Saskia Gerards

Plants release a wide set of secondary metabolites including volatile organic compounds (VOCs). Many of those compounds are considered to function as defense against herbivory, pests, and pathogens. However, little knowledge exists about the role of belowground plant VOCs for attracting beneficial soil microorganisms. We developed an olfactometer system to test the attraction of soil bacteria by VOCs emitted by Carex arenaria roots. Moreover, we tested whether infection of C. arenaria with the fungal pathogen Fusarium culmorum modifies the VOCs profile and bacterial attraction. The results revealed that migration of distant bacteria in soil towards roots can be stimulated by plant VOCs. Upon fungal infection, the blend of root VOCs changed and specific bacteria with antifungal properties were attracted. Tests with various pure VOCs indicated that those compounds can diffuse over long distance but with different diffusion abilities. Overall, this work highlights the importance of plant VOCs in belowground long-distance plant–microbe interactions.

Recent studies indicated that the production of secondary metabolites by soil bacteria can be triggered by interspecific interactions. However, little is known to date about interspecific interactions between Gram-positive and Gram-negative bacteria. In this study, we aimed to understand how the interspecific interaction between the Gram-positive Paenibacillus sp. AD87 and the Gram-negative Burkholderia sp. AD24 affects the fitness, gene expression and the production of soluble and volatile secondary metabolites of both bacteria. To obtain better insight into this interaction, transcriptome and metabolome analyses were performed. Our results revealed that the interaction between the two bacteria affected their fitness, gene expression and the production of secondary metabolites. During interaction, the growth of Paenibacillus was not affected, whereas the growth of Burkholderia was inhibited at 48 and 72 h. Transcriptome analysis revealed that the interaction between Burkholderia and Paenibacillus caused significant transcriptional changes in both bacteria as compared to the monocultures. The metabolomic analysis revealed that the interaction increased the production of specific volatile and soluble antimicrobial compounds such as 2,5-bis(1-methylethyl)-pyrazine and an unknown Pederin-like compound. The pyrazine volatile compound produced by Paenibacillus was subjected to bioassays and showed strong inhibitory activity against Burkholderia and a range of plant and human pathogens. Moreover, strong additive antimicrobial effects were observed when soluble extracts from the interacting bacteria were combined with the pure 2,5-bis(1-methylethyl)-pyrazine. The results obtained in this study highlight the importance to explore bacterial interspecific interactions to discover novel secondary metabolites and to perform simultaneously metabolomics of both, soluble and volatile compounds.

Rhizogenic Agrobacterium biovar 1 is the causative agent of hairy root disease (HRD) in the hydroponic cultivation of tomato and cucumber causing significant losses in marketable yield. In order to prevent and control the disease chemical disinfectants such as hydrogen peroxide or hypochlorite are generally applied to sanitize the hydroponic system and/or hydroponic solution. However, effective control of HRD sometimes requires high disinfectant doses that may have phytotoxic effects. Moreover, several of these chemicals may be converted to unwanted by-products with human health hazards. Here we explored the potential of beneficial bacteria as a sustainable means to control HRD. A large collection of diverse bacterial genera was screened for antagonistic activity against rhizogenic Agrobacterium biovar 1 using the agar overlay assay. Out of more than 130 strains tested only Paenibacillus strains showed antagonistic activity. Strikingly, phylogenetic analysis showed that antagonistic activity was restricted to a particular Paenibacillus clade, representing the species P. illinoisensis, P. pabuli, P. taichungensis, P. tundrae, P. tylopili, P. xylanexedens and P. xylanilyticus. Assessment of the spectrum of activity revealed that some strains were able to inhibit the growth of all 35 rhizogenic agrobacteria strains tested, while others were only active against part of the collection, suggesting a different mode of action. Preliminary characterization of the compounds involved in the antagonistic activity of two closely related Paenibacillus strains, tentatively identified as P. xylanexedens, revealed that they are water-soluble and have low molecular weight. Application of a combination of these strains in greenhouse conditions resulted in a significant reduction of HRD, indicating the great potential of these strains to control HRD.

There is increasing evidence that volatile organic compounds (VOCs) play an important role in the interactions between fungi and bacteria, two major groups of soil inhabiting microorganisms. Yet, most of the research has been focused on effects of bacterial volatiles on suppression of plant pathogenic fungi whereas little is known about the responses of bacteria to fungal volatiles. In the current study we performed a metabolomics analysis of volatiles emitted by several fungal and oomycetal soil strains under different nutrient conditions and growth stages. The metabolomics analysis of the tested fungal and oomycetal strains revealed different volatile profiles dependent on the age of the strains and nutrient conditions. Furthermore, we screened the phenotypic responses of soil bacterial strains to volatiles emitted by fungi. Two bacteria, Collimonas pratensis Ter291 and Serratia plymuthica PRI-2C, showed significant changes in their motility, in particular to volatiles emitted by Fusarium culmorum. This fungus produced a unique volatile blend, including several terpenes. Four of these terpenes were selected for further tests to investigate if they influence bacterial motility. Indeed, these terpenes induced or reduced swimming and swarming motility of S. plymuthica PRI-2C and swarming motility of C. pratensis Ter291, partly in a concentration-dependent manner. Overall the results of this work revealed that bacteria are able to sense and respond to fungal volatiles giving further evidence to the suggested importance of volatiles as signaling molecules in fungal–bacterial interactions.

It is increasingly recognized that volatile organic compounds play an import role during interactions between soil microorganisms. Here, we examined the possible involvement of volatiles in the interaction of Collimonas bacteria with soil fungi. The genus Collimonas is known for its ability to grow at the expense of living fungi (mycophagy), and antifungal volatiles may contribute to the attack of fungi by these bacteria. We analyzed the composition of volatiles produced by Collimonas on agar under different nutrient conditions and studied the effect on fungal growth. The volatiles had a negative effect on the growth of a broad spectrum of fungal species. Collimonas bacteria did also produce volatiles in sand microcosms supplied with artificial root exudates. The production of volatiles in sand microcosms was enhanced by the presence of fungi. The overall picture that we get from our study is that antifungal volatiles produced by Collimonas could play an important role in realizing its mycophagous lifestyle. The current work is also interesting for understanding the ecological relevance of volatile production by soil bacteria in general as we found strong influences of root exudates composition and incubation conditions on the spectrum of volatiles produced.

There is increasing evidence that organic volatiles play an important role in interactions between micro-organisms in the porous soil matrix. Here we report that volatile compounds emitted by different soil bacteria can affect the growth, antibiotic production and gene expression of the soil bacterium Pseudomonas fluorescens Pf0–1. We applied a novel cultivation approach that mimics the natural nutritional heterogeneity in soil in which P. fluorescens grown on nutrient-limited agar was exposed to volatiles produced by 4 phylogenetically different bacterial isolates (Collimonas pratensis, Serratia plymuthica, Paenibacillus sp., and Pedobacter sp.) growing in sand containing artificial root exudates. Contrary to our expectation, the produced volatiles stimulated rather than inhibited the growth of P. fluorescens. A genome-wide, microarray-based analysis revealed that volatiles of all four bacterial strains affected gene expression of P. fluorescens, but with a different pattern of gene expression for each strain. Based on the annotation of the differently expressed genes, bacterial volatiles appear to induce a chemotactic motility response in P. fluorescens, but also an oxidative stress response. A more detailed study revealed that volatiles produced by C. pratensis triggered, antimicrobial secondary metabolite production in P. fluorescens. Our results indicate that bacterial volatiles can have an important role in communication, trophic - and antagonistic interactions within the soil bacterial community.