Microbial Volatiles: Small Molecules with an Important Role in Intra- and Inter-Kingdom Interactions

1Many secondary metabolites have been reported to be involved in microbial interactions. One group of secondary metabolites produced by soil and plant-associated microorganisms, but largely unexplored to date, are the volatile organic compounds (VOCs). VOCs are typically small, odorous compounds (<C15) with low molecular mass (<300 Da), high vapor pressure, low boiling point, and a lipophilic moiety. These properties facilitate evaporation and diffusion aboveground and belowground through gas- and water- filled pores in soil and rhizosphere environments. Microbial volatile organic compounds (mVOCs) belong to different chemical classes including alkenes, alcohols, ketones, benzenoids, pyrazines, sulfides, and terpenes. A recent meta-analysis by provided a comprehensive overview of VOCs derived from soil-borne microbes.

2The production of mVOCs in soil is influenced by various factors including the growth stage of the microbes, nutrient availability, temperature, oxygen availability, pH, and soil moisture content. Several recent studies reported that the production of certain mVOCs can be induced or suppressed during inter-specific microbial interactions. mVOCs were often considered to be by-products of primary metabolism, but recent findings revealed that many mVOCs demonstrate biological activity. Furthermore, in bacteria, the production of certain mVOCs is dependent on the GacS/GacA two-component regulatory system. These findings clearly disagree with the opinion that mVOCs are just waste products.

3While soluble metabolites are often responsible for short distance interactions, VOCs are considered to be long-distance messengers. There are many types of microbial interactions occurring belowground such as bacteria–bacteria, fungi–fungi, fungi–bacteria, bacteria–protists, fungi–plant, bacteria–plant, and bacteria–fungi–plant interactions. However, most studies addressing belowground VOCs-mediated interactions are focused mainly on the root-emitted volatiles (recently reviewed by ).

4The knowledge we have gained from research conducted over the last few years reveals that mVOCs can have both beneficial and harmful effects on other organisms. mVOCs can provide organisms with rapid and precise ways to recognize neighboring organisms (both friends and foe) and to launch proper responses.

5The aim of this review is to summarize the current knowledge concerning the role of mVOCs in intra- and inter-kingdom interactions, to pinpoint mVOCs (e.g., terpenes) involved in microbe–microbe and microbe–plant interactions as well as to indicate challenges in studying belowground mVOCs-mediated interactions and opportunities for further studies and practical applications.

6Bacterial VOCs can have direct antagonistic effects against other bacteria. For instance, the sesquiterpene albaflavenone produced by Streptomyces albidoflavus revealed activity against Bacillus subtilis and the emission of dimethyl disulphide by two rhizospheric bacteria, Pseudomonas fluorescens and Serratia plymuthica, showed bacteriostatic effects against two plant bacterial pathogens Agrobacterium tumefaciens and Agrobacterium vitis. Pseudomonas fluorescens WR-1 produces volatiles such as benzothiazole and 1-methyl naphthalene with bacteriostatic effects against the tomato pathogen Ralstonia solanacearum. In fact, many species of Pseudomonas and Bacillus that are used as biocontrol agents against plant pathogens, have been reported to produce VOCs with antibacterial activity. For instance, a recent study revealed that VOCs produced by Bacillus spp., including benzaldehyde, 1,2-benzisothiazol-3(2 H)-one and 1,3-butadiene, had strong inhibitory activity against R. solanacearum, the causal agent of bacterial wilt disease. The mVOCs altered the transcriptional expression levels of several genes involved in motility and pathogenicity (e.g., global virulence regulator PhcA, type III secretion system, and extracellular polysaccharide [EPS] production) and induced systemic resistance by plants, which resulted in a decrease of wilt disease.

7Several reports describe the effect of VOCs in bacterial virulence. For instance, 2,3 butanediol and acetoin are required for full virulence in Pectobacterium carotovorum. The same compounds can increase the production of virulence factors in Pseudomonas aeruginosa.

8In contrast, VOCs produced by some bacteria can also have positive effects on the growth of other neighboring bacteria in the rhizosphere. For instance, VOCs from Collimonas pratensis and S. plymuthica are able to induce the growth of P. fluorescens Pf0-1. These VOCs induced expression of genes involved in motility in P. fluorescens Pf0-1 and provoked an increase in the production of secondary metabolites with antibacterial activity against Bacillus. This suggests that C. pratensis and S. plymuthica may be attracting and promoting the growth of P. fluorescens in a collaborative attempt to increase their chances against different bacterial competitors or soil fungal pathogens. Another example of the growth-promoting effect of VOCs was reported recently by which showed that VOCs released by mixtures of root exudate-consuming bacteria stimulated the activity and growth of distant nutrient-limited bacteria.

9In addition to exerting antagonistic effects toward other bacteria, VOCs can also modify the behavior of other bacteria and modulate their resistance to antibiotics. Bacterial volatiles such as ammonia, trimethylamine, hydrogen sulfide, nitric oxide, and 2-amino-acetophenone can alter biofilm formation or dispersal or affect motility of bacteria. Bacteria often make use of their motility to move to other areas with more resources and/or less competitors. In Streptomyces venezuelae, a new mode of development, so-called exploration, has been recently discovered that allows non-motile bacteria to access regions with more nutrients. S. venezuelae is able to produce hydrophilic fast growing non-branching vegetative hyphae, triggered by glucose depletion and a rise in pH, to presumably escape from poor nutrient areas. Interestingly, explorer cells can release signals for long distance communication with other members of the species to induce their exploratory growth. One of these signals is trimethylamine, which works not only as a signal to communicate with distantly located Streptomyces and induce exploratory growth but also displays antibacterial activity against B. subtilis and Micrococcus luteus, probably by rising the pH of the medium.

10Fungal VOCs can play an important role in long distance fungal–bacterial interactions and can lead to different phenotypical responses in the interacting partners. For example, VOCs emitted by Trichoderma atroviride increased the expression of a biocontrol gene (phlA) in P. fluorescens that encodes the biosynthesis of 2,4-diacetylphloroglucinol. A few recent studies demonstrated that the growth of some bacterial species can be suppressed by fungal VOCs such as the VOCs that exhibit inhibitory effects on B. cereus and B. subtilis produced by the oyster mushroom Pleurotus ostreatus.

11Recently, screened the phenotypic responses of soil bacterial strains to volatiles emitted by several fungal and oomycetal soil strains under different nutrient conditions during different growth stages. Out of the phenotypical responses tested such as growth alteration, antimicrobial activity, biofilm formation or motility, motility of bacteria (both swimming and swarming) was significantly positively or negatively affected by fungal and oomycetal VOCs. This finding could, therefore, reflect a potential strategy employed by the fungus to attract mutualistic bacteria toward itself and to repel competitors by manipulating their motility through the use of VOCs. Transcriptomics and proteomics analyses of S. plymuthica PRI-2C exposed to VOCs emitted by the fungal pathogen Fusarium culmorum, showed that S. plymuthica PRI-2C responded to the fungal VOCs with changes in gene and protein expression related to motility, signal transduction, energy metabolism, cell envelope biogenesis, and secondary metabolite production. The metabolomic analyses of S. plymuthica PRI-2C exposed to the fungal VOCs, the gene cluster comparison, and the heterologous co-expression of a terpene synthase and a methyltransferase revealed the production of the unusual terpene named sodorifen in response to fungal VOCs. These findings support the suggested importance of VOCs (and in particular terpenes) as signaling molecules in fungal–bacterial interactions.

12Many soil bacteria can produce VOCs with antifungal effects and thus contribute to the phenomenon known as soil fungistasis where fungal propagules are restricted in their ability to grow or germinate. Recently, revealed that VOCs produced by Streptomyces spp. exhibit antifungal properties against Rhizoctonia solani and may contribute to plant disease suppressiveness. showed that VOCs from the tomato rhizosphere isolate Pseudomonas donghuensis P482 have strong antifungal and anti-oomycete activity which suggests that the antagonistic capabilities of this strain against plant pathogens are due to their volatile potential. This effect of bacterial VOCs against oomycetes is not an isolated case and other Pseudomonas strains have been reported to have anti-oomycete activities. In a recent report, VOCs produced by several Lysobacter strains growing in a protein-rich medium showed anti-oomycete activity whereas non-antagonistic VOCs were produced by these strains when grown on a sugar-rich medium. This indicates that the production of volatiles is highly dependent on growth conditions and nutrient availability.

13The 1-octen-3-ol, one of the most prominent fungal VOC, known as the mushroom smell, is produced by a wide range of filamentous fungi and can function as a development signal among fungi. The same compound was described to function in Penicillium paneum as a self-inhibitor signal in spore germination. As developmental signals during population establishment, certain fungal VOCs act in a concentration-dependent manner to regulate conspecific mycelial growth and spore germination.

14Fungal VOCs can have inhibitory effects and drive antagonistic interactions among fungi. For example, the endophytic fungi Muscodor albus and Oxyporus latemarginatus can strongly inhibit the growth of several plant pathogenic fungi, including Botrytis cinerea and Rhizoctonia solani. VOCs emitted by Trichoderma spp. have a strong effect against plant pathogenic fungi such as Fusarium oxysporum, Rhizoctonia solani, Sclerotium rolfsii, Sclerotinia sclerotiorum, and Alternaria brassicicola. Similarly, VOCs such as 5-hexenoic acid, limonene, octanoic acid and 3,4-2H-dihydropyran produced by the non-pathogenic fungus F. oxysporum CanR-46 could inhibit mycelial growth of 14 fungal species including the pathogenic Verticillium dahlia. Recently, a proteomic study demonstrated that fungal VOCs can interfere with essential metabolic pathways to prevent fungal growth.

15Some fungal species can detoxify the antifungal compounds produced by their microbial competitors. For example, Fusarium graminearum can detoxify the toxic compound 6-pentyl-alpha-pyrone, emitted by Trichoderma harzianum. Fungal VOCs can be important carbon sources for fungi colonizing carbon-limited environments. Conversely, for fungi colonizing a more carbon-rich environment, VOCs may act, in a concentration-dependent manner, as semio-chemicals to mediate antagonistic and beneficial interactions between fungi.

16A very diverse and abundant group of soil microorganisms are protists (Protozoa). Due to their grazing activities, protists play an important role in the soil food web and significantly affect carbon allocation and nutrient-cycling in the soil-plant-interphase. Most soil protists are known to be key predators of bacteria and can shape bacterial communities by selective feeding. Reaching suitable prey is very energy consuming. Thus, sensing their prey over long distances in the porous soil matrix would be very beneficial for protists. A recent study by revealed that volatile organic compounds can play a key role in long-distance bacterial–protists interactions. By testing various volatile-mediated interactions between phylogenetically different soil bacteria and protists and comparing those with direct trophic interactions, they demonstrated that specific bacterial volatiles can provide early information about suitable prey. In particular, it was shown that terpenes such as β-linalool, β-pinene, germacrene D-4-ol or δ-cadinene produced by C. pratensis Ter91 can stimulate protist activity and motility suggesting that terpenes can be key components in VOCs-mediated communication between protists and bacteria. Interestingly, soil protists such as Dictyostelium discoideum produce volatile terpenes. These terpenes might be involved in defense mechanisms, for example, to repel nematode predators. Similarly, it was shown that soil bacteria can produce specific volatiles to repel protist predators.

17Besides bacterivorous protists, obligate and facultative mycophageous (fungus grazing) protists are common soil inhabitants. Mycophageous protists feed mostly on yeast and fungal spores while some specialists are able to graze directly on the hyphae of filamentous fungi. It is well known that soil fungi such as yeast produce a wide set of volatile compounds involved in various belowground interactions. Thus, although not demonstrated yet, it is plausible that fungal volatiles might play an important role in belowground communication between soil fungi and protists, as well.

18In recent years, evidence supporting the idea that plants respond strongly to mVOCs has grown. Most of the research carried out so far has investigated the impact of microbial VOCs on the model plant Arabidopsis thaliana. This has revealed that, without physical contact, microorganisms are able to drastically alter plant root system development, plant physiology, hormonal pathways, and biomass production. mVOCs can also function as a direct source of nutrients for plants, induce resistance to pathogens in plants, affect plant secondary metabolite production, directly inhibit plant pathogens and induce soil fungistasis and suppressiveness. Moreover, one single mVOC can show various functions, such as dimethyl disulfide, which improves plant growth by enhancing the availability of reduced sulfur. It also protects tobacco and corn plants against Botrytis cinerea and Cochliobolus heterostrophus by directly inhibiting pathogens and inducing systemic resistance in plants. Likewise, a characteristic compound of Trichoderma asperellum, 6-pentyl-pyrone, can increase plant defense reactions and at the same time decrease B. cinerea and Alternaria alternata sporulation.

19Many independent studies revealed that mVOCs emitted by beneficial soil microorganisms can affect plant growth but only few studies focused on how VOCs produced by soil-borne plant pathogens affect plant growth and development. These studies suggest that mVOCs from plant pathogens may modulate the trade-off between plant growth, development and defense. showed that VOCs emitted by pathogenic F. oxysporum promoted the growth of A. thaliana and Nicotiana tabacum and affected auxin transport and signaling. VOCs emitted by the pathogen Alternaria alternaria enhanced growth, early flowering and photosynthesis rates of Arabidopsis, maize and pepper by affecting the levels of plastidic cytocinin. A more recent study showed that the soil-borne pathogen Rhizoctonia solani produced an array of mVOCs that promote plant growth, accelerate development, change plant VOCs emission and reduce insect resistance. This must be a successful strategy for the pathogenic fungi since with increased root biomass and stimulation of lateral root formation there is a greater surface area for fungal colonization and infection.

20When analyzing mVOCs effects on plant growth, it is important to take into account, that microorganisms can produce high amounts of CO2 that can promote plant growth. Hence, a good experimental setup with appropriate controls are required to avoid artifacts in the results.

21Alternatively, plants are able to mediate the belowground plant–microbe interactions via root-emitted VOCs. Root-derived VOCs may serve multiple roles such as carbon sources, defense metabolites and chemo-attractants. Rhizobacteria such as Pseudomonas fluorescens and Alcaligenes xylosoxidans have been shown to metabolize α- pinene as their sole carbon source. also reported that bacteria associated with the roots of vetiver grass (Vetiveria zizanioides) use sesquiterpenes as a carbon source. Undoubtedly, plants and soil microorganisms are engaged via VOCs in long-distance interactions. However, so far, limited knowledge exists concerning the role of plant VOCs in attracting beneficial organisms and how plant-associated microorganisms affect the quantity and quality of plant volatile emission. Only recently, using a glass olfactometer system, the attraction of distant soil bacteria by VOCs emitted by plant roots was revealed. Olfactometer systems have been used successfully to study aboveground plant–herbivores interactions or belowground plant–nematode interactions. However, this is the first case to apply an olfactometer to study plant–microbe interactions. Moreover, the same study revealed that upon fungal infection, the blend of root VOCs changed and specific bacteria with antifungal properties were attracted.

22Several reports describe the chemical dialog between microbes, plants, and other organisms by the exchange of soluble compounds. Most of the studies reporting mVOCs-mediated communication belowground focus on the uni-directional responses and only a few studies reported on bi-directional mVOCs-mediated interactions. For instance, the importance of mVOCs in the dialog between the fungal plant pathogen Verticillium longisporum and its bacterial antagonist Paenibacillus polymyxa was recently revealed in both in vitro and in planta experiments. Both microorganisms responded to one another’s VOCs and this specific mVOCs-mediated interaction resulted in the inhibition of cellular metabolism and growth reduction of the fungal pathogen.

23A VOCs-mediated dialog between bacteria and fungi was also reported by where VOCs of the fungal plant pathogen Aspergillus flavus reduced the production of the major virulence factor EPS of the bacterial plant pathogen R. solanacearum. In parallel, A. flavus responded to VOCs of R. solanacearum by reducing conidia production and increasing aflatoxin production.

24Over the last decades, our understanding of the chemical complexity of mVOCs produced by many different soil microorganisms has grown. It is clear that these small and odorous molecules can modify the behavior and promote or inhibit growth of neighboring organisms (Figure 1).

25Most existing studies on mVOCs are focused on describing the uni-directional effect of mVOCs produced by a single organism and the responses of the organisms perceiving them without considering mVOCs-mediated dialog and the bi-directional responses to one another. Furthermore, microbial interactions taking place belowground are far more complex than single one-to-one interactions and involve more organisms, which can significantly affect mVOCs emission. For example, fungal-associated bacteria have been shown to affect the production of VOCs in fungi and in addition, they can affect the fungal plant-pathogenicity and repress the expression of fungal virulence genes. Therefore, a holistic approach considering the effect of mVOCs on belowground soil community is needed. For instance, using a metagenomics approach revealed that mVOCs could alter the composition of soil bacterial and fungal communities and significantly increased the relative abundance of Proteobacteria, Bacteroidetes, Firmicutes, and Ascomycota. Furthermore, mVOCs influenced genes involved in important soil functions such as N-fixation (nifH), nitrification (amoA), denitrification (nirS) and antibiotic production (NRPS).

26From the current scientific literature, it is clear that the most studied belowground mVOCs-mediated interactions are the interactions between bacteria, fungi and plants (Figure 1). There is a lack of knowledge relating to the emission of VOCs by protists, archaea or other rhizosphere organisms, such as nematodes or earthworms, indicating that these groups are currently understudied with regards to this aspect.

27Several VOCs are commonly produced and emitted by both plant roots, fungi, bacteria and protists and it is possible that these compounds act as a ‘lingua franca’ for intra- and inter-kingdom communication between these organisms. Let us take as an example only one chemical class, the terpenes. Terpenes are the largest and most diverse class of metabolites known to date. They are best known to humans as plants metabolites. However, recent studies revealed that terpenes can be produced by all kingdoms of life including prokaryotes. Recently, described a powerful bioinformatics method based on the use of Hidden Markov Models (HMMs) and Protein Families Database (PFAM) search that has allowed the discovery of terpene synthases of bacterial origin and showed that phylogenetically different bacteria can be a rich source of terpenes. Both the number, the wide distribution, and the structural diversity of terpenes provide enormous potential for mediating significant chemical interactions and communication belowground. Examples of terpene-mediated microbial interactions are presented in Figure 2 and Table 1, indicating the ecological importance of terpenes in interactions between soil micro- and macro-organisms, including plant roots.

28Despite the rapid increasing numbers of studies showing the importance of mVOCs in the long-distance belowground chemical interactions, we still do not know exactly how VOCs are recognized and perceived. VOCs receptors or other perception mechanisms have not been identified in any of the described cases. The big challenge is to determine whether VOCs are internalized and transduced by receptor-mediated processes, whether they interact with the cell membrane to initiate signal transduction cascades or whether they are simply taken up by the cell and metabolized. For plants, the current view is that due to their lipophilic nature, VOCs such as mono- and sesquiterpenes may interfere with membrane structures, thereby causing depolarization of the membranes and triggering Ca2+-signaling in plants. For further deciphering of mVOC-mediated microbe-microbe interactions, the mVOCs microbial perception mechanism needs to be elucidated. The application of methods for screening of mutant strains may be useful for that purpose, to identify microbial genes and proteins that are required for VOCs perception.

29Another big challenge is to determine what concentrations of mVOCs are produced in soil and at what distances these mVOCs are eliciting a biological response in other organisms. There is the possibility that, similar to the roles of antibiotics in nature, mVOCs could have concentration-dependent function either as weapons in intercellular chemical warfare or as signaling compounds when they are present in low concentrations.

30Concerning the implementations of mVOCs, our knowledge on the potential use of those compounds in large-scale agriculture and horticulture is still limited. In agriculture systems, mVOCs have to be applied under open-field conditions, which are very different from the in vitro conditions currently used in most studies. There are very few studies assessing the effects of mVOCs application under open conditions and they have been summarized in a recent review from. Since it was discovered that the 2,3-butanediol elicited plant growth and induced systemic resistance, several studies have applied this compound or the producing strains to the soil of open fields to test its effects under agricultural conditions and have revealed promising results. Dimethyl disulfide, frequently emitted by many bacteria, is another compound used in recent years in the novel soil fumigant PALADIN® that targets nematodes and soil-borne pathogens. However, the research concerning the application of other mVOCs in agriculture is still in its infancy. We now live in a time in which the old methods of using chemicals to protect crops need to be replaced with and, in some cases, complemented by green solutions. The traditional harmful synthetic fungicides currently used could be replaced with the so far under-explored and unique mVOCs for which significant proof of plant growth promoting effects and plant protection ability already exists. In spite of the obvious potential of mVOCs in agriculture, the field suffers from the common ‘translational gap’ because of a lack of studies evaluating other unexpected effects of those bioactive molecules on non-target beneficial soil organisms.

31All authors contributed and approved the manuscript.

32The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.