*3.1. Essential Oil*

Peppermint plants subjected to salt stress showed a reduction in EO content. Plants grown under 75 or 100-mM salt concentrations and those not treated with mVOCs revealed a 50% decrease in EO yield (*p* < 0.05) (Figure 1). When plants were treated with mVOCs under control conditions, the EO content rose approximately 3.3 times compared to plants not exposed to mVOCs (Figure 1). When plants were grown under salt stress conditions and treated with mVOC, positive effects of mVOCs on EO yields were detected. The levels of EOs increased approximately 5.6 and 6.5-fold in plants grown under 75 or 100 mM and treated with mVOCs, respectively, in relation to plants subjected to salt conditions but not treated with mVOCs, with a statistically significant interaction effect between salt stress and mVOCs being found (*p* < 0.05).

**Figure 1.** Essential oil yield in *Mentha piperita* plants grown under different salt concentrations (0, 75, and 100 mM NaCl) and exposed to *B. amyloliquefaciens* GB03 mVOCs (mean ± SE). Values followed by the same letter in a column are not significantly different according to Fisher's LSD test (*p* < 0.05).

Regarding the main compounds of the EOs, growing under salt stressed conditions resulted in a decrease in menthone and menthol (Table 1); although menthol content was approximately 3.5 times lower in plants grown under 75 or 100 mM concentrations and not treated with mVOCs (*p* < 0.05), the effect on menthol concentration was not statistically significant but followed the same trend as for menthone, which was significant. However, the pulegone concentration was not significantly different for control plants exposed to salt. For plants treated with mVOCs, the levels of menthone and pulegone increased approximately 2 and 3-fold, respectively, compared to those of the corresponding controls at each salinity level. However, the menthol concentration was not modified by mVOC exposure. In plants submitted to 75 mM NaCl and treated with GB03 mVOCs, the concentrations of menthone, menthol, and pulegone were approximately 6.7, 5.8, and 3.4-fold higher, respectively, in relation to plants subjected to salt conditions but not treated to mVOCs and similar to plants treated to mVOCs and not salt stressed. At 100 mM NaCl, the menthone and pulegone contents revealed the same tendency, with an increase observed in plants treated with mVOCs (*p* < 0.05), but the menthol concentration was not modified by the mVOCs (Table 1).


**Table 1.** Concentrations of main essential oil (EO) compounds in *Mentha piperita* grown under salt stress media (0, 75, and 100 mM NaCl) and exposed to *B. amyloliquefaciens* GB03 mVOCs emission (mean ± SE). Values are mean ± standard error (SE).

Means followed by the same letter in a given column are not significantly different according to Fisher's LSD test (*p* < 0.05).

#### *3.2. Total Phenolic Content*

The level of TPC in plants subjected to salt stress conditions increased with the severity of the NaCl concentration (*p* < 0.05), both in plants exposed and not exposed to mVOCs. In plants grown under salt conditions (75 or 100 mM), the TPC levels rose by 15 and 50%, respectively, in relation to control plants (Figure 2). In addition, the plants subjected to both concentrations of NaC and treated with GB03 VOCs registered an increase in TPC compared to non-exposed plants (*p* < 0.05), but no statistically significant interaction effect was found (*p* > 0.05). The highest TPC concentrations were detected in plants treated with salt 100 mM and mVOCs.

#### *3.3. Radical Scavenging Capacity*

The antioxidant capacity of the DPPH• radical scavenger increased 2.6 and 3.6-fold in peppermint leaves grown under 75 and 100 mM NaCl conditions, respectively (*p* < 0.05) (Figure 3). Moreover, when plants were subjected to salt conditions and treated with mVOCs, the antioxidant capacity increased (*p* < 0.05) by 50% and 30% for 75 and 100 mM NaCl, respectively, in relation to salt stressed plants not exposed to mVOCs. The highest levels of antioxidant activity were observed when plants were exposed to VOCs and grown under 100 mM NaCl conditions, with the ascorbic acid equivalents (AAE) increasing 4.75-fold with respect to control plants (not exposed to mVOCs).

**Figure 3.** Antioxidant activity expressed as ascorbic acid equivalents (AAE) in *Mentha piperita* grown under salt stress media (0, 75, and 100 mM NaCl) and exposed to *B. amyloliquefaciens* GB03 mVOCs emission (mean ± SE). Values followed by the same letter in a column are not significantly different according to Fisher's LSD test (*p* < 0.05).

#### *3.4. Lipid Peroxidation*

Oxidative damage to the membrane lipids was observed due to salt stress, as shown by the MDA levels (Figure 4), with the highest MDA levels being observed (*p* < 0.05) at the higher salt concentration. The lipid peroxidation increased 1.4 and 2-fold in 75 and 100 mM NaCl treated plants, respectively, in relation to control plants. For plants treated with mVOCs and subjected to salt stress, the MDA content was approximately 25% lower than for plants stressed and not treated with mVOCs (75 and 100 mM NaCl plants).

**Figure 4.** Malondialdehyde (MDA) content in *Mentha piperita* grown under salt stress media (0, 75, and 100 mM NaCl) and exposed to *B. amyloliquefaciens* GB03 mVOCs emission (mean ± SE). Values followed by the same letter in a column are not significantly different according to Fisher's LSD test (*p*< 0.05).

#### *3.5. Principal Component Analysis*

PCA represents a graphic image that simplifies the visualization and perception of the dataset and the variables. We used the PCA to extract and reveal the relationships among the factors (growth conditions and exposure to mVOCs) and different variables as EO, TPC, lipid peroxidation (MDA), and antioxidant capacity (AAE) in the multivariate analysis (Figure 5). The plot defined by the first two principal components was enough to explain most of the variations in the data (96.8%) and give

a cophenetic correlation coefficient of 0.997. The PCA (Figure 5) showed that 100 mM NaCl (high salt concentrations) combined with exposure to mVOCs was strongly associated with TPC content and antioxidant capacity (AAE), as revealed by the circle in Figure 5. Considering the relationships among variables, a strong positive correlation (acute angle) was observed between TPC levels and AAE. There were also positive correlations found among MDA levels with no mVOC exposure and 100 mM NaCl. In addition, in PC2, positive relationships were observed between AAE, EO, and TPC with mVOC exposure.

**Figure 5.** Principal component analysis for the physiological response of *Mentha piperita* grown under different salt stress concentrations (0, 75, and 100 mM NaCl) and *B. amyloliquefaciens* GB03 mVOCs emission. PRO: proline, TPC: total phenolic content, and MDA: lipid peroxidation were determined by estimating the amount of malondialdehyde (MDA); AEE: DPPH radical scavenging capacity.

#### **4. Discussion**

Salinity is one of the most important environmental factors diminishing plant yield, mainly in arid and semi-arid environments. The responses of plants to salt stress are intricate and affect several components, with plants having the ability to respond via signal transduction pathways by adjusting their metabolism [45,46]. These responses can differ in relation to toxic ion uptake, ion compartmentation and/or exclusion, osmotic regulation, CO2 assimilation, photosynthetic electron transport, chlorophyll content and fluorescence, ROS generation, and antioxidant defenses [45–48].

PGPR make a significant contribution to the protection against abiotic stress through their biological activities at the rhizosphere, as exopolysaccharides production (EPS), phytohormones and 1-aminocyclopropane- 1-carboxylate (ACC) deaminase synthesis, induction of the accumulation of osmolytes and antioxidants, upregulating or downregulating the stress responsive genes, and by changes in the root morphology and volatile compounds [17–21,49,50]. In addition, in recent years, an increasing number of PGPR VOC studies have demonstrated an effect against abiotic stresses [7,38,51].

In the present study, we found that when peppermint plants were subjected to salt stress, the EO yield decreased by 50% for both concentrations evaluated (75 and 100 mM NaCl). Additionally, there was a corresponding decrease in the main compounds menthone, menthol, and pulegone. Comparable effects were reported in *M. arvensis* grown under 100, 300, and 500 mM NaCl, with a reduction of 31%, 54%, and 67%, respectively [52]. In contrast, Karray-Bouraoui et al. [53] noted an enhanced *M. pulegium* EO yield of about 2.75-fold under 50-mM salt stress conditions, with a higher density of glandular trichomes on the leaves. Furthermore, Neffati and Marzouk [54] showed that the compounds of *Coriandrum sativum* L. oil were modified by salinity and were revealed to be dependent on salt level treatment. There are contradictory reports concerning changes in EO yield in relation

to salt stress. An increase in EOs and in their composition in response to low levels of salinity was reported in *Satureja hortensis* [55], in sage [56] and in thyme [57]. In contrast, other studies reported a decrease in EOs in lemon balm and in sweet marjoram [58]. Additionally, Ben Taarit et al. [59] reported that the compositions of EOs of *Salvia o*ffi*cinalis* were altered in moderate or high salt stress, in controls and in plants grown under 25 mM NaCl, with the major compound of the EOs being viridiflorol, whereas at higher levels (50 and 75 mM NaCl), 1, 8-cineole was predominant, and at 100 mM NaCl, manool was the principal compound.

The EO yield variations reported under abiotic stress could have resulted from the fact that their production is affected by different physiological, biochemical, metabolic, and genetic factors, which are complex to isolate from one another. In addition, the geographical, seasonal, developmental, and organ variations all contribute to EO yield, as do anatomical and hormonal factors [60–63]. The impact of salt stress on the EO levels probably was due to acclimation processes in stressed plants. Whereas in the initial stage of stress, the metabolism is severely affected, later, the acclimatization processes may reduce the secondary metabolite biosynthesis [64,65].

In the present study, the EO content in salt stressed plants treated with mVOCs showed a 5.6 and 6.5-fold increase with respect to their respective controls (plants grown under 75 or 100 mM NaCl and not treated with mVOCs, respectively), demonstrating that GB03 mVOCs have the capacity to reverse the negative effects of salinity on the EO yield. In fact, mVOCs induced salt tolerance in plants in a previous study of ours, with peppermint plants subjected to salt stress conditions and treated with GB03 VOCs having a higher shoot fresh weight, root dry weight, and total chlorophyll content compared to controls [38]. In this sense, the biosynthesis of terpenoids is affected by the primary metabolism—for example, the photosynthesis for carbon and energy supply. Factors that increase biomass production may have an impact on the relationships among the primary and secondary metabolisms, causing an increased biosynthesis of secondary metabolites [66]. Related to this, augmented plant biomass seems to lead to a larger availability of substrate for monoterpene biosynthesis [35,67].

We have also observed that abscisic acid (ABA) was not connected to salt tolerance generated in plants subjected to salt stress and treated with VOCs [38]. This observation suggests that GB03 VOCs protection against osmosis is ABA independent [68]. The jasmonic acid (JA) levels were similar in salt treated plants, when treated with mVOCs or not. In contrast, the salicylic acid (SA) levels were higher in plants subjected to salt and treated with mVOCs compared to plants subjected to salt conditions and not treated with mVOCs. SA is an important signal molecule for modulating plant responses to stress [38]. Chemical analysis using Solid Phase Microextraction (SPME) fibers of the VOC emissions from GB03 grown under salt conditions revealed the release of a total of seven components, belonging to the following four classes: hydrocarbons (cyclohexane, dodecane, undecane and hexadecane), ketones (acetoin), aldehydes (benzaldehyde), and ethers (2-butanone-3metioxy-3 methyl). The relative quantity of acetoin, the major VOC compound emitted by GB03, enhanced with salt concentration [38]. Concerning the complex profile of compounds, VOC emission is strongly affected by the collection methodology employed, the growth medium, and the density of the bacterium [50,69,70]. For instance, Farag et al. [71] identified a higher number of compounds from GB03 VOCs than Cappellari and Banchio [38], probably due to the different collection methodology used.

It has also been reported that plants treated with GB03 mVOCs and grown in a saline media accumulated less Na + through the regulation of the Na transporter. The GB03 VOCs decreased the Na level in *Arabidopsis* by decreasing Na uptake and/or increasing Na exudation [49]. Furthermore, they led to an acidification of the rhizosphere [72]. Certain bacterial VOCs activate closure of the stomata, reducing the water evaporation [73], and are also involved in biofilm formation, which maintains soil moisture content and increases drought tolerance in plants [51,74,75]. In addition, mVOCs emitted by PGPR also act as a biocontrol against several phytopathogens and trigger plant defense responses through the induction of systemic resistance (ISR) [24,71,76]. For example, the production of EOs is related to the defense response system [63], since numerous terpenes have antimicrobial activity [77]. Similarly, monoterpene synthesis is induced by herbivore feeding in *Minthostachys mollis* [78] and

several plant species, suggesting that these compounds protect leaves from future attacks [67,79–81]. Consequently, as mentioned above, endogenous SA levels increased in plants cultivated under salt conditions and treated with GB03, with previous observations suggesting that the biosynthesis of *M. piperita* monoterpenes is SA and JA dependent [82].

A rise in TPC levels in different tissues under salt conditions has also been described in different plant species [83–85]. A consequence of abiotic stress is superoxide production, which leads to a detoxification mechanism. Related to this, phenolics are synthesized by many plant species for protection against abiotic stress conditions, and their levels are correlated with antioxidant activity [63,86]. Salinity stress induces metabolic and physiological reactions, as well as drastically decreasing the CO2 uptake due to stomatal restrictions. As a consequence, the consumption of reduction equivalents (NADPH 2+) for CO2 fixation via the Calvin cycle decreases significantly, leading to oxidative stress and an oversupply of reduction equivalents, with the metabolic processes being moved to biosynthetic activities that consume reduction equivalents. Hence, the biosynthesis of reduced compounds, such as phenols, is increased [63,85,87]. Among the SM found in *M. piperita* are phenolic compounds such as caffeic acid, rosmarinic acid, eriocitrin, and luteolin- 7-O-glucoside [88,89], with their proportion in leaves being approximately 19–23% of dry weight [90–92]. Here, we found that peppermint plants either subjected to salt conditions and/or treated with GB03 VOCs produced a positive effect on the TPC content compared to the respective control plants. Plants grown under 100 mM NaCl and treated with VOCs revealed a higher TPC content. In fact, phenolic compounds are important and powerful agents in scavenging free radicals [93–96]. The antioxidant capacity of phenolic compounds is due to their high reactivity as hydrogen or electron donors, to the particularity of the polyphenol-derived radical to stabilize and delocalize the unpaired electron, and to their capacity to chelate transition metal ions [92,97].

In a previous study, we observed that direct inoculation as well as drought stress in *M. piperita* increased TPC and phenylalanine ammonia lyase (PAL) activity, with the latter being responsible for the synthesis of phenolic compounds [41,43]. In agreement, the TPC was observed to increase in different plant species submitted to abiotic stress [86]—for example, in *T. vulgaris* subjected to drought stress [96] and in *M. pulegium* under salt stress [98]. Conversely, Rahimi et al. [99] and Alhaithloul et al. [100] described a reduction in TPC in *M piperita* plants subjected to drought stress. However, in *Tagetes minuta* plants inoculated with *P. fluorescens* WCS417r and *Azospirillum brasilense,* and in chickpea inoculated with *P. fluorescens* [101], TPC levels increased significantly [36]. Jayapala et al. [102] reported the induction of resistance against pathogens through enhancement of the activities of defense-related enzymes and a higher accumulation of TPC in chili plants inoculated with *Bacillus* sp. Furthermore, Tahir et al. [27] revealed that *Bacillus* sp. mVOCs negatively influence the development of the pathogen *R. solanacearum* by activating ISR in tobacco plants. Molecular studies have shown that resistance is the consequence of an increase in the SM levels and defense-related enzymes, including PAL.

Phenolic compounds are antioxidants that may be required for scavenging ROS and protecting the lipid membrane from oxidative stress [12]. For example, Fagopyrum *esculentum* plants grown under media with increasing salt concentrations revealed a concentration-dependent increase in the accumulation of phenolic compounds, resulting in a higher DPPH free radical scavenging potential [103]. This effect was corroborated in the present study in plants subjected to salinity environments and treated with mVOCs, which showed a heightened antioxidant capacity, as revealed by the high levels of AAE detected in the DPPH• scavenging assay and by the low amounts of MDA. The highest levels of antioxidant activity were observed when plants were grown under 100 mM NaCl and mVOC. The GB03 mVOCs decreased the MDA levels in plants subjected to salt stress, to similar levels as those in control plants. In contrast, after water deficit treatment in peppermint plants, heightened amounts of MDA, as a cell membrane damage index, were detected [99]. Additionally, peppermint growing under control conditions was revealed to be more effective in scavenging DPPH free radicals and had a higher reducing power than when exposed to drought and heat stress. This observation provides

signals that tissues of peppermint subjected to heat and/or drought stress contain fewer antioxidants and reducing compounds [100].

The PCA analysis showed that plants subjected to high salt concentrations combined with exposure to mVOCs strongly affected the TPC content and antioxidant capacity (AAE). This relationship was also detected in drought-stressed peppermint plants inoculated with GB03 [43].

In plants that were inoculated and subjected to osmotic stress, similar results in MDA reduction were observed to those reported for cucumber plants inoculated with a consortium of PGPR under drought stress conditions [104], as well as those in white clover and *M. arvensis* inoculated under saline conditions [51,105]. The decrease in the leaf MDA content resulting from mVOC treatment suggests its ability to reduce the peroxidation of cell membrane lipids under salt stress and to protect the leaf cell from damage. Moreover, Gopinath et al. [106] reported in *Nicotiana tabacum* that when callus was exposed to volatile compounds from *Bacillus badius* M12 and the volatile, 2,3- butanediol, this led to increased antioxidant activity by the expression of SOD, a key antioxidant enzyme. In addition, treatment with mVOCs from GB03 and *Pseudomonas simiae* increased choline and glycine betaine biosynthesis in *Arabidopsis* [51,68]. These osmolytes have positive effects on enzyme and membrane integrity, along with adaptive roles in mediating osmotic adjustment in plants subjected to stress conditions [107]. In another investigation, 2,3-butanediol was found to induce plant production of nitric oxide (NO) and hydrogen peroxide [108], and it was reported that NO regulates antioxidant enzymes at the level of activity and gene expression [109]. At the same time, the plant hormone SA is required for plant growth under abiotic stress [7,17,73]. Finally, an increase in the SA levels was shown in peppermint plants subjected to salt stress and treated with GB03 VOCs [38].

#### **5. Conclusions**

Salt stresses affect the growth and productivity of crop plants and are detrimental to the plants, thereby reducing their yield. Thus, it is necessary to improve the technologies of abiotic stress management. In recent decades, several studies have shown that PGPR has the ability to ameliorate the negative effects of salt or water. However, only a few reports have been published on PGPR VOCs as elicitors of tolerance to abiotic stress in aromatic and medicinal plants. The GB03 VOCs have been shown to increase plant growth and chlorophyll content and lead to better morphological characteristics in *M. piperita* plants subjected to salt stress. The results shown in the present study establish that for peppermint plants grown in the laboratory under salt media, the volatiles emitted by GB03 significantly increased SM production and improved the antioxidant status. This suggests that the accumulation of SMs is a plant strategy to avoid oxidative damage caused by ROS, a direct result of salt stress. Bacterial volatiles are promising candidates for a rapid non-invasive technique to increase SM production in aromatic and medicinal crops growing under abiotic stress conditions. In addition, this is a potentially useful system for the production of SMs, which have remarkable biological activities and are often exploited as medicinal and food ingredients for therapeutic, aromatic, and culinary purposes. However, future studies are still necessary to elucidate how plants modulate and perceive PGPR VOC-elicited abiotic tolerance.

**Author Contributions:** L.d.R.C., J.C., and T.B.P. performed the experiments; E.B. designed the research and analyzed the data; L.d.R.C., E.B., and W.G. wrote the manuscript. All authors read, revised, and approved the final manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by grants from the Secretaría de Ciencia y Técnica de la Universidad Nacional de Río Cuarto, the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) PICT 0636/14, Argentina, and financial support was given to E.B. by the Georg Forster Research Fellowship of the Alexander von Humboldt Foundation. E.B. and W.G. are Career Members of CONICET. L.C., J.C., and T.B.P. received fellowships from CONICET.

**Acknowledgments:** This research was supported by grants from the Secretaría de Ciencia y Técnica de la Universidad Nacional de Río Cuarto, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT). E.B. and W.G. are Career Members of CONICET. E.B. obtained financial support from a Georg Forster Research Fellowship of the Alexander von

Humboldt Foundation. L.C., J.C., and T.B.P. have fellowships from CONICET. The authors are grateful to Paul Hobson, native speaker, for editorial assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.
