Pitting the olive seed microbiome.

BACKGROUND: The complex and co-evolved interplay between plants and their microbiota is crucial for the health and fitness of the plant holobiont. However, the microbiota of the seeds is still relatively unexplored and no studies have been conducted with olive trees so far. In this study, we aimed to characterize the bacterial, fungal and archaeal communities present in seeds of ten olive genotypes growing in the same orchard through amplicon sequencing to test whether the olive genotype is a major driver in shaping the seed microbial community, and to identify the origin of the latter. Therefore, we have developed a methodology for obtaining samples from the olive seed's endosphere under sterile conditions. RESULTS: A diverse microbiota was uncovered in olive seeds, the plant genotype being an important factor influencing the structure and composition of the microbial communities. The most abundant bacterial phylum was Actinobacteria, accounting for an average relative abundance of 41%. At genus level, Streptomyces stood out because of its potential influence on community structure. Within the fungal community, Basidiomycota and Ascomycota were the most abundant phyla, including the genera Malassezia, Cladosporium, and Mycosphaerella. The shared microbiome was composed of four bacterial (Stenotrophomonas, Streptomyces, Promicromonospora and Acidipropionibacterium) and three fungal (Malassezia, Cladosporium and Mycosphaerella) genera. Furthermore, a comparison between findings obtained here and earlier results from the root endosphere of the same trees indicated that genera such as Streptomyces and Malassezia were present in both olive compartments. CONCLUSIONS: This study provides the first insights into the composition of the olive seed microbiota. The highly abundant fungal genus Malassezia and the bacterial genus Streptomyces reflect a unique signature of the olive seed microbiota. The genotype clearly shaped the composition of the seed's microbial community, although a shared microbiome was found. We identified genera that may translocate from the roots to the seeds, as they were present in both organs of the same trees. These findings set the stage for future research into potential vertical transmission of olive endophytes and the role of specific microbial taxa in seed germination, development, and seedling survival.

Interspecific interactions facilitate keystone species in a multispecies biofilm that promotes plant growth.

Microorganisms colonizing plant roots co-exist in complex, spatially structured multispecies biofilm communities. However, little is known about microbial interactions and the underlying spatial organization within biofilm communities established on plant roots. Here, a well-established four-species biofilm model (Stenotrophomonas rhizophila, Paenibacillus amylolyticus, Microbacterium oxydans, and Xanthomonas retroflexus, termed as SPMX) was applied to Arabidopsis roots to study the impact of multispecies biofilm on plant growth and the community spatial dynamics on the roots. SPMX co-culture notably promoted root development and plant biomass. Co-cultured SPMX increased root colonization and formed multispecies biofilms, structurally different from those formed by monocultures. By combining 16S rRNA gene amplicon sequencing and fluorescence in situ hybridization with confocal laser scanning microscopy, we found that the composition and spatial organization of the four-species biofilm significantly changed over time. Monoculture P. amylolyticus colonized plant roots poorly, but its population and root colonization were highly enhanced when residing in the four-species biofilm. Exclusion of P. amylolyticus from the community reduced overall biofilm production and root colonization of the three species, resulting in the loss of the plant growth-promoting effects. Combined with spatial analysis, this led to identification of P. amylolyticus as a keystone species. Our findings highlight that weak root colonizers may benefit from mutualistic interactions in complex communities and hereby become important keystone species impacting community spatial organization and function. This work expands the knowledge on spatial organization uncovering interspecific interactions in multispecies biofilm communities on plant roots, beneficial for harnessing microbial mutualism promoting plant growth.

Microbiome homeostasis on rice leaves is regulated by a precursor molecule of lignin biosynthesis.

In terrestrial ecosystems, plant leaves provide the largest biological habitat for highly diverse microbial communities, known as the phyllosphere microbiota. However, the underlying mechanisms of host-driven assembly of these ubiquitous communities remain largely elusive. Here, we conduct a large-scale and in-depth assessment of the rice phyllosphere microbiome aimed at identifying specific host-microbe links. A genome-wide association study reveals a strong association between the plant genotype and members of four bacterial orders, Pseudomonadales, Burkholderiales, Enterobacterales and Xanthomonadales. Some of the associations are specific to a distinct host genomic locus, pathway or even gene. The compound 4-hydroxycinnamic acid (4-HCA) is identified as the main driver for enrichment of bacteria belonging to Pseudomonadales. 4-HCA can be synthesized by the host plant's OsPAL02 from the phenylpropanoid biosynthesis pathway. A knockout mutant of OsPAL02 results in reduced Pseudomonadales abundance, dysbiosis of the phyllosphere microbiota and consequently higher susceptibility of rice plants to disease. Our study provides a direct link between a specific plant metabolite and rice phyllosphere homeostasis opening possibilities for new breeding strategies.

Rhizosphere assembly alters along a chronosequence in the Hallstätter glacier forefield (Dachstein, Austria).

Rhizosphere microbiome assembly is essential for plant health, but the temporal dimension of this process remains unexplored. We used a chronosequence of 150 years of the retreating Hallstätter glacier (Dachstein, Austria) to disentangle this exemplarily for the rhizosphere of three pioneer alpine plants. Time of deglaciation was an important factor shaping the rhizosphere microbiome. Microbiome functions, i.e. nutrient uptake and stress protection, were carried out by ubiquitous and cosmopolitan bacteria. The rhizosphere succession along the chronosequence was characterized by decreasing microbial richness but increasing specificity of the plant-associated bacterial community. Environmental selection is a critical factor in shaping the ecosystem, particularly in terms of plant-driven recruitment from the available edaphic pool. A higher rhizosphere microbial richness during early succession compared to late succession can be explained by the occurrence of cold-acclimated bacteria recruited from the surrounding soils. These taxa might be sensitive to changing habitat conditions that occurred at the later stages. A stronger influence of the plant host on the rhizosphere microbiome assembly was observed with increased time since deglaciation. Overall, this study indicated that well-adapted, ubiquitous microbes potentially support pioneer plants to colonize new ecosystems, while plant-specific microbes may be associated with the long-term establishment of their hosts.

The edible plant microbiome: evidence for the occurrence of fruit and vegetable bacteria in the human gut.

Diversity of the gut microbiota is crucial for human health. However, whether fruit and vegetable associated bacteria contribute to overall gut bacterial diversity is still unknown. We reconstructed metagenome-assembled genomes from 156 fruit and vegetable metagenomes to investigate the prevalence of associated bacteria in 2,426 publicly available gut metagenomes. The microbiomes of fresh fruits and vegetables and the human gut are represented by members in common such as Enterobacterales, Burkholderiales, and Lactobacillales. Exposure to bacteria via fruit and vegetable consumption potentially has a beneficial impact on the functional diversity of gut microbiota particularly due to the presence of putative health-promoting genes for the production of vitamin and short-chain fatty acids. In the human gut, they were consistently present, although at a low abundance, approx. 2.2%. Host age, vegetable consumption frequency, and the diversity of plants consumed were drivers favoring a higher proportion. Overall, these results provide one of the primary links between the human microbiome and the environmental microbiome. This study revealed evidence that fruit and vegetable-derived microbes could be found in the human gut and contribute to gut microbiome diversity.

Rhizosphere bacteria show a stronger response to antibiotic-based biopesticide than to conventional pesticides.

The plant microbiota can substantially contribute to various functions related to host health, fitness, and productivity. Therefore, maintaining the integrity of the microbiota is beginning to be seen as a crucial factor in modern agriculture. Here, we evaluated the effects of two chemical pesticides (azoxystrobin and carbendazim) and an antibiotic-based biopesticide (wuyiencin) on the rhizosphere microbiome of tomato plants. It was found that all treatments resulted in changes in the bacterial community structure to varying degrees. The most pronounced changes were observed with the biopesticide, which resulted in an enrichment of Streptomyces in the microbiome. In contrast, the relative abundance of Actinobacteria decreased in samples that were treated with low and high dosages of carbendazim. Clostridia were enriched after the applications of azoxystrobin and wuyiencin. When functioning of the microbiome was assessed, it was shown that genes encoding multidrug efflux pumps and ABC transporters related to nutrient uptake were enriched. This enrichment is likely to overcome potentially negative effects linked to the exposure to the employed substances. The study provides new insights into the potential of different pesticides to modulate native plant microbiomes, and thus highlights the importance to include such evaluations when new active agents are developed.

The microbiome of Riccia liverworts is an important reservoir for microbial diversity in temporary agricultural crusts.

BACKGROUND: The microbiota of liverworts provides an interesting model for plant symbioses; however, their microbiome assembly is not yet understood. Here, we assessed specific factors that shape microbial communities associated with Riccia temporary agricultural crusts in harvested fields by investigating bacterial, fungal and archaeal communities in thalli and adhering soil from different field sites in Styria and Burgenland, Austria combining qPCR analyses, amplicon sequencing and advanced microscopy. RESULTS: Riccia spec. div. was colonized by a very high abundance of bacteria (1010 16S rRNA gene copies per g of thallus) as well as archaea and fungi (108 ITS copies per g of thallus). Each Riccia thallus contain approx. 1000 prokaryotic and fungal ASVs. The field type was the main driver for the enrichment of fungal taxa, likely due to an imprint on soil microbiomes by the cultivated crop plants. This was shown by a higher fungal richness and different fungal community compositions comparing liverwort samples collected from pumpkin fields, with those from corn fields. In contrast, bacterial communities linked to liverworts are highly specialized and the soil attached to them is not a significant source of these bacteria. Specifically, enriched Cyanobacteria, Bacteroidetes and Methylobacteria suggest a symbiotic interaction. Intriguingly, compared to the surrounding soil, the thallus samples were shown to enrich several well-known bacterial and fungal phytopathogens indicating an undescribed role of liverworts as potential reservoirs of crop pathogens. CONCLUSIONS: Our results provide evidence that a stable bacterial community but varying fungal communities are colonizing liverwort thalli. Post-harvest, temporary agricultural biocrusts are important reservoirs for microbial biodiversity but they have to be considered as potential reservoirs for pathogens as well.

The mature phyllosphere microbiome of grapevine is associated with resistance against Plasmopara viticola.

Phyllosphere microbiota represents a substantial but hardly explored reservoir for disease resistance mechanisms. The goal of our study was to understand the link between grapevine cultivars susceptibility to Plasmopara viticola, one of the most devastating leaf pathogens in viticulture, and the phyllosphere microbiota. Therefore, we analyzed a 16S rRNA gene library for the dominant phyllosphere bacterial phyla Alphaproteobacteria of seven Vitis genotypes at different developmental stages, i.e., flowering and harvesting, via amplicon sequencing. Young leaves had significantly higher Alphaproteobacterial richness and diversity without significant host-specificity. In contrast, the microbial communities of mature leaves were structurally distinct in accordance with P. viticola resistance levels. This statistically significant link between mature bacterial phyllosphere communities and resistant phenotypes was corroborated by beta diversity metrics and network analysis. Beyond direct host-driven effects via the provision of microhabitats, we found evidence that plants recruit for specific bacterial taxa that were likely playing a fundamental role in mediating microbe-microbe interactions and structuring clusters within mature communities. Our results on grape-microbiota interaction provide insights for targeted biocontrol and breeding strategies.

Viral Community Structure and Potential Functions in the Dried-Out Aral Sea Basin Change along a Desiccation Gradient.

The dried-out Aral Sea basin represents an extreme environment due to a man-made ecological disaster. Studies conducted in this unique environment revealed high levels of pollution and a specifically adapted microbiota; however, viral populations remained entirely unexplored. By employing an in-depth analysis based on the sequencing of metagenomic DNA recovered from rhizosphere samples of Suaeda acuminata (C. A. Mey.) Moq. along a desiccation gradient of 5, 10, and 40 years, we detected a diverse viral community comprising 674 viral populations (viral operational taxonomic units [vOTUs]) dominated by Caudovirales. Targeted analyses highlighted that viral populations in this habitat are subjected to certain dynamics that are driven mainly by the gradient of desiccation, the corresponding salinity, and the rhizosphere bacterial populations. In silico predictions linked the viruses to dominant prokaryotic taxa in the Aral Sea basin, such as Gammaproteobacteria, Actinomycetia, and Bacilli. The lysogenic lifestyle was predicted to be predominant in areas that dried out 5 years ago, representing the early revegetation phase. Metabolic prediction of viral auxiliary metabolic genes (AMGs) suggests that viruses may play a role in the biogeochemical cycles, stress resilience, and competitiveness of their hosts due to the presence of genes that are involved in biofilm formation. Overall, our study provides important insights into viral ecology in an extreme environment and expands our knowledge related to virus occurrence in terrestrial systems. IMPORTANCE Environmental viruses have added a wealth of knowledge to ecological studies with the emergence of metagenomic technology and approaches. They are also becoming recognized as important genetic repositories that underpin the functioning of terrestrial ecosystems but have remain moslty unexplored. Using shotgun metagenome sequencing and bioinformatic tools, we found that the viral community structure was affected during natural revegetation in the dried-up Aral Sea area, a model habitat for investigating natural ecological restoration but still understudied. In this study, we highlight the importance of viruses, elements that are overlooked, for their potential contribution to terrestrial ecosystems, i.e., nutrient cycles, stress resilience, and host competitiveness, during natural revegetation.

Impact of Cultivation and Origin on the Fruit Microbiome of Apples and Blueberries and Implications for the Exposome.

Vegetables and fruits are a crucial part of the planetary health diet, directly affecting human health and the gut microbiome. The objective of our study was to understand the variability of the fruit (apple and blueberry) microbiome in the frame of the exposome concept. The study covered two fruit-bearing woody species, apple and blueberry, two countries of origin (Austria and Finland), and two fruit production methods (naturally grown and horticultural). Microbial abundance, diversity, and community structures were significantly different for apples and blueberries and strongly influenced by the growing system (naturally grown or horticultural) and country of origin (Austria or Finland). Our results indicated that bacterial communities are more responsive towards these factors than fungal communities. We found that fruits grown in the wild and within home gardens generally carry a higher microbial diversity, while commercial horticulture homogenized the microbiome independent of the country of origin. This can be explained by horticultural management, including pesticide use and post-harvest treatments. Specific taxonomic indicators were identified for each group, i.e., for horticultural apples: Pseudomonas, Ralstonia, and Stenotrophomonas. Interestingly, Ralstonia was also found to be enriched in horticultural blueberries in comparison to such that were home and wildly grown. Our study showed that the origin of fruits can strongly influence the diversity and composition of their microbiome, which means that we are exposed to different microorganisms by eating fruits from different origins. Thus, the fruit microbiome needs to be considered an important but relatively unexplored external exposomic factor.

Function-Based Rhizosphere Assembly along a Gradient of Desiccation in the Former Aral Sea.

The desiccation of the Aral Sea represents one of the largest human-made environmental regional disasters. The salt- and toxin-enriched dried-out basin provides a natural laboratory for studying ecosystem functioning and rhizosphere assembly under extreme anthropogenic conditions. Here, we investigated the prokaryotic rhizosphere communities of the native pioneer plant Suaeda acuminata (C.A.Mey.) Moq. in comparison to bulk soil across a gradient of desiccation (5, 10, and 40 years) by metagenome and amplicon sequencing combined with quantitative PCR (qPCR) analyses. The rhizosphere effect was evident due to significantly higher bacterial abundances but less diversity in the rhizosphere compared to bulk soil. Interestingly, in the highest salinity (5 years of desiccation), rhizosphere functions were mainly provided by archaeal communities. Along the desiccation gradient, we observed a significant change in the rhizosphere microbiota, which was reflected by (i) a decreasing archaeon-bacterium ratio, (ii) replacement of halophilic archaea by specific plant-associated bacteria, i.e., Alphaproteobacteria and Actinobacteria, and (iii) an adaptation of specific, potentially plant-beneficial biosynthetic pathways. In general, both bacteria and archaea were found to be involved in carbon cycling and fixation, as well as methane and nitrogen metabolism. Analysis of metagenome-assembled genomes (MAGs) showed specific signatures for production of osmoprotectants, assimilatory nitrate reduction, and transport system induction. Our results provide evidence that rhizosphere assembly by cofiltering specific taxa with distinct traits is a mechanism which allows plants to thrive under extreme conditions. Overall, our findings highlight a function-based rhizosphere assembly, the importance of plant-microbe interactions in salinated soils, and their exploitation potential for ecosystem restoration approaches. IMPORTANCE The desertification of the Aral Sea basin in Uzbekistan and Kazakhstan represents one of the most serious anthropogenic environmental disasters of the last century. Since the 1960s, the world's fourth-largest inland body of water has been constantly shrinking, which has resulted in an extreme increase of salinity accompanied by accumulation of many hazardous and carcinogenic substances, as well as heavy metals, in the dried-out basin. Here, we investigated bacterial and archaeal communities in the rhizosphere of pioneer plants by combining classic molecular methods with amplicon sequencing as well as metagenomics for functional insights. By implementing a desiccation gradient, we observed (i) remarkable differences in the archaeon-bacterium ratio of plant rhizosphere samples, (ii) replacement of archaeal indicator taxa during succession, and (iii) the presence of specific, potentially plant-beneficial biosynthetic pathways in archaea present during the early stages. In addition, our results provide hitherto-undescribed insights into the functional redundancy between plant-associated archaea and bacteria.

Modulation of the food microbiome by apple fruit processing.

During the early life, introduction to external exposures such as consumption of solid foods contribute to the development of the gut microbiota. Among solid foods, fruit and vegetables are normally consumed during early childhood making them key components of a healthy human diet. The role of the indigenous microbiota of fruits as a source for beneficial gut microbes, especially during food processing, is largely unknown. Therefore, we investigated the apple fruit microbiota before and after processing using functional assays, advanced microscopic as well as sequencing technologies. Apple fruits carried a high absolute bacterial abundance (1.8 × 105 16S rRNA copies per g of apple pulp) and diversity of bacteria (Shannon diversity index = 2.5). We found that heat and mechanical treatment substantially affected the fruit's microbiota following a declining gradient of absolute bacterial abundance and bacterial diversity from shredded > boiled > pureed > preserved > dried apples. Betaproteobacteriales and Enterobacteriales were the two dominant bacterial orders (51.3%, 20.4% of the total 16S rRNA sequence reads) in the unprocessed apple. Boiling and air drying reduced the microbial load, but an unexpected, substantial fraction of 1/3 of the microbiota survived. Boiling and air drying shifted the microbiota leading to a relative increase in low abundant taxa such as Pseudomonas and Ralstonia (>2 log2 fold change), while others such as Bacillus decreased. Bacillus spp., frequently found in raw fruits, were shown to have specific traits, i.e. antagonist activity against opportunistic pathogens, biosurfactant production, and bile salt resistance indicating a probiotic potential. Our findings provide novel insights into food microbial changes during processing and demonstrate that food microbiome studies need a combined methodological approach. Food inhabiting microbes, currently considered being a risk factor for food safety, are a potential resource for the infant gut microbiome.

Recovery of metagenome-assembled genomes from the phyllosphere of 110 rice genotypes.

The plant microbiota plays crucial roles in sustaining plant health and productivity. Advancing plant microbiome research and designing sustainable practices for agriculture requires in-depth assessments of microorganisms associated with different host plants; however, there is little information on functional aspects of many microorganisms of interest. Therefore, we enriched microorganisms from the phyllosphere of 110 rice genotypes and subjected them to shotgun metagenomic sequencing to reconstruct bacterial genomes from the obtained datasets. The approach yielded a total of 1.34 terabases of shotgun-sequenced metagenomic data. By separately recovering bacterial genomes from each of the 110 rice genotypes, we recovered 569 non-redundant metagenome-assembled genomes (MAGs) with a completeness higher than 50% and contaminations less than 10%. The MAGs were primarily assigned to Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia. The presented data provides an extended basis for microbiome analyses of plant-associated microorganisms. It is complemented by detailed metadata to facilitate implementations in ecological studies, biotechnological mining approaches, and comparative assessments with genomes or MAGs from other studies.

The Brassica napus seed microbiota is cultivar-specific and transmitted via paternal breeding lines.

Seed microbiota influence germination and plant health and have the potential to improve crop performance, but the factors that determine their structure and functions are still not fully understood. Here, we analysed the impact of plant-related and external factors on seed endophyte communities of 10 different oilseed rape (Brassica napus L.) cultivars from 26 field sites across Europe. All seed lots harboured a high abundance and diversity of endophytes, which were dominated by six genera: Ralstonia, Serratia, Enterobacter, Pseudomonas, Pantoea, and Sphingomonas. The cultivar was the main factor explaining the variations in bacterial diversity, abundance and composition. In addition, the latter was significantly influenced by diverse biotic and abiotic factors, for example host germination rates and disease resistance against Plasmodiophora brassicae. A set of bacterial biomarkers was identified to discriminate between characteristics of the seeds, for example Sphingomonas for improved germination and Brevundimonas for disease resistance. Application of a Bayesian community approach suggested vertical transmission of seed endophytes, where the paternal parent plays a major role and might even determine the germination performance of the offspring. This study contributes to the understanding of seed microbiome assembly and underlines the potential of the microbiome to be implemented in crop breeding and biocontrol programmes.

Insights into the microbiome assembly during different growth stages and storage of strawberry plants.

BACKGROUND: Microbiome assembly was identified as an important factor for plant growth and health, but this process is largely unknown, especially for the fruit microbiome. Therefore, we analyzed strawberry plants of two cultivars by focusing on microbiome tracking during the different growth stages and storage using amplicon sequencing, qPCR, and microscopic approaches. RESULTS: Strawberry plants carried a highly diverse microbiome, therein the bacterial families Sphingomonadaceae (25%), Pseudomonadaceae (17%), and Burkholderiaceae (11%); and the fungal family Mycosphaerella (45%) were most abundant. All compartments were colonized by high number of bacteria and fungi (107-1010 marker gene copies per g fresh weight), and were characterized by high microbial diversity (6049 and 1501 ASVs); both were higher for the belowground samples than in the phyllosphere. Compartment type was the main driver of microbial diversity, structure, and abundance (bacterial: 45%; fungal: 61%) when compared to the cultivar (1.6%; 2.2%). Microbiome assembly was strongly divided for belowground habitats and the phyllosphere; only a low proportion of the microbiome was transferred from soil via the rhizosphere to the phyllosphere. During fruit development, we observed the highest rates of microbial transfer from leaves and flowers to ripe fruits, where most of the bacteria occured inside the pulp. In postharvest fruits, microbial diversity decreased while the overall abundance increased. Developing postharvest decay caused by Botrytis cinerea decreased the diversity as well, and induced a reduction of potentially beneficial taxa. CONCLUSION: Our findings provide insights into microbiome assembly in strawberry plants and highlight the importance of microbe transfer during fruit development and storage with potential implications for food health and safety.

Enhanced survival of multi-species biofilms under stress is promoted by low-abundant but antimicrobial-resistant keystone species.

Multi-species biofilms are more resistant against stress compared to single-species biofilms. However, the mechanisms underlying this common observation remain elusive. Therefore, we studied biofilm formation of well-known opportunistic pathogens (Acinetobacter baumanii, Enterococcus faecium, Escherichia coli, Staphylococcus haemolyticus and Stenotrophomonas maltophilia) in various approaches. Synergistic effects in their multi-species biofilms were observed. Using metatranscriptomics, changes in the gene expression of the involved members became evident, and provided explanations for the improved survivability under nutrient limitation and exposure to disinfectants. Genes encoding proteins for vitamin B6 synthesis and iron uptake were linked to synergism in the multi-species biofilm under nutrient-limited conditions. Our study indicates that sub-lethal concentrations of an alcohol-based disinfectant enhance biofilm yields in multi-species assemblages. A reduction of the dominant taxa in the multi-species biofilm under disinfectant pressure allowed minor taxa to bloom. The findings underline the importance of minor but antimicrobial-resistant species that serve as "protectors" for the whole assemblage due to upregulation of genes involved in defence mechanisms and biofilm formation. This ultimately results in an increase in the total yield of the multi-species biofilm. We conclude that inter-species interactions may be crucial for the survival of opportunistic pathogens; especially under conditions that are typically found under hospital settings.

Bog ecosystems as a playground for plant-microbe coevolution: bryophytes and vascular plants harbour functionally adapted bacteria.

BACKGROUND: Bogs are unique ecosystems inhabited by distinctive, coevolved assemblages of organisms, which play a global role for carbon storage, climate stability, water quality and biodiversity. To understand ecology and plant-microbe co-occurrence in bogs, we selected 12 representative species of bryophytes and vascular plants and subjected them to a shotgun metagenomic sequencing approach. We explored specific plant-microbe associations as well as functional implications of the respective communities on their host plants and the bog ecosystem. RESULTS: Microbial communities were shown to be functionally adapted to their plant hosts; a higher colonization specificity was found for vascular plants. Bryophytes that commonly constitute the predominant Sphagnum layer in bogs were characterized by a higher bacterial richness and diversity. Each plant group showed an enrichment of distinct phylogenetic and functional bacterial lineages. Detailed analyses of the metabolic potential of 28 metagenome-assembled genomes (MAGs) supported the observed functional specification of prevalent bacteria. We found that novel lineages of Betaproteobacteria and Actinobacteria in the bog environment harboured genes required for carbon fixation via RuBisCo. Interestingly, several of the highly abundant bacteria in both plant types harboured pathogenicity potential and carried similar virulence factors as found with corresponding human pathogens. CONCLUSIONS: The unexpectedly high specificity of the plant microbiota reflects intimate plant-microbe interactions and coevolution in bog environments. We assume that the detected pathogenicity factors might be involved in coevolution processes, but the finding also reinforces the role of the natural plant microbiota as a potential reservoir for human pathogens. Overall, the study demonstrates how plant-microbe assemblages can ensure stability, functioning and ecosystem health in bogs. It also highlights the role of bog ecosystems as a playground for plant-microbe coevolution. Video abstract.

Explorative assessment of coronavirus-like short sequences from host-associated and environmental metagenomes.

The ongoing COVID-19 pandemic has not only globally caused a high number of causalities, but is also an unprecedented challenge for scientists. False-positive virus detection tests not only aggravate the situation in the healthcare sector, but also provide ground for speculations. Previous studies have highlighted the importance of software choice and data interpretation in virome studies. We aimed to further expand theoretical and practical knowledge in bioinformatics-driven virome studies by focusing on short, virus-like DNA sequences in metagenomic data. Analyses of datasets obtained from different sample types (terrestrial, animal and human related samples) and origins showed that coronavirus-like sequences have existed in host-associated and environmental samples before the current COVID-19 pandemic. In the analyzed datasets, various Betacoronavirus-like sequences were detected that also included SARS-CoV-2 matches. Deepening analyses indicated that the detected sequences are not of viral origin and thus should not be considered in virome profiling approaches. Our study confirms the importance of parameter selection, especially in terms of read length, for reliable virome profiling. Natural environments are an important source of coronavirus-like nucleotide sequences that should be taken into account when virome datasets are analyzed and interpreted. We therefore suggest that processing parameters are carefully selected for SARS-CoV-2 profiling in host related as well as environmental samples in order to avoid incorrect identifications.

Microbiome-Assisted Breeding to Understand Cultivar-Dependent Assembly in Cucurbita pepo.

Recently, it was shown that long-term plant breeding does not only shape plant characteristics but also impacts plant-associated microbiota substantially. This requires a microbiome-integrative breeding approach, which was not yet shown. Here we investigate this for the Styrian oil pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by analyzing the microbiome of six genotypes (the complete pedigree of a three-way cross-hybrid, consisting of three inbred lines and one open pollinating cultivar) in the seed and rhizosphere as well as the progeny seeds. Using high-throughput amplicon sequencing targeting the 16S rRNA and the ITS1 genes, the bacterial and fungal microbiomes were accessed. Seeds were found to generally carry a significantly lower microbial diversity compared to the rhizosphere and soil as well as a different microbial composition, with an especially high fraction of Enterobacteriaceae (40-83%). Additionally, potential plant-beneficial bacterial taxa, including Bacillaceae, Burkholderiaceae, and Pseudomonadaceae, were found to be enriched in progeny seeds. Between genotypes, more substantial changes can be observed for seed microbiomes compared to the rhizosphere. Moreover, rhizosphere communities were assembled for the most part from soil. Interestingly, bacterial signatures are mainly linked from seed to seed, while fungal communities are shaped by the soil and rhizosphere. Our findings provide a deep look into the rhizosphere and seed microbiome assembly of pumpkin-associated communities and represent the first steps into microbiome-driven breeding for plant-beneficial microbes.

Antimicrobial-specific response from resistance gene carriers studied in a natural, highly diverse microbiome.

BACKGROUND: Antimicrobial resistance (AMR) is a major threat to public health. Microorganisms equipped with AMR genes are suggested to have partially emerged from natural habitats; however, this hypothesis remains inconclusive so far. To understand the consequences of the introduction of exogenic antimicrobials into natural environments, we exposed lichen thalli of Peltigera polydactylon, which represent defined, highly diverse miniature ecosystems, to clinical (colistin, tetracycline), and non-clinical (glyphosate, alkylpyrazine) antimicrobials. We studied microbiome responses by analysing DNA- and RNA-based amplicon libraries and metagenomic datasets. RESULTS: The analyzed samples consisted of the thallus-forming fungus that is associated with cyanobacteria as well as other diverse and abundant bacterial communities (up to 108 16S rRNA gene copies ng-1 DNA) dominated by Alphaproteobacteria and Bacteroidetes. Moreover, the natural resistome of this meta-community encompassed 728 AMR genes spanning 30 antimicrobial classes. Following 10 days of exposure to the selected antimicrobials at four different concentrations (full therapeutic dosage and a gradient of sub-therapeutic dosages), we observed statistically significant, antimicrobial-specific shifts in the structure and function but not in bacterial abundances within the microbiota. We observed a relatively lower response after the exposure to the non-clinical compared to the clinical antimicrobial compounds. Furthermore, we observed specific bacterial responders, e.g., Pseudomonas and Burkholderia to clinical antimicrobials. Interestingly, the main positive responders naturally occur in low proportions in the lichen holobiont. Moreover, metagenomic recovery of the responders' genomes suggested that they are all naturally equipped with specific genetic repertoires that allow them to thrive and bloom when exposed to antimicrobials. Of the responders, Sphingomonas, Pseudomonas, and Methylobacterium showed the highest potential. CONCLUSIONS: Antimicrobial exposure resulted in a microbial dysbiosis due to a bloom of naturally low abundant taxa (positive responders) with specific AMR features. Overall, this study provides mechanistic insights into community-level responses of a native microbiota to antimicrobials and suggests novel strategies for AMR prediction and management. Video Abstract.