated taxa in the groundwater microbiome and specifically of taxa assigned to Aeromonas and Bacillus. A field experiment simulating high surface runoff around a groundwater well confirmed the increased abundance of surface soil microorganisms in the well water, indicating vulnerability of groundwater towards surface microbial intrusion during extreme weather events. As such events are likely to increase due to climate change, novel water management tools such as microbial community analysis could help ensure drinking water safety.

Genome analysis of sponge symbiont 'Candidatus Halichondribacter symbioticus' shows genomic adaptation to a host-dependent lifestyle.

The marine sponge Halichondria panicea inhabits coastal areas around the globe and is a widely studied sponge species in terms of its biology, yet the ecological functions of its dominant bacterial symbiont 'Candidatus Halichondribacter symbioticus' remain unknown. Here, we present the draft genome of 'Ca. H. symbioticus' HS1 (2.8 Mbp, ca. 87.6% genome coverage) recovered from the sponge metagenome of H. panicea in order to study functions and symbiotic interactions at the genome level. Functional genome comparison of HS1 against closely related free-living seawater bacteria revealed a reduction of genes associated with carbohydrate transport and transcription regulation, pointing towards a limited carbohydrate metabolism, and static transcriptional dynamics reminiscent of other bacterial symbionts. In addition, HS1 was enriched in sponge symbiont specific gene families related to host-symbiont interactions and defence. Similarity in the functional gene repertoire between HS1 and a phylogenetically more distant symbiont in the marine sponge Aplysina aerophoba, based on COG category distribution, suggest a convergent evolution of symbiont specific traits and general metabolic features. This warrants further investigation into convergent genomic evolution of symbionts across different sponge species and habitats.

Co-cultivation of the marine sponge Halichondria panicea and its associated microorganisms.

Marine sponges host bacterial symbionts with biotechnological potential, yet isolation of true sponge symbionts remains difficult due to their host dependency. Moreover, attempts to grow sponges for their pharmacologically-active compounds outside of their habitat often results in a shift of their microbial community. In this study we evaluate suitable sponge cultivation methods that allow maintenance of both the marine sponge Halichondria panicea and its associated bacteria in an ex situ environment. In addition, we present a method for co-cultivation of sponge explants and microbes separated by a membrane in a multi-chamber device. Tests on ex situ cultivation of H. panicea under different controlled conditions showed that only high water exchange rates in the aquarium enabled maintenance of its dominant symbiont "Candidatus Halichondribacter symbioticus" at a high relative abundance in the sponge body, a prerequisite for co-cultivation. The bacterial enrichment retrieved from co-cultivation contained bacteria from nine different classes in addition to sequences corresponding to "Ca. H. symbioticus". This represents an increase of the cultivable bacterial classes from H. panicea compared to standard isolation techniques on solid media plates. The current study provides insights into sponge-microbe maintenance under ex situ conditions and proposes a new method for the isolation of sponge-associated bacteria.

Pelagibaculum spongiae gen. nov., sp. nov., isolated from a marine sponge in South-West Iceland.

A Gram-stain-negative, motile, mesophilic, aerobic, rod-shaped bacterium, designated Hp12T, was isolated from a marine sponge in the intertidal zone off the coast of Seltjarnarnes (64° 16' N 22° 00' W), Iceland. Strain Hp12T grew optimally at 20-22 °C, at pH 7-8 and in the presence of 1-2 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences placed strain Hp12T in the class Gammaproteobacteria, related to members of the genus Alcanivorax in the order Oceanospirillales with 90.3-88.5 % sequence similarity. The strain had a draft genome size of 4.99 Mbp with a DNA G+C content of 43.0 mol%. Cellular fatty acids were dominated by C16 : 1 ω7c, C18 : 1 ω7c and C16 : 0. The predominant polar lipids were phosphatidylglycerol and phosphatidylethanolamine. The major respiratory lipoquinones were ubiquinone Q8 and menaquinone MK8. From the taxonomic information and phenotypic properties obtained in this study, it is proposed that strain Hp12T be placed into a novel genus and species named Pelagibaculum spongiae gen. nov., sp. nov. The type strain of Pelagibaculum spongiae is Hp12T (=DSM 104963T=CECT 9367T).

Bacterial diversity in the marine sponge Halichondria panicea from Icelandic waters and host-specificity of its dominant symbiont "Candidatus Halichondribacter symbioticus".

Marine sponges can harbour diverse bacteria that contribute to host metabolism and defence. Identifying these stable members of sponge bacterial communities remains a necessary step in understanding their ecological roles and underlying co-evolutionary processes. In this study, we applied high-throughput sequencing of 16S rRNA gene amplicons, ribosomal nucleotide variant analysis and fluorescence in situ hybridisation to characterise the core members of the bacterial community in the marine sponge Halichondria panicea from Icelandic waters. We show that the core bacterial community across all samples consisted of a single, dominant bacterial taxon, for which we propose a candidate status 'Candidatus Halichondribacter symbioticus'. Comparison against public databases showed that 'Ca. H. symbioticus' is both a highly abundant specialist in H. panicea and a low abundant opportunist in other sponge species. Additionally, H. panicea with and without 'Ca. H. symbioticus' co-exist in similar locations in the North Atlantic. This dichotomy paired with the presence of geographically distinct ribosomal sequence variants of the symbiont make H. panicea an interesting sponge species for studying sponge-symbiont co-evolution and functional interactions.

Biotechnological Potential of Cold Adapted Pseudoalteromonas spp. Isolated from 'Deep Sea' Sponges.

The marine genus Pseudoalteromonas is known for its versatile biotechnological potential with respect to the production of antimicrobials and enzymes of industrial interest. We have sequenced the genomes of three Pseudoalteromonas sp. strains isolated from different deep sea sponges on the Illumina MiSeq platform. The isolates have been screened for various industrially important enzymes and comparative genomics has been applied to investigate potential relationships between the isolates and their host organisms, while comparing them to free-living Pseudoalteromonas spp. from shallow and deep sea environments. The genomes of the sponge associated Pseudoalteromonas strains contained much lower levels of potential eukaryotic-like proteins which are known to be enriched in symbiotic sponge associated microorganisms, than might be expected for true sponge symbionts. While all the Pseudoalteromonas shared a large distinct subset of genes, nonetheless the number of unique and accessory genes is quite large and defines the pan-genome as open. Enzymatic screens indicate that a vast array of enzyme activities is expressed by the isolates, including ß-galactosidase, ß-glucosidase, and protease activities. A ß-glucosidase gene from one of the Pseudoalteromonas isolates, strain EB27 was heterologously expressed in Escherichia coli and, following biochemical characterization, the recombinant enzyme was found to be cold-adapted, thermolabile, halotolerant, and alkaline active.