Volatile sensation: The chemical ecology of the earthy odorant geosmin.

Geosmin may be the most familiar volatile compound, as it lends the earthy smell to soil. The compound is a member of the largest family of natural products, the terpenoids. The broad distribution of geosmin among bacteria in both terrestrial and aquatic environments suggests that this compound has an important ecological function, for example, as a signal (attractant or repellent) or as a protective specialized metabolite against biotic and abiotic stresses. While geosmin is part of our everyday life, scientists still do not understand the exact biological function of this omnipresent natural product. This minireview summarizes the current general observations regarding geosmin in prokaryotes and introduces new insights into its biosynthesis and regulation, as well as its biological roles in terrestrial and aquatic environments.

Biosynthesis, evolution and ecology of microbial terpenoids.

Covering: through June 2021Terpenoids are the largest class of natural products recognised to date. While mostly known to humans as bioactive plant metabolites and part of essential oils, structurally diverse terpenoids are increasingly reported to be produced by microorganisms. For many of the compounds biological functions are yet unknown, but during the past years significant insights have been obtained for the role of terpenoids in microbial chemical ecology. Their functions include stress alleviation, maintenance of cell membrane integrity, photoprotection, attraction or repulsion of organisms, host growth promotion and defense. In this review we discuss the current knowledge of the biosynthesis and evolution of microbial terpenoids, and their ecological and biological roles in aquatic and terrestrial environments. Perspectives on their biotechnological applications, knowledge gaps and questions for future studies are discussed.

Exploring the Volatiles Released from Roots of Wild and Domesticated Tomato Plants under Insect Attack.

Plants produce volatile organic compounds that are important in communication and defense. While studies have largely focused on volatiles emitted from aboveground plant parts upon exposure to biotic or abiotic stresses, volatile emissions from roots upon aboveground stress are less studied. Here, we investigated if tomato plants under insect herbivore attack exhibited a different root volatilome than non-stressed plants, and whether this was influenced by the plant's genetic background. To this end, we analyzed one domesticated and one wild tomato species, i.e., Solanum lycopersicum cv Moneymaker and Solanum pimpinellifolium, respectively, exposed to leaf herbivory by the insect Spodoptera exigua. Root volatiles were trapped with two sorbent materials, HiSorb and PDMS, at 24 h after exposure to insect stress. Our results revealed that differences in root volatilome were species-, stress-, and material-dependent. Upon leaf herbivory, the domesticated and wild tomato species showed different root volatile profiles. The wild species presented the largest change in root volatile compounds with an overall reduction in monoterpene emission under stress. Similarly, the domesticated species presented a slight reduction in monoterpene emission and an increased production of fatty-acid-derived volatiles under stress. Volatile profiles differed between the two sorbent materials, and both were required to obtain a more comprehensive characterization of the root volatilome. Collectively, these results provide a strong basis to further unravel the impact of herbivory stress on systemic volatile emissions.

Volatile-mediated antagonism of soil bacterial communities against fungi.

Competition is a major type of interaction between fungi and bacteria in soil and is also an important factor in suppression of plant diseases caused by soil-borne fungal pathogens. There is increasing attention for the possible role of volatiles in competitive interactions between bacteria and fungi. However, knowledge on the actual role of bacterial volatiles in interactions with fungi within soil microbial communities is lacking. Here, we examined colonization of sterile agricultural soils by fungi and bacteria from non-sterile soil inoculums during exposure to volatiles emitted by soil-derived bacterial communities. We found that colonization of soil by fungi was negatively affected by exposure to volatiles emitted by bacterial communities whereas that of bacteria was barely changed. Furthermore, there were strong effects of bacterial community volatiles on the assembly of fungal soil colonizers. Identification of volatile composition produced by bacterial communities revealed several compounds with known fungistatic activity. Our results are the first to reveal a collective volatile-mediated antagonism of soil bacteria against fungi. Given the better exploration abilities of filamentous fungi in unsaturated soils, this may be an important strategy for bacteria to defend occupied nutrient patches against invading fungi. Another implication of our research is that bacterial volatiles in soil atmospheres can have a major contribution to soil fungistasis.

Microbial and volatile profiling of soils suppressive to Fusarium culmorum of wheat.

In disease-suppressive soils, microbiota protect plants from root infections. Bacterial members of this microbiota have been shown to produce specific molecules that mediate this phenotype. To date, however, studies have focused on individual suppressive soils and the degree of natural variability of soil suppressiveness remains unclear. Here, we screened a large collection of field soils for suppressiveness to Fusarium culmorum using wheat (Triticum aestivum) as a model host plant. A high variation of disease suppressiveness was observed, with 14% showing a clear suppressive phenotype. The microbiological basis of suppressiveness to F. culmorum was confirmed by gamma sterilization and soil transplantation. Amplicon sequencing revealed diverse bacterial taxonomic compositions and no specific taxa were found exclusively enriched in all suppressive soils. Nonetheless, co-occurrence network analysis revealed that two suppressive soils shared an overrepresented bacterial guild dominated by various Acidobacteria. In addition, our study revealed that volatile emission may contribute to suppression, but not for all suppressive soils. Our study raises new questions regarding the possible mechanistic variability of disease-suppressive phenotypes across physico-chemically different soils. Accordingly, we anticipate that larger-scale soil profiling, along with functional studies, will enable a deeper understanding of disease-suppressive microbiomes.

Airborne medicine: bacterial volatiles and their influence on plant health.

Like most other eukaryotes, plants do not live alone but in close association with a diverse microflora. These plant-associated microbes contribute to plant health in many different ways, ranging from modulation of hormonal pathways to direct antibiosis of plant pathogens. Over the last 15 yr, the importance of volatile organic compounds as mediators of mutualistic interactions between plant-associated bacteria and their hosts has become evident. This review summarizes current knowledge concerning bacterial volatile-mediated plant protection against abiotic and biotic stresses. It then discusses the translational potential of such metabolites or of their emitters for sustainable crop protection, the possible ways to harness this potential, and the major challenges still preventing us from doing so. Finally, the review concludes with highlighting the most pressing scientific gaps that need to be filled in order to enable a better understanding of: the molecular mechanisms underlying the biosynthesis of bacterial volatiles; the complex regulation of bacterial volatile emission in natural communities; the perception of bacterial volatiles by plants; and the modes of actions of bacterial volatiles on their host.

Phylogenomic analyses and distribution of terpene synthases among Streptomyces.

Terpene synthases are widely distributed among microorganisms and have been mainly studied in members of the genus Streptomyces. However, little is known about the distribution and evolution of the genes for terpene synthases. Here, we performed whole-genome based phylogenetic analysis of Streptomyces species, and compared the distribution of terpene synthase genes among them. Overall, our study revealed that ten major types of terpene synthases are present within the genus Streptomyces, namely those for geosmin, 2-methylisoborneol, epi-isozizaene, 7-epi-α-eudesmol, epi-cubenol, caryolan-1-ol, cyclooctat-9-en-7-ol, isoafricanol, pentalenene and α-amorphene. The Streptomyces species divide in three phylogenetic groups based on their whole genomes for which the distribution of the ten terpene synthases was analysed. Geosmin synthases were the most widely distributed and were found to be evolutionary positively selected. Other terpene synthases were found to be specific for one of the three clades or a subclade within the genus Streptomyces. A phylogenetic analysis of the most widely distributed classes of Streptomyces terpene synthases in comparison to the phylogenomic analysis of this genus is discussed.

LAESI mass spectrometry imaging as a tool to differentiate the root metabolome of native and range-expanding plant species.

MAIN CONCLUSION: LAESI-MSI, an innovative high-throughput technique holds a unique potential for untargeted detection, profiling and spatial localization of metabolites from intact plant samples without need for extraction or extensive sample preparation. Our understanding of chemical diversity in biological samples has greatly improved through recent advances in mass spectrometry (MS). MS-based-imaging (MSI) techniques have further enhanced this by providing spatial information on the distribution of metabolites and their relative abundance. This study aims to employ laser-ablation electrospray ionization (LAESI) MSI as a tool to profile and compare the root metabolome of two pairs of native and range-expanding plant species. It has been proposed that successful range-expanding plant species, like introduced exotic invaders, have a novel, or a more diverse secondary chemistry. Although some tests have been made using aboveground plant materials, tests using root materials are rare. We tested the hypothesis that range-expanding plants possess more diverse root chemistries than native plant species. To examine the root chemistry of the selected plant species, LAESI-MSI was performed in positive ion mode and data were acquired in a mass range of m/z 50-1200 with a spatial resolution of 100 µm. The acquired data were analyzed using in-house scripts, and differences in the spatial profiles were studied for discriminatory mass features. The results revealed clear differences in the metabolite profiles amongst and within both pairs of congeneric plant species, in the form of distinct metabolic fingerprints. The use of ambient conditions and the fact that no sample preparation was required, established LAESI-MSI as an ideal technique for untargeted metabolomics and for direct correlation of the acquired data to the underlying metabolomic complexity present in intact plant samples.

Fungus-associated bacteriome in charge of their host behavior.

Bacterial-fungal interactions are widespread in nature and there is a growing number of studies reporting distinct fungus-associated bacteria. However, little is known so far about how shifts in the fungus-associated bacteriome will affect the fungal host's lifestyle. In the present study, we describe for the first time the bacterial community associated with the saprotrophic fungus Mucor hiemalis, commonly found in soil and rhizosphere. Two broad-spectrum antibiotics that strongly altered the bacterial community associated with the fungus were applied. Our results revealed that the antibiotic treatment did not significantly reduce the amount of bacteria associated to the fungus but rather changed the community composition by shifting from initially dominating Alpha-Proteobacteria to dominance of Gamma-Proteobacteria. A novel approach was applied for the isolation of fungal-associated bacteria which also revealed differences between bacterial isolates obtained from the original and the antibiotic-treated M. hiemalis. The shift in the composition of the fungal-associated bacterial community led to significantly reduced fungal growth, changes in fungal morphology, behavior and secondary-metabolites production. Furthermore, our results showed that the antibiotic-treated isolate was more attractive and susceptible to mycophagous bacteria as compared to the original isolate. Overall, our study highlights the importance of the fungus-associated bacteriome for the host's lifestyle and interactions and indicate that isolation with antibacterials is not sufficient to eradicate the associated bacteria.

Lessons from 1,3-Hydride Shifts in Sesquiterpene Cyclizations.

Stereospecifically labelled precursors were subjected to conversion by seven bacterial sesquiterpene cyclases to investigate the stereochemistry of their initial 1,10-cyclisation-1,3-hydride shift cascades. Enzymes with products of known absolute configuration showed a coherent stereochemical course, except for (-)-α-amorphene synthase, for which the obtained results are better explained by an initial 1,6-cyclisation. The link between the absolute configuration of the product and the stereochemical course of the 1,3-hydride shifts enabled assignment of the absolute configurations of three enzyme products, which were confirmed independently through the absolute configuration of the common byproduct germacrene D-4-ol.

Exploring the genomic traits of fungus-feeding bacterial genus Collimonas.

BACKGROUND: Collimonas is a genus belonging to the class of Betaproteobacteria and consists mostly of soil bacteria with the ability to exploit living fungi as food source (mycophagy). Collimonas strains differ in a range of activities, including swimming motility, quorum sensing, extracellular protease activity, siderophore production, and antimicrobial activities. RESULTS: In order to reveal ecological traits possibly related to Collimonas lifestyle and secondary metabolites production, we performed a comparative genomics analysis based on whole-genome sequencing of six strains representing 3 recognized species. The analysis revealed that the core genome represents 43.1 to 52.7% of the genomes of the six individual strains. These include genes coding for extracellular enzymes (chitinase, peptidase, phospholipase), iron acquisition and type II secretion systems. In the variable genome, differences were found in genes coding for secondary metabolites (e.g. tripropeptin A and volatile terpenes), several unknown orphan polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), nonribosomal peptide synthetase (NRPS) gene clusters, a new lipopeptide and type III and type VI secretion systems. Potential roles of the latter genes in the interaction with other organisms were investigated. Mutation of a gene involved in tripropeptin A biosynthesis strongly reduced the antibacterial activity against Staphylococcus aureus, while disruption of a gene involved in the biosynthesis of the new lipopeptide had a large effect on the antifungal/oomycetal activities. CONCLUSIONS: Overall our results indicated that Collimonas genomes harbour many genes encoding for novel enzymes and secondary metabolites (including terpenes) important for interactions with other organisms and revealed genomic plasticity, which reflect the behaviour, antimicrobial activity and lifestylesof Collimonas spp.

Non-random species loss in bacterial communities reduces antifungal volatile production.

The contribution of low-abundance microbial species to soil ecosystems is easily overlooked because there is considerable overlap between metabolic abilities (functional redundancy) of dominant and subordinate microbial species. Here we studied how loss of less abundant soil bacteria affected the production of antifungal volatiles, an important factor in the natural control of soil-borne pathogenic fungi. We provide novel empirical evidence that the loss of soil bacterial species leads to a decline in the production of volatiles that suppress root pathogens. By using dilution-to-extinction for seven different soils we created bacterial communities with a decreasing number of species and grew them under carbon-limited conditions. Communities with high bacterial species richness produced volatiles that strongly reduced the hyphal growth of the pathogen Fusarium oxysporum. For most soil origins loss of bacterial species resulted in loss of antifungal volatile production. Analysis of the volatiles revealed that several known antifungal compounds were only produced in the more diverse bacterial communities. Our results suggest that less abundant bacterial species play an important role in antifungal volatile production by soil bacterial communities and, consequently, in the natural suppression of soil-borne pathogens.

Effect of microplastic pollution on the gut microbiome of anecic and endogeic earthworms.

Microplastic (MP) pollution constitutes an emerging type of pollution threatening both aquatic and terrestrial ecosystems. The impact on aquatic ecosystems has been extensively studied, but the effect on terrestrial ecosystems and their inhabitants is mostly underexplored. In this study, we explored the effect of MP pollution on gut bacterial microbiome of endogeic (Aporrectodea caliginosa) and anecic (Lumbricus terrestris) earthworms. The experiments were performed in sandy soil with 0.2% of low-density polyethylene MPs (LDPE MPs). We observed that the endogeic earthworms had 100% survival, while anecic earthworms survived 25 days in the control (i.e. in absence of MPs) and 21 days in the treatment with LDPE MPs. The main driver of shifts in the diversity and composition of the bacterial communities in the gut of tested earthworms was the lifestyle of the worms, followed by the presence of MPs. The bacterial microbiome diversity was significantly different among the two types of earthworms, and the highest bacterial diversity was found in the gut of the endogeic earthworms. The effect of MPs on gut bacterial microbiome was clearly observed in the changes in the relative abundance of several phyla and families of the bacterial communities in both types of earthworms, although it was most evident in the anecic earthworms. The Actinobacteriota, Proteobacteria, and Firmicutes were the main groups enhanced in the MP treatments, suggesting enrichment of the bacterial communities with potential plastic degraders.

Strategies for tailoring functional microbial synthetic communities.

Natural ecosystems harbor a huge reservoir of taxonomically diverse microbes that are important for plant growth and health. The vast diversity of soil microorganisms and their complex interactions make it challenging to pinpoint the main players important for the life support functions microbes can provide to plants, including enhanced tolerance to (a)biotic stress factors. Designing simplified microbial synthetic communities (SynComs) helps reduce this complexity to unravel the molecular and chemical basis and interplay of specific microbiome functions. While SynComs have been successfully employed to dissect microbial interactions or reproduce microbiome-associated phenotypes, the assembly and reconstitution of these communities have often been based on generic abundance patterns or taxonomic identities and co-occurrences but have only rarely been informed by functional traits. Here, we review recent studies on designing functional SynComs to reveal common principles and discuss multidimensional approaches for community design. We propose a strategy for tailoring the design of functional SynComs based on integration of high-throughput experimental assays with microbial strains and computational genomic analyses of their functional capabilities.

Impact of bacterial and fungal inoculants on the resident rhizosphere microbiome and the volatilome of tomato plants under leaf herbivory stress.

Various studies have addressed the impact of microbial inoculants on the composition of the resident microbiome. How microbial inoculants impact plant metabolism and interact with the resident rhizobiota under herbivory stress remains elusive. Here, we investigated the impact of two bacterial and two fungal inoculants, inoculated as single species and as a synthetic community, on the rhizosphere microbiome and volatilome of tomato plants (Solanum lycopersicum) comparing nonstress conditions to exposed to leaf herbivory by Spodoptera exigua. Based on amplicon sequencing analysis, rhizobacterial community composition was significantly affected by all four inoculants and the magnitude of this effect was dependent on herbivory stress. Fungal community composition was altered by the microbial inoculants but independent of herbivory stress. The rhizosphere volatilome was impacted by the microbial inoculation and differences between treatments were evened under herbivory stress. Each microbial inoculant caused unique changes in the volatilome of stressed plants but also shared similar responses, in particular the enhanced production of dimethyl disulfide and benzothiazole. In conclusion, the introduction of microbial inoculants in the tomato rhizosphere caused unique as well as common changes in the rhizosphere microbiome and volatilome, but these changes were minor compared to the microbiome changes induced by herbivory stress.

Enhanced surface colonisation and competition during bacterial adaptation to a fungus.

Bacterial-fungal interactions influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Here, we use a co-culture experimental evolution approach to investigate bacterial adaptation to the presence of a fungus, using a simple model of bacterial-fungal interactions encompassing the bacterium Bacillus subtilis and the fungus Aspergillus niger. We find in one evolving population that B. subtilis was selected for enhanced production of the lipopeptide surfactin and accelerated surface spreading ability, leading to inhibition of fungal expansion and acidification of the environment. These phenotypes were explained by specific mutations in the DegS-DegU two-component system. In the presence of surfactin, fungal hyphae exhibited bulging cells with delocalised secretory vesicles possibly provoking an RlmA-dependent cell wall stress. Thus, our results indicate that the presence of the fungus selects for increased surfactin production, which inhibits fungal growth and facilitates the competitive success of the bacterium.

Exploring the Interspecific Interactions and the Metabolome of the Soil Isolate Hylemonella gracilis.

Microbial community analysis of aquatic environments showed that an important component of its microbial diversity consists of bacteria with cell sizes of ~0.1 µm. Such small bacteria can show genomic reductions and metabolic dependencies with other bacteria. However, so far, no study has investigated if such bacteria exist in terrestrial environments like soil. Here, we isolated soil bacteria that passed through a 0.1-µm filter. The complete genome of one of the isolates was sequenced and the bacterium was identified as Hylemonella gracilis. A set of coculture assays with phylogenetically distant soil bacteria with different cell and genome sizes was performed. The coculture assays revealed that H. gracilis grows better when interacting with other soil bacteria like Paenibacillus sp. AD87 and Serratia plymuthica. Transcriptomics and metabolomics showed that H. gracilis was able to change gene expression, behavior, and biochemistry of the interacting bacteria without direct cell-cell contact. Our study indicates that in soil there are bacteria that can pass through a 0.1-µm filter. These bacteria may have been overlooked in previous research on soil microbial communities. Such small bacteria, exemplified here by H. gracilis, can induce transcriptional and metabolomic changes in other bacteria upon their interactions in soil. In vitro, the studied interspecific interactions allowed utilization of growth substrates that could not be utilized by monocultures, suggesting that biochemical interactions between substantially different sized soil bacteria may contribute to the symbiosis of soil bacterial communities. IMPORTANCE Analysis of aquatic microbial communities revealed that parts of its diversity consist of bacteria with cell sizes of ~0.1 µm. Such bacteria can show genomic reductions and metabolic dependencies with other bacteria. So far, no study investigated if such bacteria exist in terrestrial environments such as soil. Here, we show that such bacteria also exist in soil. The isolated bacteria were identified as Hylemonella gracilis. Coculture assays with phylogenetically different soil bacteria revealed that H. gracilis grows better when cocultured with other soil bacteria. Transcriptomics and metabolomics showed that H. gracilis was able to change gene expression, behavior, and biochemistry of the interacting bacteria without direct contact. Our study revealed that bacteria are present in soil that can pass through 0.1-µm filters. Such bacteria may have been overlooked in previous research on soil microbial communities and may contribute to the symbiosis of soil bacterial communities.

Air Ambulance: Antimicrobial Power of Bacterial Volatiles.

We are currently facing an antimicrobial resistance crisis, which means that a lot of bacterial pathogens have developed resistance to common antibiotics. Hence, novel and innovative solutions are urgently needed to combat resistant human pathogens. A new source of antimicrobial compounds could be bacterial volatiles. Volatiles are ubiquitous produced, chemically divers and playing essential roles in intra- and interspecies interactions like communication and antimicrobial defense. In the last years, an increasing number of studies showed bioactivities of bacterial volatiles, including antibacterial, antifungal and anti-oomycete activities, indicating bacterial volatiles as an exciting source for novel antimicrobial compounds. In this review we introduce the chemical diversity of bacterial volatiles, their antimicrobial activities and methods for testing this activity. Concluding, we discuss the possibility of using antimicrobial volatiles to antagonize the antimicrobial resistance crisis.

Antimicrobial volatiles emitted by members of the nest microbiome of social spiders.

Microbes produce and respond to a range of structurally and functionally diverse volatiles. Many microbial volatiles have antimicrobial properties. Since volatiles can diffuse through complex 3D systems like spider nests, they are promising pathogen protection for social arthropods. Here, we analyzed the volatilomes of five nest microbiome members of the Namibian, social spider Stegodyphus dumicola, namely the bacteria Massilia sp. IC2-278, Massilia sp. IC2-477, Sphingomonas sp. IC-11, Streptomyces sp. IC-207, and the fungus Aureobasidium sp. CE_32, and tested their antimicrobial activity against two putative spider pathogens, namely Bacillus thuringiensis and Purpureocillium lilacinum. Most nest microbiome members released volatilomes with antibacterial and/or antifungal activities under in vitro conditions. The analysis of their volatilomes using GC/Q-TOF revealed that they include numerous antimicrobial volatiles. We tested the antimicrobial activity of five pure volatile compounds found in the volatilomes and revealed that all of them were antibacterial and/or antifungal. We could not identify the same antimicrobial volatiles as in a previous in situ study, but our results indicate that social spider-associated microorganisms as a source of antimicrobial volatiles are important for pathogen inhibition. Additionally, we showed the influence of the volatilomes on the antibiotic sensitivity of B. thuringiensis offering novel approaches to counter antibiotic resistance.

Plastic mulch film residues in agriculture: impact on soil suppressiveness, plant growth, and microbial communities.

Plastic mulch film residues have been accumulating in agricultural soils for decades, but so far, little is known about its consequences on soil microbial communities and functions. Here, we tested the effects of plastic residues of low-density polyethylene and biodegradable mulch films on soil suppressiveness and microbial community composition. We investigated how plastic residues in a Fusarium culmorum suppressive soil affect the level of disease suppressiveness, plant biomass, nutrient status, and microbial communities in rhizosphere using a controlled pot experiment. The addition of 1% plastic residues to the suppressive soil did not affect the level of suppression and the disease symptoms index. However, we did find that plant biomasses decreased, and that plant nutrient status changed in the presence of plastic residues. No significant changes in bacterial and fungal rhizosphere communities were observed. Nonetheless, bacterial and fungal communities closely attached to the plastisphere were very different from the rhizosphere communities with overrepresentation of potential plant pathogens. The plastisphere revealed a high abundance of specific bacterial phyla (Actinobacteria, Bacteroidetes, and Proteobacteria) and fungal genera (Rhizoctonia and Arthrobotrys). Our work revealed new insights and raises emerging questions for further studies on the impact of microplastics on the agroecosystems.