Keywords

8.1 Introduction

The plant rhizosphere is a highly competitive environment with plants releasing as much as 40% of their photosynthetic carbon through their roots with these secreted nutrients enabling the creation of a ‘hot-spot’ of microbial activity (including rhizobacterial activity) (Kai et al. 2016). Rhizobacteria enhance plant growth through a number of different mechanisms, some known, others as yet unknown (Velivelli et al. 2015). Over the past four decades these microbes have been commonly referred to as ‘Plant Growth-Promoting Rhizobacteria ’ (PGPR) (Kloepper and Schroth 1978; Ryu et al. 2005a). Different mechanisms are involved in the enhancement of plant growth by rhizobacteria. Some of these mechanisms are termed direct, others indirect. Examples of direct mechanisms can include the biosynthesis of chemicals analogous to plant hormones involved in the plant growth process such as indole-3-acetic acid (Shao et al. 2015). The optimisation of plant nutrient-uptake is also facilitated through phosphorus solubilisation (Oteino et al. 2015), nitrogen fixation (Singh 2014) and/or by modulating the levels of ethylene in the plant through the activity of enzymes such as aminocyclopropane-1-carboxylic acid (ACC) deaminase (Glick 2014). Indirect mechanisms include the synthesis of non-volatile antibiotics such as pyoluterin, surfactin and fengycin (Dimkić et al. 2017); competition for nutrients mediated by siderophore production for enhanced iron-uptake from soil (Ahmed and Holmström 2014); the secretion of lytic enzymes (e.g. chitinase, β-1,3-glucanase) and the modulation of plant immunity via activation of the induced systemic resistance (ISR) pathway (Compant et al. 2005; Lugtenberg and Kamilova 2009; Velivelli et al. 2015; Tahir et al. 2017a) Fig. 8.1. In recent years, there has been increased interest in the effects of rhizobacterial volatiles on plants (Weisskopf et al. 2016). The metabolic activity of the soil microbiota involves the synthesis of a broad variety of infochemicals, of which volatile organic compounds (VOCs) comprise a large proportion (Kanchiswamy et al. 2015; Velivelli et al. 2015). VOCs are characterised as having a relatively low molecular weight (<300 Da), a low boiling point and high vapour pressure (Vespermann et al. 2007; Velivelli et al. 2014). Microbial-emitted volatiles (mVOCs) belong to a number of different chemical classes including, but not limited to; alcohols, ketones, alkenes and terpenes (Schulz-Bohm 2017) and to date bacteria have been found to produce over 1000 VOCs (Sharifi and Ryu 2018a). The profile of VOCs emitted depends to a large extent on the external environment, be that soil properties or media components (Fincheira and Quiroz 2018). Infochemicals are of great importance, because volatiles can facilitate both the intra and inter-kingdom interaction between many organisms including plants and microbes (Farag et al. 2017). Due to their capacity to disperse in the atmosphere and to circulate through permeable soil structures, volatiles can exert their effects on plants above and below ground (Sharifi and Ryu 2018a). There are two types of VOCs—organic (e.g. 2,3-butanediol) and inorganic (e.g. HCN, CO2). The complex blends of VOCs that rhizobacteria are capable of generating have been the focus of numerous studies over the past decade (Yuan et al. 2017; Song and Ryu 2018; Tahir et al. 2017b; Blom et al. 2011a) These VOC blends can have beneficial or detrimental effects on the growth of plants, fungi and other associated organisms within the environment of the respective VOC-emitter (Effmert et al. 2012) Table 8.1. Exposure to BVCs can enhance plant growth under certain conditions, but can induce phytotoxic effects in others (Rath et al. 2018). Different blends of volatiles have been implicated in seed germination, flowering time and number, and in fruit and seed production (Sharifi and Ryu 2018a). A variety of chemical signalling molecules are produced by both rhizobacteria and plants when grown together, demonstrating that active communication exists between these kingdoms during plant development (Leach et al. 2017; Farag et al. 2017). For example, microbe-derived compounds are detected by plants, which can then adapt their defence and growth responses to specific types of microorganism. Furthermore, for the exploitation of BVCs in agronomical contexts, we need to determine both their activity and validity-for-use in the field (Rosier et al. 2018). Therefore, it is essential to have a comprehensive understanding of the biocontrol mechanisms of these agents so as to facilitate efficient and effective agronomic application.

Fig. 8.1
figure 1

Representation of interactions of bacterial volatile organic compounds (VOCs) on plants (reproduced with permission from Velivelli et al. 2014)

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Table 8.1 List of some of volatile-mediated effects of bacteria on plants, fungi and nematodes
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8.2 Rhizobacterial Volatiles and Plant Growth

The direct physical interaction between rhizobacteria and their respective plant host underpins most PGPR-plant interactions. However, an emerging field over the past number of years has examined the long-distance relationships which plants and rhizobacteria can achieve via the medium of VOCs (Velivelli et al. 2014). The first observation of this phenomenon was by Choong-Min Ryu and co-workers and this has led to the opening of many new avenues in the field of plant–microbe interactions. Where physical contact with the plant is not possible, certain rhizobacteria rely on the production of BVCs, a classic example being the volatile alcohol, 2,3-butanediol or more specifically its stereoisomer ‘2R, 3R- butanediol’ to stimulate plant development or activate ISR (Ryu et al. 2003, 2004; Lee et al. 2012; Fincheira and Quiroz 2018) Fig. 8.1. The identification of various plant growth promoting infochemicals and the determination of their structures and their associated functions have been ground-breaking moments in the study of plant-microbe interactions and pinning down the roles of VOCs in the intricate signalling systems between plants and rhizobacteria has been the key area of interest among a number of research groups (Bailly and Weisskopf 2017; Sharifi and Ryu 2018a). Nevertheless, the contribution of rhizobacterial VOCs to plant development and the importance of these compounds in agricultural systems are still topics of significant debate and speculation. In addition, only a handful of VOCs that are secreted by rhizobacteria have been identified to date. Therefore, a comprehensive understanding of the biological and ecological functions of BVCs—let alone a full understanding of their potential uses—has yet to be achieved.

However, according to Fincheira and Quiroz (2018), mVOCs can influence plant growth in at least four ways: 1. Modulation of nutrients; 2. Alteration of hormone levels; 3. Influencing plant metabolism and 4. Changing sugar concentrations.

Most importantly, future studies will need to address the types of responses and signalling cascades that are induced in plants by BVCs. Research focusing on the effects of plant exposure to different BVCs has uncovered a wide range of effects, including significant plant growth, the induction of ISR and even plant phytotoxicity. In particular, 2,3-butanediol (Ryu et al. 2003, 2004) dimethylhexadecylamine (Velázquez-Becerra et al. 2011), 2-pentylfuran (Zou et al. 2010), indole (Blom et al. 2011a; Yu and Lee 2013; Fincheira and Quiroz 2018) and dimethyl disulphide (DMDS) (Groenhagen et al. 2013), are amongst some of the infochemicals that have been shown to increase plant growth, whereas negative effects are at least in part due to the presence of high levels of hydrogen cyanide (HCN) (Blom et al. 2011b), DMDS and ammonia (Kai et al. 2010; Weise et al. 2013). To determine the extent to which BVCs stimulate plant growth, Choong-Min Ryu and co-workers used two-compartment Petri dishes, hereby by referred to as ‘I-plates’, to physically separate Arabidopsis thaliana from rhizobacteria under laboratory conditions. In this way, the dispersal of non-volatile metabolites through the medium was prevented, allowing for only the exchange of volatile organic compounds.

The researchers observed that plant growth was most strongly stimulated by the bacterial strains Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937a. When the volatiles produced by these two strains were examined, it was found that these bacteria produced the compounds 3-hydroxy-2-butanone (acetoin) and 2, 3-butanediol, which were not detected in bacterial strains that were unable to induce volatile-mediated plant growth. The exogenous application of these two compounds in pure solutions induced similar effects in a dose-dependent manner, and Bacillus spp. mutants defective in 2,3-butanediol and acetoin synthesis showed no plant growth-promotion, which confirmed the role of these compounds in mediating plant growth. A set of hormonal mutant A. thaliana lines impaired in specific regulatory pathways was then tested to identify the signalling networks necessary for these growth-promoting activities. It was found that exposure to volatiles from the B. subtilis GB03 strain did not promote growth in cytokinin receptor-deficient (cre1) or cytokinin/ethylene-insensitive (ein2) mutants. On the other hand, the B. subtilis GB03 volatiles did promote growth in ethylene-insensitive (etr1), auxin-transporter-deficient/ethylene-insensitive (eir1), gibberellic acid-insensitive (gai2), and brassinosteroid-insensitive (cbb1) mutants, suggesting that the promotion of growth elicited by GB03 VOCs is mediated by the cytokinin-signalling pathway (Ryu et al. 2003). Further experiments demonstrated that disease severity caused by the necrotrophic bacterial pathogen Erwinia carotovora subsp. carotovora was significantly decreased when A. thaliana seedlings were exposed to VOCs produced by B. subtilis GB03 and B. amyloliquefaciens IN937a. This phenomenon, called ‘induced systemic resistance’ (ISR), occurred in as little as 4 days. The exogenous application of pure 2,3-butanediol induced similar effects in a dose-dependent manner. Seedlings exposed to Bacillus mutants defective in 2,3-butanediol synthesis showed no disease protection, which confirmed the priming activity of this compound in ISR-induction. A set of mutant A. thaliana lines impaired in specific regulatory pathways, including a jasmonic acid (JA)-insensitive (coi1), an ethylene-insensitive (ein2), a salicylic acid (SA)-degrading line (NahG), and a line that is SA-insensitive or non-expressor of pathogenesis-related (PR) genes (npr1), was then tested to identify the signalling networks necessary for ISR. Pre-exposure to VOCs from B. subtilis GB03 did not trigger ISR in the ethylene-insensitive line (ein2) and did not show pathogen resistance. In addition, to further test whether these VOCs induced known signalling pathways in A. thaliana, transgenic plants with β-glucuronidase (GUS) fusions to Pr-1a (a gene activated by SA), Pdf1.2 (a gene activated by JA and ethylene), and Jin14 (a gene activated by JA) were exposed to VOCs released by B. subtilis GB03 (Ryu et al. 2004). Of these lines, the JA/ethylene-activated Pdf1.2-GUS line showed increased GUS activity compared with untreated control plants.

The plants carrying an ectopic copy of the JA-activated Jin14 gene were unaffected by B. subtilis GB03 VOCs; thus, ethylene signalling may be required for the activation of ISR in A. thaliana, independently of the SA and JA signalling pathways. Surprisingly, the VOCs of B. amyloliquefaciens IN937a functioned independently of all of the signaling pathways, indicating that some VOCs utilised alternative pathways to trigger ISR (Ryu et al. 2004), and are still not known. Meanwhile, a proteomics study revealed that ethylene biosynthetic enzymes were significantly up-regulated in A. thaliana plants exposed to B. subtilis GB03 VOCs, and a transcriptomic analysis showed the up-regulation of genes related to ethylene biosynthesis (SAM-2, ACS4, ACS12 and ACO2) as well as ethylene response genes (ERF1, CHIB and GST1) following exposure to B. subtilis GB03 VOCs (Kwon et al. 2010; Velivelli et al. 2015). It was demonstrated that the long-chain volatiles produced by Paenibacillus polymyxa E681 stimulated plant growth in A. thaliana and induced systemic resistance against Pseudomonas syringae pv. maculicola ES4326 (Lee et al. 2012; Velivelli et al. 2014).

A set of hormonal mutant A. thaliana lines impaired in specific regulatory pathways was tested to identify the signalling networks necessary for plant growth promoting activities. Lee et al. (2012) found that exposure to volatiles from P. polymyxa E681 did not promote growth in cytokinin/ethylene-insensitive (ein2) mutants. On the other hand, the P. polymyxa E681 volatiles did promote growth in jasmonic acid-insensitive line (coi1), a salicyclic acid-degrading line (NahG), and gibberellic acid-insensitive line (gai2) mutants, indicating that cytokinin/ethylene signalling is essential for the promotion of plant growth in response to P. polymyxa E681 volatiles. Further experiments demonstrated that the severity of the disease caused by the hemibiotrophic bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 was significantly decreased where A. thaliana seedlings were pre-exposed to VOCs produced by P. polymyxa E681. In addition, to further test whether P. polymyxa E681 VOCs induced known signalling pathways in A. thaliana, transgenic plants with β-glucuronidase (GUS) fusions to Pr-1a (a gene activated by SA), and Pdf1.2 (a gene activated by JA and ethylene), were exposed to VOCs released by P. polymyxa E681. Of these lines, the SA-activated Pr-1a-GUS line showed increased GUS activity compared with untreated control plants; thus indicating SA signalling may be required for the activation of ISR. A further transcriptomic study of A. thaliana exposed to VOCs from P. polymyxa E681 followed by pathogen challenge revealed the induction of salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signalling marker genes, PR1, ChiB, and VSP2, respectively. When the volatiles produced by P. polymyxa E681 were examined, it was found that this rhizobacteria produced the long-chain volatile compound tridecane. The exogenous application of pure tridecane induced PR1 and VSP2 in a dose-dependent manner after pathogen challenge; thus indicating that SA/ET signalling is essential for the activation of ISR in response to tridecane. The researchers performed additional tests to demonstrate whether the observed growth promotion of A. thaliana when exposed to P. polymyxa E681 was correlated to carbon dioxide (CO2). Plant growth was still enhanced when exposed to barium hydroxide (Ba(OH)2) which traps CO2, indicating that some other unknown VOCs are involved in the promotion of growth (Lee et al. 2012; Jeong et al. 2019).

In addition to the impact of BVCs, it has been speculated that increased plant growth could be due to the increase in CO2 concentrations that is seen to rise when using the sealed petri dish method. Based on co-cultivation studies involving Arabidopsis and Serratia odorifera in a closed system, it was observed that growth promotion was closely linked to carbon dioxide enrichment. In particular, the growth of A. thaliana was stimulated in a closed system, where carbon dioxide was the dominant component of the volatile mixture (390–3000 ppm). By contrast, in an open system under ambient carbon dioxide concentrations, volatiles with negative effects on plant growth became dominant. It is possible that activation of the tricarboxylic acid cycle (TCA) triggers the emission of carbon dioxide, although this molecule does not accumulate to higher-than-ambient concentrations in an open system (Kai and Piechulla 2009). Similarly, the growth of Physcomitrella patens (moss) was stimulated in a closed system in which carbon dioxide was the dominant component of an S. odorifera-derived volatile mixture, growth of this moss was inhibited in an open system due to the negative influence of volatiles (Kai and Piechulla 2010). At most, high levels of carbon dioxide have been observed to increase plant biomass by as much as 25%, although this was primarily the result of increased starch accumulation rather than biomass expansion. However, it is likely that volatiles are present at much higher concentrations in closed systems than in any found under natural circumstances (Van der Kooij et al. 1999; Ward and Strain 1999; Blom et al. 2011a).

In another study, C16 hexadecane, a long-chain hydrocarbon emitted by P. polymyxa E681, also protected Arabidopsis plants from infection by the necrotrophic pathogen Pectobacterium carotovorum and the hemibiotrophic pathogen Pseudomonas syringae pv. maculicola ES4326 (Park et al. 2013). Certain volatiles produced by rhizobacteria regulate plant auxin homeostasis, and can promote growth in Arabidopsis. Genes for auxin biosynthesis were up-regulated when aerial parts of the plant were exposed to B. subtilis GB03. As observed in a transgenic (DR5:auxin-responsive reporter) Arabidopsis line expressing a DR5::GUS fusion, exposure to B. subtilis GB03 volatiles induced a decrease in auxin accumulation in leaves and an increase in roots, indicative of basipetal auxin transport activation. The decrease in auxin accumulation in leaves resulted in enhanced leaf cell elongation, whereas the increase in auxin accumulation in roots led to the development of lateral roots. Thus, despite the fact that auxin is not produced by B. subtilis GB03, auxin signalling must be present in the root architecture response elicited by one or more BVC produced by B. subtilis GB03. Auxin accumulation to the sites of synthesis was impeded by the application of the auxin transport inhibitor 1-napthylphthalamic acid (NPA), which prevented the B. subtilis GB03-induced reduction in shoot auxin levels and the associated growth promotion. Moreover, modifications in cell wall-loosening were observed during transcriptional analysis, which might explain the accelerated cell expansion and leaf growth associated with exposure to B. subtilis GB03 volatiles (Zhang et al. 2007). Further experiments revealed that exposure to B. subtilis GB03 volatiles caused Arabidopsis to increase both its photosynthetic activity and chlorophyll content.

Exposure to BVCs also enhanced endogenous sugar accumulation and also led to the partial suppression of sugar sensing in plants. In contrast to wild-type plants, enhanced photosynthetic capacity (that was not additionally increased by exposure to B. subtilis GB03) was observed in the two glucose-insensitive Arabidopsis mutants, gin1 and gin2, which lack hexokinase-dependent sugar signalling. Photosynthesis is promoted by BVCs through repression of the hexokinase-dependent sugar signalling pathway. Exposure to B. subtilis GB03 causes an overlap in sugar/ABA sensing in plants, as ABA-synthetic transcripts, ABA-responsive genes and ABA levels in leaves become reduced. Furthermore, the increase in photosynthetic efficiency and chlorophyll content induced by B. subtilis GB03 can be abolished by exogenous ABA treatment. Therefore, to enhance photosynthesis, some modulate endogenous sugar/ABA signalling and use soil symbionts as regulators of energy procurement by plants (Zhang et al. 2008b). Common photosynthetic markers, which are enhanced by high carbon dioxide levels, include increase of photosynthetic efficiency, chlorophyll content, and sugar accumulation (Kai and Piechulla 2009). Indeed, Arabidopsis exhibited enhanced photosynthetic capacity (e.g. chlorophyll content) and iron accumulation following protracted exposure to B. subtilis GB03 (Xie et al. 2009). Under normal growth conditions, B. subtilis GB03 volatiles triggered a rise in the mRNA levels of Fe-deficiency-induced transcription factor 1 (FIT1), as well as two of its downstream targets, ferric reductase FRO2 and the iron transporter gene IRT1. However, in Arabidopsis fit1-2 knockout mutants, volatile-induced increases in iron assimilation and photosynthetic efficiency are impaired, suggesting that volatile-induced iron assimilation is mediated by FIT1 (Zhang et al. 2009). To stimulate iron assimilation, Sinorhizobium meliloti VOCs induced acidification of the Medicago truncatula rhizosphere, which triggers enhanced photosynthetic activity (e.g. chlorophyll content), an indicator of nutritional Fe status in plants (Carmen Orozco-Mosqueda et al. 2013). B. subtilis GB03 volatiles enhance salt tolerance through the sodium transporter HKT1, which is down-regulated in roots and up-regulated in shoots, resulting in a plant-wide reduction in Na+ accumulation versus control plants not exposed to GB03 volatiles (Zhang et al. 2008a). It was demonstrated that tolerance to abiotic stress could be induced in Arabidopsis by Pseudomonas chlororaphis O6 and the results suggested that this phenomenon was largely due to production of the volatile compound 2,3-butanediol. A P. chlororaphis O6 mutant defective in 2,3-butanediol production showed no drought resistance upon bacterial colonisation, which confirmed the role of this compound in induction of drought tolerance.

Interestingly, it was shown that 2,3-butanediol produced by this rhizobacterium facilitated both stomatal closure and drought tolerance through an ABA-1- and OST-1 kinase-dependent manner. A set of mutant A. thaliana lines impaired in specific regulatory pathways, including a mutant with reduced ABA synthesis (aba1) and a mutant deficient in the protein kinase mediating stomatal regulation in response to drought (ost-1) showed no drought tolerance upon P. chlororaphis O6 root colonisation. When drought-stressed plants were exposed to 2,3-butanediol, the plants accumulated greater levels of SA than unexposed plants, indicating that the SA signalling pathways are involved in P. chlororaphis O6-induced drought tolerance (Cho et al. 2008). Arbidopsis plants treated with Bacillus subtilis FB17 significantly reduced the severity of the disease caused by the hemibiotrophic bacterial pathogen Pseudomonas syringae pv. tomato DC3000. This phenomenon, called ‘induced systemic resistance’ (ISR), also occurred when Arabidopsis plants were exposed to acetoin. B. subtilis FB17 mutants defective in acetoin biosynthesis showed reduced disease protection, and this result confirmed the priming activity of this compound in ISR. A set of mutant A. thaliana lines impaired in specific regulatory pathways, including a jasmonic acid (JA) mutant (jar1-1), an ethylene mutant (etr1-3), a salicylic acid (SA) deficient mutant ( NahG ), and a line that is SA-insensitive or non-expressor of pathogenesis-related (PR) genes (npr1-1), was then tested to identify the signalling networks necessary for ISR. Of these lines, treatment with B. subtilis FB17 and acetoin did not trigger ISR in the etr1-3, NahG and npr1-1 lines and did not show the pathogen resistance against Pseudomonas syringae pv. tomato DC3000; thus indicating that ISR elicitation is mediated via NPR1 and SA/ET signalling pathways to activate ISR in Arabidopsis independently of JA signalling pathway (Rudrappa et al. 2010).

In species such as B. subtilis, 2,3-butanediol synthesis is mediated by the transformation of pyruvate into acetocholate by the enzyme ‘acetocholate synthase’. Following this, acetocholate decarboxylase converts alpha-acetocholate into acetoin which is subsequently converted to 2,3-butanediol via catalysis mediated by the acetoin reductase/2,3-butanediol dehydrogenase (AR/BDH) (Nicholson 2008).

The conversion of glucose into 2,3-butanediol and acetoin occurs under hypoxic conditions and serves as an electron sink for the generation of NAD+ when aerobic respiration is restricted. Furthermore, low partial oxygen pressure (as generally exists in soil surrounding roots) induces the bacterial acetoin pathway that controls the production of 2,3-butanediol. Consequently, it is likely that 2,3-butanediol and/or other biologically active molecules are produced by certain root-colonising rhizobacteria at concentrations appropriate to elicit plant reactions (Ryu et al. 2003, 2004). The Methyl Red Voges Proskauer (MR-VP) medium is generally used to determine the ability of bacteria to ferment 2,3-butanediol (Nicholson 2008). As a by-product of the fermentation pathway employed by some rhizobacteria to avoid acidification, the biosynthesis of 2,3-butanediol is often induced on low pH Murashige and Skoog (MS) medium containing sucrose (Ryu et al. 2003). The growth of Penicillium spp. was suppressed both in vitro and in vivo by volatiles released by Bacillus spp., and citrus fruit inoculated with Penicillium crustosum showed reduced disease incidence and severity due to the presence of acetoin. Furthermore, it was observed that longer exposure times led to stronger volatile-mediated antifungal effects; this was attributed to the extended incubation period within the closed system, leading to the restriction of oxygen over time (Arrebola et al. 2010).

On the other hand, it was also demonstrated that the acetoin pathway is optimal in the lifecycle of the necrotrophic bacterial pathogen Pectobacterium carotovorum subsp. carotovorum WPP14. Mutants defective in the 2,3-butanediol pathway were unable to alkalinise growth media and also showed reduced virulence on potato tubers (Marquez-Villavicencio et al. 2011). Furthermore, the capacity of Bacillus megaterium XTBG34 to promote growth in A. thaliana was validated by Zou and Co-workers. In particular, a number of compounds produced by this organism, including 2-pentylfuran, were identified by GC/MS analysis, and they showed that plant growth was significantly enhanced by 2-pentylfuran in a dose-dependent manner. The lowest dose at which this compound could enhance plant growth was 0.1 µg, and maximum growth was achieved with 10 µg; by contrast, doses greater than 10 µg inhibited growth (Zou et al. 2010). Santoro and Co-workers examined the effects of BVCs on growth promotion and the enhanced biosynthesis of essential oils (EO), such as pulegone and menthone in Mentha piperita (peppermint). The results of this study indicated that BVCs exhibit species-specific effects on plants. BVCs not only trigger secondary metabolite production but also impact pathway flux during certain stages of monoterpene metabolism (Santoro et al. 2011). It was demonstrated that volatiles released by Proteus vulgaris JBLS202 stimulate growth in Chinese cabbage and GC/MS analysis showed that indole was the primary headspace volatile compound produced by this bacteria. They showed that plant growth was significantly enhanced by indole in a dose-dependent manner. When plants were exposed to 0.63 µg of synthetic indole, growth was significantly enhanced. Indole and its derivatives are known to be involved in the auxin signalling pathway (Yu and Lee 2013).

Blom and colleagues analysed the effects of different bacterial strains cultured on four distinct media on the growth of A. thaliana . Of the bacterial strains tested, one strain promoted growth on all four media tested. GC/MS analysis revealed the presence of a range of compounds, including indole, 1-hexanol and pentadecane, and they showed that plant growth was affected in a concentration-dependent manner by these compounds. Indole promoted growth at low concentrations but showed lethal effects when used at high concentrations. Furthermore, when 1-hexanol was applied in moderate amounts, it showed weak growth promotion, and pentadecane promoted growth when applied at high concentrations (Blom et al. 2011a).

In another study, rhizobacterial strains were isolated from the rhizosphere of lemon plants (Citrus aurantifolia) and then analysed to determine whether their VOCs had an effect on the development of A. thaliana roots. Using a simple experimental system involving I-plates, the authors observed several morphological changes in root architecture due to VOCs. It is interesting to note that some rhizobacterial strains stimulated primary root growth and lateral root development. Several compounds were detected by GC/MS, including aldehydes, ketones and alcohols. However, short-chain alcohols, such as 2,3-butanediol and acetoin were not identified in this study, indicating that other VOCs can also trigger plant growth (Gutiérrez-Luna et al. 2010). Exposure to A. thaliana plants with Bacillus megaterium UMCV1 modified the architecture of the root system in this plant. In particular, the authors observed an inhibition of primary root growth as well as increases in lateral root number, lateral root growth, and root hair length, and they found that reduced cell elongation and cell proliferation in the root meristem was the cause of the inhibition of primary root growth. The analysis of Arabidopsis mutant lines defective in either ethylene (etr1 and ein2) or auxin (aux1-7, axr4, eir1) signalling revealed that, modifications in root architecture caused by B. megaterium UMCV1 may involve either auxin- or ethylene-independent mechanisms. Furthermore, transgenic Arabidopsis line expressing a DR5:uidA (a reporter line for auxin and ethylene-inducible gene expression) GUS fusion showed reduced expression in root tips (López-Bucio et al. 2007).

Velázquez-Becerra and Co-workers tested the effects of Arthrobacter agilis UMCV2 volatiles on alfalfa ( Medicago sativa ), and they found that A. agilis volatiles decreased taproot growth and increased lateral root formation, indicating that the BVCs emitted by this bacterium play an important role in root development. Analysis of BVCs produced by this organism revealed a range of compounds, at least one of which, N-N-dimethyl-hexadecanamine, may act as a growth-promoting trigger, affecting root development in Medicago sativa in a dose-dependent manner (Velázquez-Becerra et al. 2011).

A study by Tahir and colleagues determined the effect of BVCs from Bacillus subtilis SYST2 on tomato was examined. Two compounds, albuterol and 1,3-propanediol were identified as having a positive effect on plant growth with observed increases in auxin and cytokinin in the plant tissues and noticeable increases in expansin gene transcripts. Variations in VOC concentrations and/or plant exposure times can dictate whether an inoculation has positive or negative effects on primary root growth and/or lateral root formation (Tahir et al. 2017b). Arabidopsis plants exposed to Burkholderia ambifaria volatiles show enhanced biomass, greater numbers of secondary roots and shorter main roots. Analysis of the VOCs produced by this bacterium revealed a range of compounds, including dimethyl disulphide (DMDS ), acetophenone and 3-hexanone, 4-methyl-2-pentanone, 4-octanone, and 2,5-dimethyl pyrazine. These compounds were shown to affect plant growth in a concentration-dependent manner, and indeed, treatment with very high amounts could inhibit plant growth. Plants showed greater biomass when exposed to some concentrations of dimethyl disulphide (DMDS), acetophenone and 3-hexanone. By contrast, high concentrations of 4-methyl-2-pentanone and 4-octanone were lethal to the plants. Finally, 2,5-dimethyl pyrazine promoted growth at lower concentrations, whereas higher concentrations were deleterious to Arabidopsis plants (Groenhagen et al. 2013).

Many studies have shown that BVC interact with the host root system and in addition to its role as a structural support for the plant, it is also crucial for the acquisition of water and nutrients from the soil. From an ecological perspective, BVC-mediated alterations in the root system proteome and root architecture may have beneficial effects through increasing bacterial root colonisation and optimising symbiotic interactions (Yaoyao et al. 2017). These symbiotic relationships mediate biochemical interactions which stimulate root growth and development which leads to enhanced levels of bioavailable nutrients for the plant (Velivelli et al. 2015; Hérnandez-Calderón et al. 2018). In reciprocation, PGPR gain access to richer sources of nutrition and carbon through root exudates produced by a healthy plant host (Gutiérrez-Luna et al. 2010; Velivelli et al. 2015).

8.3 Inhibitory Effects of Rhizobacterial Volatiles on Plants

In addition to promoting plant growth, it has been demonstrated that certain BVC have negative effects on plants such as A. thaliana (Vespermann et al. 2007). One of the most important sources of nitrogen is ammonia, although this compound has recently been shown to play a number of other biological roles. When S. odorifera 4Rx13 is grown on a peptone-rich medium, it produces high levels of ammonia, and when this plant was exposed to A. thaliana plants in an I-plate, the bacterium caused the neighbouring plant medium to become alkalinised, leading to reduced plant growth (Weise et al. 2013). Under conditions of low oxygen, such as in a closed system, the production of HCN by some Pseudomonas sp. is enhanced (Athukorala et al. 2010). Deleterious effects were observed when A. thaliana was exposed to HCN, with a four-fold reduction in growth following exposure to 1 µmol HCN (Blom et al. 2011b). In has also been shown that rhizobacterial volatile compounds such as ammonia and DMDS have negative effects in higher concentrations on A. thaliana growth (Kai et al. 2010).

8.4 Effect of Rhizobacterial Volatiles on Fungi and Other Organisms

For truly sustainable agriculture, the strategies we employ to combat plant diseases must become more environmentally-friendly with lower inputs of synthetic chemicals. The use of beneficial microbes as a biological input to sustainable agricultural systems offers an alternative, and potentially more environmentally stable approach, to conventional agri-chemical-based solutions for the suppression of plant pathogens and the treatment of plant diseases in an integrated pest management system (Velivelli et al. 2015). Despite their powerful antifungal activities, non-volatile antibiotics are unable to spread over long distances, making them only effective at preventing infection by pathogenic microbes/fungi when applied directly to plant roots. The ability of BVC to suppress the growth and proliferation of plant pathogens has attracted ample attention with regard to biological applications and rhizobacterial VOCs have displayed antagonistic activity against pathogenic fungi, which may classify them as novel antibiotic compounds (Velivelli et al. 2015). The best known example of one such inorganic volatile metabolite is hydrogen cyanide (HCN).

Hydrogen cyanide (HCN) is a secondary metabolite produced by some gram-negative Pseudomonas spp. upon the hydrolysis of glycine by HCN synthase. Pseudomonas fluorescens CHA0 was shown to inhibit the development of Thielaviopsis basicola, which causes black root rot in tobacco plants, through the production of HCN (Bailly and Weisskopf 2017). The biocontrol potential of these antibiotics has been experimentally validated through the use of mutant rhizobacterial strains with altered antibiotic production. A hydrogen cyanide negative mutant (hcn), P. fluorescens CHA0 strain was no longer able to protect tobacco against black root rot (Voisard et al. 1989; Blumer and Haas 2000). A more recent study (Rijavec and Lapanje 2016) proposed that the main contribution of HCN to biocontrol is more indirect and is related to the sequestration of metals and the associated beneficial increase of nutrients to the plant and rhizobacteria.

The volatile inorganic compound ammonia, which is released by the rhizobacteria Enterobacter cloacae, suppressed the growth of Pythium ultimum in dual-culture assays, thus describing its possible role in the biological control of Pythium pre-emergence damping-off (Howell et al. 1988). In addition to HCN and ammonia, the antifungal nature of the organic volatiles has been demonstrated in several experiments. Pseudomonas spp. isolated from canola and soybean plants were reported to produce volatile antibiotics including; n-decanal , nonanal, 2-ethyl-1-hexanol, benzothiazole and dimethyl trisulfide, that inhibit the fungal pathogen Sclerotinia sclerotiorum in I-plate assays (Fernando et al. 2005). Furthermore, the growth of S. sclerotiorum was also suppressed in antifungal bioassays performed in sealed plates containing pure synthetic volatiles such as furfural, benzaldehyde, 1-octanol, 1-octen-3-ol, 3,7-dimethyl-1-ol, 6-octadien-3-ol, 2-ethyl-1-hexanol (Liu et al. 2009).

The growth of Fusarium oxysporum f. sp. cubense was suppressed in a divided plate assay by BVC produced by Bacillus amyloliquefaciens NJN-6. The volatile organic compounds emitted by this organism were diverse and included; benzothiazole, phenol, 2,3,6-trimethyl-phenol, 2-ethyl-1-hexanol, 2-undecanol, 2-nonanone, 2-decanone, nonanal, naphthalene, naphthalene, 2-methyl and naphthalene 1-methyl. The application of pure, synthetic volatiles in the same bioassay revealed strong antifungal activities against F. oxysporum f. sp. cubense (Yuan et al. 2012).

Jasmonic acid BVC produced by B. subtilis significantly inhibited the spore germination of B. cinerea in an I-plate assay. An analysis of VOCs revealed a range of compounds, such 4-Hydroxybenzaldehyde, 2-nonanone, Ammonium acetate, 1,2,4,5-Tetramethyl-pyrazine, 9-Methyl-nonadecane, 2,6,11,15-Tetramethyl-hexadecane, 2,6,10,15-Trimethyl-tetradecane and 8-Hexyl-pentadecane; however, the authors did not evaluate the antagonistic potential of these compounds against Botrytis cinerea (Chen et al. 2008). The BVC 2-nonanone showed an inhibitory effect towards B. cinerea fungal decay of strawberries in closed containers, thus suggesting its potential role in reducing post-harvest diseases of agricultural products, an area in which there is a significant increase in research activity worldwide (Almenar et al. 2007; Sharifi and Ryu 2018b).

The growth of the soil-borne pathogenic fungi Rhizoctonia solani was strongly inhibited in an I plate assay by BVC emitted by a number of common soil bacterial genera such as Bacillus spp., Pseudomonas spp., Serratia spp., and Stenotrophomonas spp. Further molecular analysis revealed a wide array of compounds including: β-phenylethanol, trans-9-hexadecene-1-ol, undecene, undecadiene, dodecanal, benzylnitrile, benzyloxybenzonitrile, and dimethyl trisulfide (Kai et al. 2007). Dimethyl disulphide was shown to inhibit the growth of Fusarium culmorum in a dual-culture assay and this inhibitory effect on mycelial growth was observed to occur in a dose-dependent manner; less obvious effects were also observed with the use of pure 1-undecene (Kai et al. 2009). Antifungal volatile metabolites produced by A. agilis UMCV2 inhibited the growth of B. cinerea on sealed plates. The volatile organic compound, dimethylhexadecylamine (DMHDA), which is released by the rhizobacteria A. agilis, inhibited the growth of both B. cinerea and P. cinnamomi in dual-culture assays when provided to the culture medium at low concentrations (Velázquez-Becerra et al. 2013). It has been demonstrated that BVC produced by Streptomyces plantesis F-1 could inhibit the growth of R. solani, B. cinerea and S. sclerotiorum. Exposure to S. plantesis F-1 BVC significantly reduced the incidence and severity of leaf blight/seedling blight caused by R. solani, leaf blight of oilseed rape caused by S. sclerotiorum and fruit rot of strawberry caused by B. cinerea; thus indicating its possible role as a biofumigant in the biological control of fungal diseases. The analysis of volatile organic compounds from this organism revealed diverse compounds, including but not limited to phenylethyl alcohol, phenol, 2,5-bis(1,1-dimethylethyl)-, (+)-epi-bicyclesesquiphellandrene and cyclohexane carboxylic acid; however, the potential role of these compounds remains to be investigated. As suggested by the complex nature of BVC, significant growth inhibition may require the synergistic activity of multiple compounds or the activity of extremely potent infochemicals normally present at low concentrations (Wan et al. 2008). It has been demonstrated that the volatile metabolites produced by fungistatic soils suppress the growth of Paecilomyces lilacinus, Pochonia chlamydosporia and Clonostachys rosea. In particular, VOCs produced by fungistatic soils, including trimethylamine, benzaldehyde, and N,N-dimethyloctylamine elicit strong antifungal activity in a sealed petri plate assay containing known amounts of fungal spore suspension, autoclaved soil and/or pure synthetic compounds (Chuankun et al. 2004).

In an I plate assay, the volatile organic compound, O-anisaldehyde, which is released by Bacillus atrophaeus CAB-1 inhibited the growth of B. cinerea; thus suggesting its possible role in soil fungistatis and the subject of further study (Zhang et al. 2013). The pathogenic activity of various fungal pathogens was suppressed by volatiles emitted by P. polymyxa strain BMP-11. The mycelial growth of various fungal pathogens, including R. solani, was suppressed in sealed Petri dish antifungal bioassays involving pure, synthetic volatiles such as 1-octen-3-ol, benzothiazole and citronellol; mycelial morphological deformities were also observed (Zhao et al. 2011). Antifungal metabolites produced by B. subtilis inhibited the growth of two strains of R. solani in sealed I plate assays. Interestingly, these two fungal strains reacted differently to exposure to the B. subtilis BVC mixtures, indicating that BVC-mediated interactions between bacteria and fungi can be species/genus specific. Therefore, it is possible that complex volatile mixtures may trigger significantly different responses in different fungi, which may for example be due to differentially conserved molecular activities of fungi for detoxifying metabolites.

Fungal growth modifications due to BVC exposure are not uncommon and indeed are closely linked to growth medium and inoculum dose, and in soil, volatile emissions have been linked to nutrient availability, pH, temperature and oxygen availability (Schulz-Bohm 2017). Rhizobacterial strains emit volatiles that can have distinct effects on fungi or inhibit different fungal types to varying degrees. This could be due to the fact that different BVC blends are released by various rhizobacteria, and inhibitory effects may be induced due to the synergistic effects of several compounds within those respective blends (Velivelli et al. 2015). For example, the fungistatic BVC emitted by Bacillus cereus were shown to more strongly repress a Trichoderma viride strain than a Gelasinospora cerealis strain. Similarly, the BVC released by a strain of Aerobacter aerogenes were not as effective against F. oxysporum f. sp. conglutinans as they were against T. viride and Penicillium sp. (Fiddaman and Rossall 1994).

Using an I plate assay, it was shown that exposure to Burkholderia ambifaria VOCs reduced the growth of fungi, and this inhibitory effect was observed to be stronger against Alterneria alternata than it was for R. solani. In particular, the growth of A. alternata and R. solani was decreased by higher doses of dimethyl trisulphide, 2-nonanone, 1-phenyl-1,2-propanedione, and 2-undecanone. Moreover, R. solani was also suppressed by acetophenone, phenylpropan-1-one, DMDS, 4-octanone and S-methyl methanthiosulphonate. The growth of Fusarium solani was not reduced by any of the volatiles tested, indicating that fungi react to BVCs differently (Groenhagen et al. 2013; Velivelli et al. 2015).

Bacterial volatile patterns can also be affected by growth media. For example, BVC emission profiles differed based on whether the media (nutrient broth) they were grown on contained glucose. The growth of R. solani on nutrient broth (NB) was more strongly suppressed by the volatiles produced from Xanthomonas campestris pv. vesicatoria 85-10 when the latter was grown on NB than when it was grown on nutrient broth with glucose (NBG). A. nidulans and F. solani exhibited similar growth patterns. The inhibition of A. nidulans was stronger when X. campestris pv. vesicatoria 85-10 was grown on NB than when it was grown on NBG. When X. campestris pv. vesicatoria 85-10 was grown on NB, the growth of A. nidulans and F. solani was inhibited by 85% and 14%, respectively, whereas growth inhibition on NBG was only 11% and 3.5%, respectively. The BVCs of X. campestris pv. vesicatoria 85-10 showed only weak effects on F. solani, indicating species-specific activity. These results demonstrate that when grown on glucose-containing media, X. campestris pv. vesicatoria 85-10 BVCs have only a weak effect on fungi, indicating that nutrient levels influence growth inhibition. There are several potential explanations for the observed reduction in growth suppression by X. campestris pv. vesicatoria when grown on NBG: (1) growth on NB media stimulates the production of a larger amount of suppressive volatiles; (2) the production of suppressive volatiles relies on peptone-rich NB media; (3) the production of suppressive volatiles is restricted by glucose through a mechanism such as catabolite repression; or (4) there is a delay in the production of suppressive volatiles on NBG media (Weise et al. 2012). Fiddaman and Rossall (1994) observed that B. subtilis only produced suppressive BVCs in the presence of D-glucose, and not L-glucose (Fiddaman and Rossall 1994). Previously, Fiddaman and Rossall (1993) had assumed that agar containing high levels of glucose, i.e. PDA (potato dextrose agar) and SGA (Sabouraud’s glucose agar) with B. subtilis would stimulate significant antifungal activity in vitro. By contrast, limited or no in vitro antifungal activity was produced by agar containing little or no glucose (VJA (V8 juice agar), NA (nutrient agar) or 10% TSA (tryptic soy agar) (Fiddaman and Rossall 1993).

Serratia plymuthica strains emitting DMDS as the primary headspace BVC, were seen to inhibit the growth of Agrobacterium tumefaciens and Agrobacterium vitis strains in dual-culture assays. When Solanum lycopersicum plants were inoculated with S. plymuthica, it inhibited the growth of Agrobacterium, and resulted in the emission of DMDS by the S. lycopersicum plants (Dandurishvili et al. 2011). It was demonstrated that S. plymuthica HRO-C48 inhibited the growth of R. solani using I plates (Kai et al. 2007). When oilseed rape cv. Talent was treated with S. plymuthica HRO-C48, disease severity of Verticillium dahlia was significantly reduced. This strain also produces DMDS and is registered and distributed by RhizoStar®, E-nema GmbH Raisdorf, Germany (Müller et al. 2009). This strain was shown to suppress V. dahliae in strawberry and R. solani in lettuce (Kurze et al. 2001; Grosch et al. 2005). The development of DMDS has been targeted as a possible alternative to the fumigant methyl bromide. DMDS was observed to supress phytopathogenic nematodes and fungi in the soil, offering strong evidence for the effect of DMDS against plant-pathogenic fungi (Fritsch 2005) and nematodes (Coosemans 2005). Based on these findings, rhizobacteria that produce DMDS can be classified as natural fumigants and this biocontrol capacity is closely associated with VOC production. Therefore, antifungal VOCs should be considered as significant tools for the control of plant pathogens, in addition to more conventional agrichemicals. At present, Paladin®, a new and effective soil fumigant based on DMDS, had already been registered in the USA, (http://www.arkema.com/en/media/news/news-details/Arkema-receives-U.S.-registration-for-Paladin-soil-fumigant-00001/) and the development of new soil fumigants based on dimethyl disulphide (DMDS) is progressing in Europe.

The growth of F. oxysporum was suppressed in an I-plate assay by the BVCs produced by endophytic bacteria. A range of compounds was observed during the analysis of these volatiles, such as 2-pentanone 3-methyl, methanethiol and 3-undecene. However, the authors did not evaluate the antifungal properties of the individual compounds on F. oxysporum (Ting et al. 2011). It has been demonstrated that the volatile metabolites produced by endophytic Burkholderia tropics strains inhibited the growth of four phytopathogenic fungi: Colletotrichum gloeosporioides, Fusarium culmorum, F. oxysporum and Sclerotium rolfsii. Further analysis revealed the existence of numerous compounds, including α-pinene, ocimene, limonene and fencona, indicating that these compounds may also play important antifungal roles (Tenorio-Salgado et al. 2013). Considering the effects of BVC on the development of fungi and plants, an obvious question is whether these effects extend to animals living within the soil as well.

In an I-plate assay, volatiles released by Bacillus megaterium YMF3.25 were shown to inhibit the hatching of nematode eggs and reduce nematode infections. GC-MS analysis revealed a variety of compounds, such as benzeneacetaldehyde, 2-nonanone, decanal , 2-undecanone and DMDS that exhibited strong nematicidal activities against both juveniles and eggs. Additional research will be required to create integrated management systems for lowering root-knot nematode inocula and enhancing crop yields in the field, perhaps by improving bacterial formulation. (Huang et al. 2010).

An analysis of Lysinibacillus mangiferahumi BVCs showed nematicidal activity against the root-knot nematode Meloidogyne incognita. Volatile analysis revealed the presence of numerous compounds, including 2-octanol, cyclohexene, 3-chloro-4-fluorobenzaldehyde, dibutyl phthalate, 2-nitro-2-chloropropane, dimethachlore, and DMDS. When nematodes were exposed to these pure volatiles in a three-compartment Petri dish assay, the compounds showed growth-inhibitory effects towards nematodes (Yang et al. 2012). Similarly, BVCs produced by soil bacteria showed nematicidal activity against Panagrellus redivivus and Bursaphelenchus xylophilus in three-compartment Petri dish bioassays. Further analysis revealed significant difference in the nematicidal activity of the VOCs, indicating that VOC-mediated interactions between bacteria and nematodes can be species-specific/isolate-specific. Numerous distinct volatiles were identified, including phenol, octanol, benzaldehyde, benzene, acetaldehyde, decanal , 2-nonanone, 2-decanone, cyclohexene, and dimethyl disulphide. Finally, exposure to pure synthetic VOCs using the same bioassay revealed growth-inhibitory activities against both B. xylophilus and P. redivivus (Gu et al. 2007).

In a recent study by Cho et al. (2017) it was observed that a novel antifungal volatile, caryolan-1-ol, produced by Streptomyces spp., was effective at inhibiting the growth of the fungal pathogen B. cinerea. By using a homozygous profiling (HOP) assay in Saccharomyces cerevisiae, in which both copies of non-essential genes are deleted, it was determined that caryolan-1-ol most likely inhibits fungal growth by targeting membrane lipid processes and intracellular transport systems in fungi (Cho et al. 2017)

For many bacterial phytopathogens an integral component of their ability to infect susceptible plant hosts is the capacity to be motile, in order to actively seek out sites of colonisation and subsequent infection. To this end, Tahir and colleagues investigated the potential of BVCs from six Bacillus spp., to inhibit the motility of the bacterial phytopathogen Ralstonia solanacaerum (Rsc) TBBS1, the causative agent of bacterial wilt disease in tobacco. They observed a particularly strong inhibitive effect of BVCs from two strains; Bacillus amyloliquefaciens FZB42 and Bacillus artrophaeus LSSC22. Three BVCs; 1,2-benzisothiazol-3(2 H)-one, Benzaldehyde and 1,3-butadiene all negatively influenced cell viability, colony size, motility and chemotaxis. Transcriptomic investigation of this activity identified alterations in the expression of pathogenesis-related genes such as PhcA which is a global regulator of virulence factors in Rsc, as well as genes involved in type III and IV secretion systems, production of extracellular polysaccharides and chemotaxis. Defence genes in tobacco were also up-regulated with over-expression of the proteins EDS1 and NPR1 suggesting the activation of the SA pathway in the ISR response to Rsc-challenge (Tahir et al. 2017c). Interestingly, in a separate study by Tahir and colleagues it was observed that the BVCs produced by Rsc could not elicit a significant plant growth promoting effect. However they did inhibit the growth-promoting potential of B. subtilis SYST2 BVCs when plants were exposed to BVCs emitted by both SYST2 and Rsc. Co-culture of both bacteria together revealed that they inhibited the growth of one another, but the effect of inhibition by Rsc of SYST2 was not as great as SYST2 versus Rsc (Tahir et al. 2017c)

8.5 Species-Specific Rhizobacterial Volatiles

The qualitative and quantitative complexity of the VOC profiles of various rhizobacteria can vary significantly. For example, common rhizobacterial strains such as B. subtilis GB03 and B. amyloliquefaciens IN937a, release a mixture of volatile compounds (e.g. 2,3-butanediol and acetoin) that promote growth in Arabidopsis thaliana, whereas other rhizobacterial strains that do not promote growth via VOCs produce different mixtures of compounds, indicating that synthesis of VOCs is a strain-specific phenomenon. Studies have shown that whereas some compounds are isolate-specific, others are produced by more than one type of bacteria (Ryu et al. 2003; Kai et al. 2007). Furthermore, BVC profiles may be affected by growth phase and environmental conditions. For example, different qualitative and quantitative VOC profiles are generated by growth on nutrient broths with and without glucose in Stenotrophomonas rhizophila P69. In particular, the production of dimethyl pyrazine and beta-phenylethanol occurs under both growth conditions, whereas trimethyl pyrazine, tetramethyl-pyrazine and beta-phenylethyl acetate are produced when glucose is absent from the growth medium (Kai et al. 2009).

The effect of different bacterial strains cultured on four distinct types of media on the growth of A. thaliana were analysed by Blom and Co-workers. They found that more nutrient-rich media Luria-Bertani (LB) caused plant death, whereas all other media tested—including two less nutrient-rich media, Murashige and Skoog (MS) and the soil mimicking Angle medium—promoted growth (Blom et al. 2011a). MS and LB have significantly different compositions; MS is a mineral medium that contains sugar as a carbon (C) source and has low pH and agar concentrations, whereas LB is a nutrient-rich medium containing hydrolysed proteins with relatively high pH and agar concentrations. Therefore, it is perhaps unsurprising that the same bacteria cultured on these two media types can emit different types of BVCs that elicit different plant responses. Furthermore, these two media types also affect the growth kinetics (maximal volatile production, which is assumed to take place during the stationary phase of bacterial growth) of bacterial strain on these two media, with LB supporting faster growth than MS (Bailly and Weisskopf 2012). The primary factors that determine the qualitative and quantitative distribution of compounds in highly complex mixtures are the metabolic abilities of the bacterial species, as well as the nutrients available in the specific growth conditions. Indeed, differences in the types of BVCs produced by pathogenic and non-pathogenic mycobacteria grown on different types of media have been observed; furthermore, variations were sometimes observed, not only between different media but also within individual analyses (Nawrath et al. 2012). However, it is important to note that results obtained under artificial conditions may not accurately reflect natural conditions and an in-depth understanding of this area of plant-microbe research cannot be acquired solely from in vitro experiments of VOC-mediated interactions between all organisms involved in these interactions. Results obtained in controlled, artificial environments may not, and indeed most likely do not, represent interactions which would be observed in the field (Velivelli et al. 2015).

This issue of the interpretation of results from field versus lab is relevant for metabolic experiments utilising artificial growth media and nutrient sources and also for interactions where BVCs are outside of a detectable range. Such concentrations of BVCs observed under in vitro conditions are unlikely to be naturally present in the soil. Nevertheless, in vitro experiments contribute greatly to our understanding of the molecular interplay between bacterial volatiles and other organisms. This can give an insight as to what may be observed within these complex relationships in the field (Ryu et al. 2003; Blom et al. 2011a; Effmert et al. 2012; Park et al. 2015; Besset-Manzoni et al. 2017; Brilli et al. 2019; Song et al. 2019). Furthermore, not all plant species respond similarly to the same group of volatiles produced by a given bacterial strain, which could be due to several reasons: (1) fundamental differences in the pathways used by plants to respond to BVCs (2) differences in reactive sites and (3) differences in the capacity to metabolise volatiles (Santoro et al. 2011). The volatiles produced by a given bacterial strain elicited different responses in different fungi; in other words, differences exist in the interactions between different fungi and bacteria. For example, although Arabidopsis growth was significantly promoted by DMDS; the responses of different fungal species to this compound varied considerably: the growth of R. solani was suppressed, whereas A. alternata and F. solani were unaffected (Groenhagen et al. 2013). Therefore, it is necessary to employ a number of different approaches and to test bacteria under distinct growth conditions to determine comprehensive bacterial emission patterns (Farag et al. 2017).

8.6 Analytical Approaches to Identify VOCs

The capture of VOCs which are emitted as a result of microbial metabolic activity is the first and most crucial step in the analysis of biological VOCs, and research in this area of ‘separation science’ has advanced significantly in the last half a century (Velivelli et al. 2015). SPME-GC/MS and PTR-MS are just two of the numerous approaches designed for the capture and subsequent identification of volatiles. Although each approach has its own advantages, no single method is currently capable of completely surveying bacterial-produced volatile profiles, either in terms of quantity or quality. To successfully identify volatiles, a number of sampling techniques are generally employed, such as purge and trap, solid phase microextraction (SPME) followed by Gas chromatography/Mass spectrometry (GC/MS) identification. The purge-and-trap method involves passing a given volume of purified air over the sample, collecting it onto an absorbent filter and then either directly releasing it using an organic solvent to rinse the filter or transferring it straight to the GC/MS (Ryu et al. 2003, 2004; Yuan et al. 2012). Solid phase microextraction (SPME) has become the method of choice for the extraction of bacterial volatiles in recent years because it minimises preparation time and has greater sensitivity than other extraction techniques.

The process of SPME for the analysis of bacterial volatiles is relatively fast and can be conducted under low oxygen conditions. Although SPME has many advantages over other methods, it is necessary to carefully consider the particular fibre coatings used in each experiment, as they can either absorb or exclude specific analytes based on polarity or size, leading to reduced sensitivity. For instance, non-polar metabolites are absorbed by Polydimethylsiloxane (PDMS) fibre, whereas short-chain polar compounds are absorbed by divinylbenzene/carboxen/PDMS (DCP) fibre. With respect to rhizobacterial volatiles, the best recovery is offered by DCP fibre, as it absorbs polar low molecular weight volatiles (Farag et al. 2006). Yuan and Co-workers tested three different fibres from Supelco—PDMS, 7 µm, stable flex DCP, 50/30 µm, and polydimethylsiloxane/divinylbenzene (PDMS-DVB, 65 µm)—and they found that DCP fibre performed best (Yuan et al. 2012). The volatile extraction methods discussed above are often used in combination with other analytical techniques, such as GC/MS. Considering its effectiveness for both separation and identification, GC/MS is the foremost method for the detection of bacterial volatiles. GC-MS can be used to separate, identify and quantify the volatiles present in a given sample. However, this technique has one significant drawback; namely, it does not allow for the identification of new compounds. Furthermore it can be difficult to obtain quantitative results using SPME, as volatile compounds compete for binding sites within the SPME fibre. Small bacterial molecules are characterised by high polarity and a strong tendency to co-elute. As a result, overlapping MS spectra can be produced, limiting the precision of volatile detection and adversely affecting the process of matching peak quality against database entries.

The identification of existing volatiles is carried out using software programs and libraries of different mass spectra such as NIST/or reference standards. Nevertheless, as similar mass spectra can be produced by related compounds, particular care must be taken in performing these analyses (Kai et al. 2009). A recently developed technique with the advantage of real-time analysis without requiring sample preparation is PTR-MS. In proton transfer reaction-mass spectrometry (PTR-MS), the headspace air is drawn directly into the instrument, where interactions occur between protonated water (H3O+) and any molecules with a proton affinity greater than that of water. Next, a quadruple mass spectrometer in mass-to-charge (m/z) ratio is used to mass analyse and identify the resulting protonated organic molecules. Although PTR-MS cannot accurately detect the individual volatiles produced, it is advantageous because it ensures real-time emission observation.

In addition, PTR-MS cannot be used to distinguish between analytes of the same mass because it employs single reagent ions (Spinelli et al. 2012). PTR-MS was used to determine the volatile profiles of various bacteria and infected plants, and it allowed for the observation of pathogenic emissions in real time (Spinelli et al. 2011). Several researchers have analysed the volatiles emitted by bacteria, and a comparison of these signature compounds indicates that GC/MS analysis can be used to distinguish between different bacterial species. Indeed, the VOC-profile data obtained from GC/MS could be harnessed to develop so-called ‘electronic-nose’ (e-nose) sensor technology for the detection of different bacterial species, or they may be useful for real-time disease monitoring in agricultural settings and post-harvest monitoring where e-nose technology could detect ring and brown rot in commercial potato storage with a detection efficiency of 90% of samples (Biondi et al. 2014). Currently, the identification of bacterial species, including phytopathogenic bacterial isolates, is achieved through ‘classical’ molecular techniques such as polymerase chain reaction followed by dideoxy sequencing of 16S rRNA and these methods, although reliable, are expensive (Velivelli et al. 2015). The efficacy of e-nose technology has been demonstrated for the detection of a single phytopathogen within a wider microbial community; using a metal-oxide-based semiconductor sensor the pathogen that causes fire blight (E. amylovora)—could be differentiated based on volatile mixes alone from other bacteria (Spinelli et al. 2012). However, this technique has one significant drawback; it does not identify and quantify each compound; rather, it uses metal-oxide semiconductor sensors to characterise an aroma based on the signature profile that is generated.

The e-nose distinguishes between Botrytis- and Sclerotinia-induced rots on both green and yellow kiwifruits. In addition, it was successfully used to detect asymptomatic apple and pear plants that had been infected with Erwinia amylovora. More research is needed before e-nose applications in open fields becomes standard practice (Spinelli et al. 2010). In 2015, Cernava et al. described a novel assay for the detection of bacterial volatiles from lichen-associated bacteria using headspace analysis and indicator assays (for HCN production) followed by the determination of the effect of the VOCs on the growth of E. coli and B. cinerea. The initial test was combined with a qPCR-based quantification assay (Cernava et al. 2015).

8.7 Discussion and Conclusive Remarks

Research into volatile-mediated plant–bacteria interactions is relatively new, and this field has the potential to yield significant agricultural applications, particularly in the context of growth promotion and induced systemic resistance (ISR) (Liu and Brettell 2019; Romera et al. 2019). This niche of plant-bacteria research is still emerging and at present, the biological effects of BVCs have only been assessed in a relatively small cohort of plant species with Arabidopsis thaliana being one of the main candidates, along with more economically relevant plants such as Capsicum annum and Nicotiana tabacum (Choi et al. 2014; Kim et al. 2015). How these bacterial volatiles will affect the growth and disease-suppression in other major crops remains to be seen. In general, I plates are used to perform experiments on the effects of bacterial volatiles on plants and fungi. This setup allows for the exchange of volatile compounds while preventing non-volatile metabolites from dispersing throughout the respective growth media. However, following identification of the bio-active compounds, further in vitro laboratory tests should be conducted under conditions that are either in, or similar to, field conditions (e.g. nutrient media that closely resembles soil environment), as opposed to the highly artificial Petri dishes used in the majority of existing studies (Choi et al. 2014; Brilli et al. 2019; Song et al. 2019).

To gain a comprehensive understanding of the role of BVCs, it is necessary to study mutant strains that do not produce such compounds. Adverse effects from chemical treatments and difficulties in establishing optimal treatment concentrations restrict the use of chemicals for growth promotion and resistance induction in plants. Therefore, future research programs should focus on measuring VOC levels produced by rhizobacteria in soil ecosystems (Ryu et al. 2005b; Sharifi and Ryu 2018a). An understanding of the relevant signalling pathways and the extent to which they resemble plant responses to pathogens, and whether the physiological and molecular plant responses vary between bacterial species is crucial to aid our understanding and will determine future technology transfer in this area (Velivelli et al. 2015). In addition, genetically altering rhizobacterial strains to produce greater amounts of volatiles that promote plant growth or that induce systemic resistance may represent promising avenues of future research. However, the use of GM organisms is becoming increasingly controversial in terms of human health and the environment. Phytopathogenic fungi—which are responsible for the majority of economically important crop diseases—are inhibited by BVCs, indicating that BVCs have the potential for use in agriculture as biological control agents. However, research on the effects of bacterial volatiles is still in the early stages (Sharifi and Ryu 2016).

In particular, it has yet to be determined whether the effects of volatiles are limited to specific plant tissues or if they affect plant development in general. Also, an understanding (positive or negative) of the impact of bacterial volatiles on plant metabolism is essential. Furthermore, it will be necessary to address whether effects observed in the laboratory transfer to the field (Velivelli et al. 2015). An alternative strategy that may prove beneficial for agriculture is the application of bio-active but affordable compounds, such as 2,3-butanediol, to aerial plant components to stimulate growth and ISR. Additional limitations on the use of rhizobacteria volatiles such as 2,3-butanediol and acetoin include the fact that they evaporate very quickly following application in the open field, however solutions are being developed to tackle this problem such as the use of microcapsules to slowly release BVCs to the environment (Song and Ryu 2013; Sharifi and Ryu 2018b). Nevertheless, significant positive effects for BVCs have been comprehensively analysed under growth-chamber, greenhouse and open-field conditions (Velivelli et al. 2015; Choi et al. 2014). For example, pathogen growth in A. thaliana by Pseudomonas syringae was found to be considerably suppressed by the direct application of acetoin to roots under growth-chamber conditions (Rudrappa et al. 2010). Under greenhouse conditions, the soil-drench application of volatile metabolites, such as DMDS (released by B. cereus C1L), inhibited the activities of Botrytis cinerea and Cochliobolus heterostrophus against tobacco and corn plants, respectively (Huang et al. 2012). Pre-treatment of cucumber plants with 3-pentanol and 2-butanone showed protective benefits against the biotrophic bacterial pathogen P. syringae in open-field trials. (Song and Ryu 2013; Velivelli et al. 2015). Notwithstanding the challenges of transitioning from laboratory to field with respect to the application of mVOCs (Bailly and Weisskopf 2017) the studies conducted to date should lead to new discoveries regarding the use of VOCs to control microbial pathogens under open-field conditions and can be expected to act as big players in any second green revolution.