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Volatile organic compounds from rhizobacteria increase biosynthesis of essential oils and growth parameters in peppermint (Mentha piperita)
Plant Physiology and Biochemistry 49 (2011) 1177e1182
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Research article
Volatile organic compounds from rhizobacteria increase biosynthesis of essential oils and growth parameters in peppermint (Mentha piperita) Maricel Valeria Santoro a, Julio Zygadlo b, Walter Giordano a, Erika Banchio a, * a b
Dpto. Biología Molecular, FCEFQyN, Universidad Nacional de Río Cuarto, Campus Universitario, 5800 Río Cuarto, Argentina Instituto Multidisciplinario de Biología Vegetal, Universidad Nacional de Córdoba, Avda. Velez Sarsfield 1600, 5000 Córdoba, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 May 2011 Accepted 27 July 2011 Available online 5 August 2011
Volatile organic compounds (VOCs), characterized by low molecular weight and high vapor pressure, are produced by all organisms as part of normal metabolism, and play important roles in communication within and between organisms. We examined the effects of VOCs released by three species of plant growth-promoting rhizobacteria (Pseudomonas fluorescens, Bacillus subtilis, Azospirillum brasilense) on growth parameters and composition of essential oils (EO) in the aromatic plant Mentha piperita (peppermint). The bacteria and plants were grown on the same Petri dish, but were separated by a physical barrier such that the plants were exposed only to VOCs but not to solutes from the bacteria. Growth parameters of plants exposed to VOCs of P. fluorescens or B. subtilis were significantly higher than those of controls or A. brasilense-treated plants. Production of EOs (monoterpenes) was increased 2-fold in P. fluorescens-treated plants. Two major EOs, (þ)pulegone and ()menthone, showed increased biosynthesis in P. fluorescens-treated plants. Menthol in A. brasilense-treated plants was the only major EO that showed a significant decrease. These findings suggest that VOCs of rhizobacteria, besides inducing biosynthesis of secondary metabolites, affect pathway flux or specific steps of monoterpene metabolism. Bacterial VOCs are a rich source for new natural compounds that may increase crop productivity and EO yield of this economically important plant species. Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords: Aromatic plants Mentha piperita Rhizobacteria Volatile organic compounds Essential oil Pulegone Menthone
1. Introduction The rhizosphere and rhizoplane are colonized more intensively by microorganisms than are other soil compartments. Some of these microorganisms stimulate plant growth by means other than simple secretion of nutrients. For instance, some rhizobacteria fix atmospheric nitrogen, which becomes available to the plant, thereby promoting plant growth in nitrogen-deficient soils. Other rhizobacteria directly promote plant growth through production of hormones, and are collectively termed “plant growth-promoting rhizobacteria” (PGPR). Because of interactions of plants, pathogens, antagonists, and environmental factors, effects of PGPR are complex and cumulative [1]. Treatment of plants with PGPR has been shown to increase shoot growth, total biomass, seed weight, early flowering, yields of grain, fodder, fruit, etc. [2]. Numerous
Abbreviation: EOs, essential oils; PGPR, plant growth-promoting rhizobacteria; R%, relative percentage; VOCs, volatile organic compounds. * Corresponding author. Fax: þ54 358 4676232. E-mail addresses: [email protected], [email protected] (E. Banchio). 0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.07.016
mechanisms have been proposed to explain how rhizobacteria promote plant growth, including increased nitrogen fixation, production of auxin, gibberellins, cytokinins, and ethylene, solubilization of phosphorus, oxidation of sulfur, increased availability of nitrates, extracellular production of antibiotics, lytic enzymes, and hydrocyanic acid, increased root permeability, competition for available nutrients and root sites, suppression of harmful rhizobacteria, and enhanced uptake of essential nutrients [3]. In addition to the above mechanisms, studies during the past 5 years have shown that rhizobacteria are capable of releasing functional volatile organic compounds (VOCs) [4e6]. This group of compounds is characterized by relatively low molecular mass (<300 Da), low boiling point, and lipophilic properties. VOCs are ideal “infochemicals” (i.e., chemicals that mediate interactions between organisms) because they occur in the biosphere in a wide range of concentrations, and can act over long distances [7]. Because they are capable of diffusing through aqueous solutions as well as through the atmosphere, volatiles can mediate interactions both above and below the ground [5]. Thus, they can have major effects on nearby organisms, and on development of organisms in an ecosystem. A few examples of the beneficial functions of VOCs are helping pollinators to locate flowers, attracting predators of
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herbivores, and directly killing or inhibiting growth of pathogenic organisms. Economically important agricultural crops include not only traditional food-, forage-, and fiber-producing species, but also species with secondary metabolites having desired aromatic or therapeutic qualities, or providing source material for the perfume and chemical industries. Lipid (oil) constituents of certain species are used as chiral auxiliaries in synthetic organic chemistry, and in microbial transformation of common chemical structures to yield highly functionalized substances of economic value [8]. Peppermint (Mentha piperita L.; family Labiatae) is an important and commonly-used flavoring agent world-wide. Fresh or dried leaves of Mentha species are used as condiments, and essential oils (EOs) of these plants, which are stored in glandular hairs, are used as flavorings for foods and beverages, as fragrances, and as fungicides or insecticides in many pharmaceutical and industrial products [9,10]. Menthol, a crystalline compound obtained from peppermint oil, has a pleasant flavor and aroma, and a cooling anesthetic effect, and is used in confectioneries, pharmaceuticals, oral health care products, cosmetics, teas, and tobacco products. Methods to enhance growth of economically important plants undergo constant evolution and improvement. Many recent studies have attempted to manipulate rhizobacterial populations by inoculation of beneficial bacteria, to promote growth of crop plants. This approach has potential environmental benefits since it reduces use of agricultural chemicals, and is compatible with sustainable management practices. “Organic agriculture” is an increasingly popular production system which avoids or minimizes use of synthetic fertilizers, pesticides, and growth regulators, relying instead on bio-fertilization, crop rotation, crop residues, mechanical cultivation, and biological pest control to maintain soil productivity [11]. It is particularly desirable to avoid use of synthetic compounds in production of aromatic and medicinal plants, which are typically consumed without further processing after harvest. In this study, we evaluated effects of VOCs produced by soil bacteria on growth parameters of M. piperita, and on qualitative and quantitative composition of EOs.
in our previous study on sweet basil and sweet marjoram [12,13]. Effects of the VOC emission on plant development varied depending on the PGPR species (Fig. 1; Table 1). Clear differences among treatments were detectable after 30 days growth. VOCs emitted by Pseudomonas fluorescens or Bacillus subtilis caused significant (p < 0.05) increases (2.5- and 1.3-fold, respectively) in number of shoot ramifications and in root length (Table 1). None of the treatments resulted in significant change of leaf number, shoot length, or node number. Exposure to B. subtilis VOCs caused a 2-fold increase (p < 0.05) in shoot fresh weight; similar effects were observed for P. fluorescens treatment (Fig. 1). Similarly, root dry weight in B. subtilis-treated plants was 3.5-fold higher than in controls, and significantly (p < 0.05) higher than in plants exposed to VOCs of other PGPR species (Fig. 1). Increased shoot fresh weight for B. subtilis-treated plants was due to a combination of w2-fold increase in leaf area (Fig. 2) (p < 0.05), and elongation of internodes (data not shown). In P. fluorescens- and Azospirillum brasilense-treated plants, EO yields were respectively 4.46 and 3.22 mg/mg fresh weight, w2-fold higher than in controls (Fig. 3). Yields of major EOs (þ)pulegone, ()menthone, ()menthol, and (þ)menthofuran were generally higher in treated plants compared to controls (Fig. 4). Pulegone concentration was significantly increased (3.14-fold; p < 0.05) only by P. fluorescens treatment. Menthone was increased 15.4- and 13.5-fold (p < 0.05) in P. fluorescens- and A. brasilense-treated plants. Menthofuran was increased significantly in P. fluorescenstreated plants. The only decreases in EO yield (w5-fold) were observed for menthol and menthofuran in A. brasilense-treated plants. Exposure to PGPR VOCs led to changes of relative percentage (R%), as well as yield, of EOs (Table 2). R% for pulegone, the main EO component, increased to 59.9% in P. fluorescens-treated plants, compared to 45.3% in controls. R% for menthone also increased in all cases. R% for menthol was lower in P. fluorescens- and A. brasilense-treated plants (6.1%; 5.9%) than in controls (9.6%), but was higher for B. subtilis-treated plants (11.3%). The only EO showing a significant R% decrease in A. brasilense-treated plants was menthofuran.
2. Results 3. Discussion To investigate whether bacterial (PGPR) VOCs affected growth of M. piperita, plants and bacteria were grown on the same dish with a physical separation such that VOCs but not solutes from the bacteria could reach the plant. Results were consistent with those
Many studies have demonstrated the ability of soil-dwelling organisms to synthesize and release volatile organic compounds (VOCs), which can travel short or long distances through soil [14].
30
400 ab
b
b
300
200
20
ab
mg
mg
ab
a
ab 10 a
100
0
0
root dry weight
shoot fresh weight Control
P. fluorescens
B. subtilis
A. brasilensis
Fig. 1. Shoot fresh weight and root dry weight of M. piperita exposed to VOCs from three PGPR species. Letters above bars indicate significant differences according to Fisher’s LSD test (p < 0.05).
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Table 1 Effect of exposure to VOCs from three PGPR species on plant growth parameters of Mentha piperita. Data shown are mean SE. Values within a column followed by the same letter are not significantly different according to Fisher’s LSD test (p < 0.05). Treatment
Leaves (n )
Node (n )
Ramification (n )
Shoot length (cm)
Root length (cm)
Control P. fluorescens WCS417r B. subtilis GB03 A. brasilense SP7
23.48 2.23 33.81 3.61 34.91 5.63 28.22 4.17
7.26 0.29 8.04 0.67 7.23 0.32 8.05 0.37
1.14 0.13a 2.68 0.39b 2.92 0.46b 1.24 0.26a
4.58 0.35 5.05 0.45 5.70 0.54 4.50 0.61
6.32 0.37a 8.77 0.89b 8.61 0.28b 7.44 0.79ab
Little is known regarding the ability and efficiency of rhizobacteria to release VOCs. The present study showed that many growth parameters of peppermint (M. piperita) are increased upon exposure to VOCs from the bacteria P. fluorescens and B. subtilis, consistent with previous reports [12,15]. In our I-plate assays, only low molecular weight VOCs had such effect. In the study by Ryu et al. [15], two volatile components from B. subtilis were identified as strong promoters of growth in Arabidopsis thaliana (family Brassicaceae): 3-hydroxy-2-butanone (acetoin) and (2R,3R)butanediol. In the present study, root dry weight was increased 3.5- and 2.7fold, respectively, by exposure to B. subtilis and P. fluorescens. This may be attributed in part to root elongation (Table 1) induced by auxin. In a study of A. thaliana, Zhang et al. [16] presented transcriptional and histochemical data indicating that VOCs from B. subtilis promote growth by increasing auxin accumulation in roots. Shoot fresh weight was also significantly increased by exposure to bacterial VOCs. Since leaf number did not increase, this effect may be due to increases of ramification number (Table 1) and/or leaf area (Fig. 2). The above findings rule out the possibility that growth promotion resulted simply from increased plant hydration. Microarray data presented by Zhang et al. [17] suggest that exposure to B. subtilis VOCs enhanced photosynthetic efficiency, chlorophyll content, and cell expansion. Our previous study of sweet basil (Ocimum basilicum) showed that exposure to B. subtilis VOCs promoted shoot and root length, leaf number, leaf area, and shoot fresh weight [13]. The present results for M. piperita seem contradictory to previous findings by Vespermann et al. [5] and Kai et al. [6] that VOCs released by P. fluorescens strongly inhibited root and leaf growth of A. thaliana. However, use of different Pseudomonas species or isolates, and variations in growth conditions and testing systems, may account for the different results; bioactive compounds emitted by Pseudomonas species have not yet been identified. Regarding the effect of rhizobacterial VOCs on formation of plant secondary compounds, we observed an almost 2-fold increase in
accumulation of EOs in plants treated with P. fluorescens as compared to B. subtilis, A. brasilensis, or control. The major EO components of M. piperita (i.e., (þ)pulegone, ()menthone, () menthol, (þ)menthofuran) showed increased accumulation upon exposure to P. fluorescens, but not B. subtilis. In our previous study, in contrast, O. basilicum plants exposed to B. subtilis VOCs showed increased accumulation and content of major EOs [12]. Relative percentages (R%) of EOs were also changed by exposure to bacterial VOCs. In P. fluorescens-treated plants, R% of pulegone was 30% higher than in controls, and R% of menthofuran was reduced to 0.8%, as compared to 1.16% in controls or A. brasilensetreated plants. The enhanced levels of EOs were not due to increased biomass, suggesting that bacterial VOCs induce biosynthesis of secondary metabolites. The observed increase of monoterpenes in M. piperita may have resulted from induction of systemic resistance (ISR). ISR occurs upon stimulation of a plant’s defense mechanisms. Ryu et al. [15] observed that rhizobacterial VOCs caused ISR in Arabidopsis. ISR has been shown to increase plant cell wall strength, and to alter plant physiology and metabolic responses, leading to enhanced synthesis of defensive chemicals upon challenge by pathogens and/ or abiotic stress factors [18]. Terpenes play important roles in plant defenses, and help the plant’s photosynthetic apparatus recover from brief exposure to high temperature. Isoprene appears to physically stabilize thylakoid membranes at high temperature, and to quench reactive oxygen species, e.g., ozone, that cause membrane damage [19]. Several monoterpenes are synthesized de novo [20,21]. in aromatic and other types of plants [22,23]. in response to herbivory, apparently to prevent damage from further attacks [10]. Increased synthesis of EOs is a defensive response to colonization by microorganisms, since several EOs have antimicrobial properties [8]. Biosynthesis of terpenoids depends on primary metabolism, e.g., photosynthesis, on oxidative pathways for carbon, and on energy supply [24]. Bacterial VOCs are capable of improving photosynthetic efficiency [15,17]. In the present study, (þ)pulegone, the major EO of M. piperita, was enhanced >3-fold by exposure to
12
6 b
10
Total Essential Oil Yield (ug/mg fresh weight)
b
cm2
8 a 6
a
4 2
b
5
ab 4 3
a a
2 1
0 leaf area Control
P. fluorescens
B. subtilis
0 A. brasilensis
Fig. 2. Leaf area of 30-day old M. piperita plants exposed to VOCs from three PGPR species. Letters above bars indicate significant differences according to Fisher’s LSD test (p < 0.05).
Control
P. fluorescens
B. subtilis
A. brasilensis
Fig. 3. Essential oil (EO) yield in M. piperita exposed to VOCs from three PGPR species. Letters above bars indicate significant differences according to Fisher’s LSD test (p < 0.05).
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0,4
4
a
a
0,3
3 a
2 a a
fresh weight ug/mg
fresh weight ug/mg
b
a
0,2 bc c ab
0,1
1
c b
a
ab
bc
a
0
0
(-)-menthone
(+)-pulegone Control
P. fluorescens
(-)-menthol
B. subtilis
(+)-menthofuran
A. brasilensis
Fig. 4. Concentrations of major EO components in M. piperita exposed to VOCs from three PGPR species. Letters above bars indicate significant differences according to Fisher’s LSD test (p < 0.05).
rhizobacterial VOCs. Pulegone is a potent inhibitor of acetylcholinesterase (AchE) in invertebrates [10,25], and has been shown to cause a repellent effect [26], toxicity [10,27e30], or interference with development and reproduction [31] in various insect species. Pulegone can also provide a biochemical barrier to host plant utilization, by eliminating herbivore symbionts [10]. Menthone and pulegone compounds seem to be antagonistic, which may further enhance toxic effects [28]. (þ)Pulegone is a central intermediate in biosynthesis of ()menthol. Depending on environmental conditions, this branch point metabolite may be reduced to ()menthone en route to menthol by pulegone reductase, or oxidized to (þ) menthofuran by menthofuran synthase [32]. The observed changes in EO levels suggest that VOCs from soil bacteria, besides inducing biosynthesis of secondary metabolites in M. piperita, influence pathway flux or specific steps of monoterpene metabolism. Increased pulegone content in EOs may enhance pulegone reductase enzyme activity, and favor somehow reduction of pulegone to menthone, increasing cytochrome P450 (þ)MFS enzyme activity, rather than oxidation to menthofuran. Full interaction of bacteria with plants (inoculation) may induce secondary metabolite responses [13,33,34]. Inoculation of marjoram (Origanum majorana) with P. fluorescens increased yield and accumulation of major EOs. Plant growth can be promoted by bacterial effects including growth hormone production, VOC production, solubilization of phosphates, oxidation of sulfur, increased nitrate availability, increased root permeability, bacterial volatiles, and combinations of these effects. It is noteworthy that A. brasilense VOCs did not significantly affect on plant growth parameters or EO yields, and caused a decrease in menthol level. B. subtilis VOCs showed a growthpromoting effect in both O. basilicum and M. piperita, but caused increased yield of EOs only in the former species. These findings Table 2 Variation in relative percentage (R%) of major EOs in M. piperita treated with VOCs from three PGPR species. Data shown are mean SE. Treatment
(þ)Pulegone ()menthone ()Menthol
(þ)Menthofuran
Control P. fluorescens WCS417r B. subtilis GB03 A. brasilense SP7
45.29 9.90 59.95 7.71
1.65 0.68 2.23 0.78
9.53 2.80 6.11 1.89
1.16 0.09 1.44 0.31
50.07 9.93 56.48 9.51
2.59 1.13 2.92 1.05
11.3 2.89 5.91 2.01
1.39 0.35 0.88 0.25
suggest that effects of bacterial VOCs on plants are species-specific. I.e., VOCs from a given bacterial strain do not cause the same effects, or to the same degree, in all plant species; responses are characteristic of a specific plantebacteria combination. Possible explanations for this phenomenon are: (i) different plants respond to different component(s) of VOC mixtures; (ii) reactive sites are different; (iii) plant differ in their ability to metabolize VOCs. Various rhizobacteria show great variability in qualitative and quantitative complexity of their VOC profiles. The number of different compounds produced and emitted by a particular strain may be as high as 60 [4]. For most VOCs, the exact chemical structure remains to be elucidated [6]. Manifestation of characteristic volatile compound or profile is attributable to specific metabolic pathways active in the bacterium. Depending on the growth conditions or medium, the “bouquet” of released VOCs can vary for a particular species. Occurrence of VOC-mediated interactions between bacteria and plants are clearly demonstrated by results of this and previous studies. Such interactions are species-specific. Certain VOCs are capable of enhancing growth and productivity of plant species. Functional analyses in future studies will clarify roles of VOCs in plant interactions on the population or community level, and in maintaining ecological balance. VOCs from bacteria show a diversity and complexity comparable to those from plants and fungi, and are rich sources for new natural compounds. Biological functions of most bacterial VOCs are not yet well understood. In analogy to VOCs from plants and fungi, we expect that they may serve as: (a) “infochemicals” for communication within and among organisms; (b) cell-to-cell communication signals; (c) carbon release valves: (d) growth-promoting or -inhibiting agents. The present results indicate that VOCs from soil bacteria, released underground, play crucial roles in rhizosphere interactions. Structural elucidation and biological activities of these volatile compounds are subjects of ongoing and future studies. 4. Materials and methods 4.1. Bacterial strains, culture conditions, media, and treatments Three strains previously reported as PGPR (P. fluorescens WCS417r, B. subtilis GB03, A. brasilense SP7) [35] were assessed for their effects. These strains were grown respectively on media
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termed TSA (15 g/l tryptone, 5 g/l soy peptone, 5 g/l NaCl), and LB (10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl), for routine use, and maintained in nutrient broth with 20% glycerol at 80 C for longterm storage. For experiments, bacteria were grown on nutrient agar, single colonies were transferred to 100 ml flasks containing culture medium, and grown aerobically on a rotating shaker (200 rpm) for 48 h at 28 C. Bacterial suspension was diluted in sterile distilled water to final concentration 109 CFU/ml, and 20 ml of the resulting suspension was used for each treatment. 5e10 Plants were used for each treatment, and experiments were replicated 4. 4.2. Micropropagation of plants Young shoots from M. piperita plants grown in Traslasierra Valley (Córdoba province, Argentina) were surface disinfected by soaking 1 min in 17% sodium hypochlorite solution, and rinsed 3 in sterile distilled water. Disinfected shoots were cultured in 100 ml of MS culture medium containing 0.7% (w/v) agar and 1.5% (w/v) sucrose [36]. All culture media contained 30 g/l sucrose and 7.5 g/l agar. Stage I. Initial shoot-tip culture. After 30 days, apical meristems with foliar primordia, not showing contamination, were aseptically removed from terminal buds of shoots obtained in the previous step. Explants were cultured in test tubes, in 40 ml MS medium with 0.66 mg/l indolebutyric acid. Stage II. Growth and in vitro multiplication. Plantlets obtained from tips were multiplied by single node culture, and MS medium was adjusted to 5.6e5.8 pH prior to autoclaving (20 min, 121 C). Explants were placed in a growth chamber with controlled conditions of light (16/8-h light/dark cycle), temperature (22 2 C), and relative humidity (w70%). Stage III. Exposure to volatiles. One node, from aseptically cultured plantlet, was placed on one side of a specialized plastic Petri dish (90 15 mm) containing a center partition (I-plate, Fisher Scientific). Both sides of the dish contained 50% strength MS solid medium. 20 ml Suspension culture of various PGPR strains in sterile distilled water was applied drop-wise to the side of the dish opposite the plant node. By this method, plants were exposed to bacterial VOCs without physical contact. Dishes were sealed with parafilm, arranged in a completely randomized design, and placed in a growth chamber with controlled conditions of light (16/8-h light/dark cycle), temperature (22 2 C), and relative humidity (w70%). Plants were harvested after 30 days. 10 plants were used for each treatment, and experiments were replicated 4. 4.3. Plant growth measurement Plants were removed from the partitioned dish, roots were rinsed with water to remove MS medium, and growth-promoting effects of bacterial treatments were evaluated in terms of shoot and root lengths, numbers of nodes, leaves, and shoot ramifications, shoot fresh weight, root dry weight, and leaf area. Total leaf surface area was measured using integrated digital image analysis. Images (JPG file, 640 480 pixels) of plant trays from above were taken by digital camera at a distance of w80 cm. JPG files were adjusted using tools under the Image menu of Adobe PhotoshopÒ 3. Color was removed to obtain a grayscale-only image, and pixels were converted to cm as a unit of measurement. A “standard” dish was included within the image for calibrating the pixel conversion. All leaves in the image were outlined, and the “measure” option under “Analyze” menu was used to estimate surface area [37]. 4.4. Extraction of essential oils (EOs) Each shoot sample was weighed, subjected to hydrodistillation in a micro Clevenger-like apparatus for 30 min, and volatile fraction
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was collected in dichloromethane. Internal standard was added (0.1 ml dodecalactone in 50 ml ethanol). M. piperita plants contain w3% volatile oils, consisting of >50 different compounds. EOs, accounting for w60% of total oil volume, were (þ)pulegone, ()menthone, ()menthol, and (þ)menthofuran. These compounds were quantified in relation to dodecalactone, which was added during the distillation procedure. FID response factors for each compound generated essentially equivalent areas (difference < 5%). Chemical analyses were performed using a PerkineElmer Clarus 600 gas chromatograph (GC) equipped with CBP-1 capillary column (30 m 0.25 mm, film thickness 0.25 mm) and mass-selective detector. Analytical conditions: injector and detector temperatures 250 and 270 C, respectively; oven temperature programmed from 60 C (3 min) to 240 C at 4 /min; carrier gas ¼ helium at constant 0.9 ml/min flow; source 70 eV. Oil components were identified based on mass spectra and retention times, in comparison to standard [12]. GC analysis was performed using a PerkineElmer Clarus 500 GC, fitted with 30 m 0.25 mm fused silica capillary column coated with Supelcowax 10 (film thickness 0.25 mm). GC operating conditions: oven temperature programmed from 60 C (3 min) to 240 C at 4 /min, injector and detector temperatures 250 C; detector FID; carrier gas ¼ nitrogen at 0.9 ml/min constant flow. 4.5. Statistical analyses Data were subjected to analysis of variance (ANOVA) followed by comparison of multiple treatment levels with control, using post hoc Fisher’s LSD (least significant difference) test. Infostat software version 2.0 (Group Infostat, Universidad Nacional de Córdoba, Argentina) was used for all statistical analyses. 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). EB, JZ, and WG are Career Members of the CONICET. MS has a fellowship from the CONICET-MCyT Cba. The authors are grateful to Steve Anderson for English editing of the MS. References [1] O.O. Babalola, Beneficial bacteria of agricultural importance, Biotechnol. Lett. 32 (2010) 1559e1570. [2] L.C. Van Loon, Plant response to plant growth-promoting rhizobacteria, Eur. J. Plant Pathol. 119 (2007) 243e254. [3] R.S. Niranjan, H.S. Shetty, M.S. Reddy, Plant growth promoting rhizobacteria: potential green alternative for plant productivity. in: Z.A. Siddiqui (Ed.), PGPR: Biocontrol and Biofertilization. Springer, Netherlands, 2006, pp. 197e216. [4] M. Kai, U. Effmert, G. Berg, B. Piechulla, Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen Rhizoctonia solani, Arch. Microbiol. 187 (2007) 351e360. [5] Vespermann, M. Kai, B. Piechulla, Rhizobacterial volatiles affect the growth of fungi and Arabidopsis thaliana, Appl. Environ. Microbiol. 73 (2007) 5639e5641. [6] M. Kai, M. Haustein, F. Molina, A. Petri, B. Scholz, B. Piechulla, Bacterial volatiles and their action potential, Appl. Microbiol. Biotechnol. 81 (2009) 1001e1012. [7] R.E. Wheatley, The consequences of volatile organic compound mediated bacterial and fungal interactions, Antonie van Leeuwenhoek 81 (2002) 357e364. [8] N.S. Sangwan, A.H.A. Farooqi, F. Shabih, R.S. Sangwan, Regulation of essential oil production in plants, Plant Growth Regul. 34 (2001) 3e21. [9] D. Ram, M. Ram, R. Singh, Optimization of water and nitrogen application to menthol mint (Mentha arvensis L.) through sugarcane trash mulch in a sandy loam soil of semi-arid subtropical climate, Bioresour. Technol. 97 (2006) 886e893. [10] P. Harrewijn, A.M. Van Oosten, P.G. Piron, Natural Terpenoids as Messengers. A Multidisciplinary Study of Their Production, Biological Functions and Practical Applications. Kluwer Academic Publishers, London, U.K., 2001, p. 440. [11] K. Lind, G. Lafer, K. Schloffer, G. Innerhoffer, H. Meister, Organic Fruit Growing. CABI Publishing, Wallingford, UK, 2004, p. 281.
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[12] E. Banchio, X. Xie, H. Zhang, P.W. Paré, Soil bacteria elevate essential oil accumulation and emissions in sweet Basil, J. Agric. Food Chem. 5 (2009) 653e657. [13] E. Banchio, P. Bogino, M. Santoro, L. Torres, J. Zygadlo, W. Giordano, Systemic induction of monoterpene biosynthesis in Origanum majoricum by soil bacteria, J. Agric. Food Chem. 58 (2010) 650e665. [14] K. Wenke, M. Kai, B. Piechulla, Belowground volatiles facilitate interactions between plant roots and soil organisms, Planta 231 (2010) 499e506. [15] C.M. Ryu, M.A. Faraq, C.H. Hu, M.S. Reddy, J.W. Kloepper, P.W. Pare, Bacterial volatiles induce systemic resistance in Arabidopsis, Plant Physiol. 134 (2004) 1017e1026. [16] H. Zhang, M.S. Kim, V. Krishnamachari, P. Payton, Y. Sun, M. Grimson, M.A. Farag, C.M. Ryu, R. Allen, I.S. Melo, P.W. Pare, Rhizobacterial volatile emissions regulate auxin homeostasis and cell expansion in Arabidopsis, Planta 226 (2007) 839e851. [17] H. Zhang, X. Xie, M.S. Kim, D.A. Kornyeyev, S. Holaday, P.W. Pare, Soil bacteria augment Arabidopsis photosynthesis by decreasing glucose sensing and abscisic acid levels in planta, Plant J. 65 (2008) 264e273. [18] V. Ramomoorthy, R. Viswanathan, T. Raguchander, V. Prakasam, R. Samiyappan, Induction of systemic resistance by plant growth promoting rhizobacteria in crop plant against pest and diseases, Crop Prot. 20 (2001) 1e11. [19] E. Pichersky, J. Gershenzon, The formation and function of plant volatiles: perfumes for pollinator attraction and defense, Curr. Opin. Plant Biol. 5 (2002) 237e243. [20] G. Valladares, A. Zapata, J. Zygadlo, E. Banchio, Phytochemical induction by herbivores could affect quality of essential oils from aromatic plants, J. Agric. Food Chem. 50 (2002) 4059e4061. [21] E. Banchio, J. Zygadlo, G. Valladares, Quantitative variations in the essential oil of Minthostachys mollis (Kunth.) Griseb. in response to insects with different feeding habits, J. Agric. Food Chem. 53 (2005) 6903e6906. [22] H.J. McAuslane, H.T. Alborn, J.P. Toth, Systemic induction of terpenoid aldehydes in cotton pigment glands by feeding of larval Spodoptera exigua, J. Chem. Ecol. 23 (1997) 2861e2879. [23] P.W. Pare, J.H. Tumlinson, Induced synthesis of plant volatiles, Nature 385 (1997) 30e31. [24] N. Singh, R. Luthra, R.S. Sangwan, Oxidative pathways of essential oil biosynthesis in the developing Cymbopogon flexuosus leaf, Plant Physiol. Biochem. 28 (1990) 703e710.
[25] S.E. Lee, J.C. Peterson, J.R. Coats, Fumigant toxicity of monoterpenoids to several stored product insects, J. Stored Prod. Res. 39 (2003) 77e85. [26] J.R. Mason, Evaluation of D-pulegone as an avian repellent, J. Wildl. Manag. 54 (1990) 130e135. [27] A. Fournet, A. Rojas de Arias, B. Charles, J. Bruneton, Chemical constituents of essential oils of Muna, Bolivian plants traditionally used as pesticides, and their insecticidal properties against Chagas disease vectors, J. Ethnopharmacol. 52 (1996) 145e149. [28] G. Franzios, M. Mirotsou, E. Hatziapostolou, J. Kral, Z.G. Scouras, P. MavraganiTsipidou, Insecticidal and genotoxic activities of mint essential oil, J. Agric. Food Chem. 45 (1997) 2690e2694. [29] M.D. Ellis, F.P. Baxendale, Toxicity of seven monoterpenoids to tracheal mites (Acari: Tarsonemidae) and their honey bee (Hymenoptera: Apidae) hosts when applied as fumigants, J. Econ. Entomol. 90 (1997) 1087e1091. [30] A. Lamiri, S. Lhaloui, B. Benjilali, M. Berrada, Insecticidal effects of essential oils against Hessian fly, Mayetiola destructor (Say), Field Crops Res. 71 (2001) 9e15. [31] L.A. Hummelbrunner, M.B. Isman, Acute, sublethal, antifeedant, and synergistic effects of monoterpenoid essential oil compounds on the tobacco cutworm, Spodoptera litura (Lep., Noctuide), J. Agric. Food Chem. 49 (2001) 715e720. [32] M. Soheil, R.B. Croteau, Menthofuran regulates essential oil biosynthesis in peppermint by controlling a downstream monoterpene reductase, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 14481e14486. [33] N. Tabanca, T. Ozek, K.H.C. Baser, G. Tumen, Comparison of the essential oils of Origanum majorana L. and Orignaum majoricum Cambess, J. Essent. Oil Res. 16 (2004) 248e252. [34] E. Banchio, P. Bogino, J. Zygadlo, W. Giordano, Plant growth promoting rhizobacteria improve growth and essential oil yield in Origanum majorana L. Biochem. Syst. Ecol. 36 (2008) 766e771. [35] J.W. Kloepper, Plant growth promoting rhizobacteria as biological control agents. in: Metting (Ed.), Soil Microbial Ecology e Applications in Agricultural and Environmental Management. Marcel Dekker, New York, 1993, pp. 255e274. [36] T. Murashige, F. Skoog, A revised medium for rapid growth and bio assay with tobacco tissue culture, Physiol. Plant. 15 (1962) 473e497. [37] M.E. O’Neal, D.A. Landis, R. Isaacs, An inexpensive, accurate method for measuring leaf area and defoliation through digital image analysis, J. Econ. Entomol. 95 (2002) 1190e1194.
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Research article
Volatile organic compounds from rhizobacteria increase biosynthesis of essential oils and growth parameters in peppermint (Mentha piperita) Maricel Valeria Santoro a, Julio Zygadlo b, Walter Giordano a, Erika Banchio a, * a b
Dpto. Biología Molecular, FCEFQyN, Universidad Nacional de Río Cuarto, Campus Universitario, 5800 Río Cuarto, Argentina Instituto Multidisciplinario de Biología Vegetal, Universidad Nacional de Córdoba, Avda. Velez Sarsfield 1600, 5000 Córdoba, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 May 2011 Accepted 27 July 2011 Available online 5 August 2011
Volatile organic compounds (VOCs), characterized by low molecular weight and high vapor pressure, are produced by all organisms as part of normal metabolism, and play important roles in communication within and between organisms. We examined the effects of VOCs released by three species of plant growth-promoting rhizobacteria (Pseudomonas fluorescens, Bacillus subtilis, Azospirillum brasilense) on growth parameters and composition of essential oils (EO) in the aromatic plant Mentha piperita (peppermint). The bacteria and plants were grown on the same Petri dish, but were separated by a physical barrier such that the plants were exposed only to VOCs but not to solutes from the bacteria. Growth parameters of plants exposed to VOCs of P. fluorescens or B. subtilis were significantly higher than those of controls or A. brasilense-treated plants. Production of EOs (monoterpenes) was increased 2-fold in P. fluorescens-treated plants. Two major EOs, (þ)pulegone and ()menthone, showed increased biosynthesis in P. fluorescens-treated plants. Menthol in A. brasilense-treated plants was the only major EO that showed a significant decrease. These findings suggest that VOCs of rhizobacteria, besides inducing biosynthesis of secondary metabolites, affect pathway flux or specific steps of monoterpene metabolism. Bacterial VOCs are a rich source for new natural compounds that may increase crop productivity and EO yield of this economically important plant species. Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords: Aromatic plants Mentha piperita Rhizobacteria Volatile organic compounds Essential oil Pulegone Menthone
1. Introduction The rhizosphere and rhizoplane are colonized more intensively by microorganisms than are other soil compartments. Some of these microorganisms stimulate plant growth by means other than simple secretion of nutrients. For instance, some rhizobacteria fix atmospheric nitrogen, which becomes available to the plant, thereby promoting plant growth in nitrogen-deficient soils. Other rhizobacteria directly promote plant growth through production of hormones, and are collectively termed “plant growth-promoting rhizobacteria” (PGPR). Because of interactions of plants, pathogens, antagonists, and environmental factors, effects of PGPR are complex and cumulative [1]. Treatment of plants with PGPR has been shown to increase shoot growth, total biomass, seed weight, early flowering, yields of grain, fodder, fruit, etc. [2]. Numerous
Abbreviation: EOs, essential oils; PGPR, plant growth-promoting rhizobacteria; R%, relative percentage; VOCs, volatile organic compounds. * Corresponding author. Fax: þ54 358 4676232. E-mail addresses: [email protected], [email protected] (E. Banchio). 0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.07.016
mechanisms have been proposed to explain how rhizobacteria promote plant growth, including increased nitrogen fixation, production of auxin, gibberellins, cytokinins, and ethylene, solubilization of phosphorus, oxidation of sulfur, increased availability of nitrates, extracellular production of antibiotics, lytic enzymes, and hydrocyanic acid, increased root permeability, competition for available nutrients and root sites, suppression of harmful rhizobacteria, and enhanced uptake of essential nutrients [3]. In addition to the above mechanisms, studies during the past 5 years have shown that rhizobacteria are capable of releasing functional volatile organic compounds (VOCs) [4e6]. This group of compounds is characterized by relatively low molecular mass (<300 Da), low boiling point, and lipophilic properties. VOCs are ideal “infochemicals” (i.e., chemicals that mediate interactions between organisms) because they occur in the biosphere in a wide range of concentrations, and can act over long distances [7]. Because they are capable of diffusing through aqueous solutions as well as through the atmosphere, volatiles can mediate interactions both above and below the ground [5]. Thus, they can have major effects on nearby organisms, and on development of organisms in an ecosystem. A few examples of the beneficial functions of VOCs are helping pollinators to locate flowers, attracting predators of
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herbivores, and directly killing or inhibiting growth of pathogenic organisms. Economically important agricultural crops include not only traditional food-, forage-, and fiber-producing species, but also species with secondary metabolites having desired aromatic or therapeutic qualities, or providing source material for the perfume and chemical industries. Lipid (oil) constituents of certain species are used as chiral auxiliaries in synthetic organic chemistry, and in microbial transformation of common chemical structures to yield highly functionalized substances of economic value [8]. Peppermint (Mentha piperita L.; family Labiatae) is an important and commonly-used flavoring agent world-wide. Fresh or dried leaves of Mentha species are used as condiments, and essential oils (EOs) of these plants, which are stored in glandular hairs, are used as flavorings for foods and beverages, as fragrances, and as fungicides or insecticides in many pharmaceutical and industrial products [9,10]. Menthol, a crystalline compound obtained from peppermint oil, has a pleasant flavor and aroma, and a cooling anesthetic effect, and is used in confectioneries, pharmaceuticals, oral health care products, cosmetics, teas, and tobacco products. Methods to enhance growth of economically important plants undergo constant evolution and improvement. Many recent studies have attempted to manipulate rhizobacterial populations by inoculation of beneficial bacteria, to promote growth of crop plants. This approach has potential environmental benefits since it reduces use of agricultural chemicals, and is compatible with sustainable management practices. “Organic agriculture” is an increasingly popular production system which avoids or minimizes use of synthetic fertilizers, pesticides, and growth regulators, relying instead on bio-fertilization, crop rotation, crop residues, mechanical cultivation, and biological pest control to maintain soil productivity [11]. It is particularly desirable to avoid use of synthetic compounds in production of aromatic and medicinal plants, which are typically consumed without further processing after harvest. In this study, we evaluated effects of VOCs produced by soil bacteria on growth parameters of M. piperita, and on qualitative and quantitative composition of EOs.
in our previous study on sweet basil and sweet marjoram [12,13]. Effects of the VOC emission on plant development varied depending on the PGPR species (Fig. 1; Table 1). Clear differences among treatments were detectable after 30 days growth. VOCs emitted by Pseudomonas fluorescens or Bacillus subtilis caused significant (p < 0.05) increases (2.5- and 1.3-fold, respectively) in number of shoot ramifications and in root length (Table 1). None of the treatments resulted in significant change of leaf number, shoot length, or node number. Exposure to B. subtilis VOCs caused a 2-fold increase (p < 0.05) in shoot fresh weight; similar effects were observed for P. fluorescens treatment (Fig. 1). Similarly, root dry weight in B. subtilis-treated plants was 3.5-fold higher than in controls, and significantly (p < 0.05) higher than in plants exposed to VOCs of other PGPR species (Fig. 1). Increased shoot fresh weight for B. subtilis-treated plants was due to a combination of w2-fold increase in leaf area (Fig. 2) (p < 0.05), and elongation of internodes (data not shown). In P. fluorescens- and Azospirillum brasilense-treated plants, EO yields were respectively 4.46 and 3.22 mg/mg fresh weight, w2-fold higher than in controls (Fig. 3). Yields of major EOs (þ)pulegone, ()menthone, ()menthol, and (þ)menthofuran were generally higher in treated plants compared to controls (Fig. 4). Pulegone concentration was significantly increased (3.14-fold; p < 0.05) only by P. fluorescens treatment. Menthone was increased 15.4- and 13.5-fold (p < 0.05) in P. fluorescens- and A. brasilense-treated plants. Menthofuran was increased significantly in P. fluorescenstreated plants. The only decreases in EO yield (w5-fold) were observed for menthol and menthofuran in A. brasilense-treated plants. Exposure to PGPR VOCs led to changes of relative percentage (R%), as well as yield, of EOs (Table 2). R% for pulegone, the main EO component, increased to 59.9% in P. fluorescens-treated plants, compared to 45.3% in controls. R% for menthone also increased in all cases. R% for menthol was lower in P. fluorescens- and A. brasilense-treated plants (6.1%; 5.9%) than in controls (9.6%), but was higher for B. subtilis-treated plants (11.3%). The only EO showing a significant R% decrease in A. brasilense-treated plants was menthofuran.
2. Results 3. Discussion To investigate whether bacterial (PGPR) VOCs affected growth of M. piperita, plants and bacteria were grown on the same dish with a physical separation such that VOCs but not solutes from the bacteria could reach the plant. Results were consistent with those
Many studies have demonstrated the ability of soil-dwelling organisms to synthesize and release volatile organic compounds (VOCs), which can travel short or long distances through soil [14].
30
400 ab
b
b
300
200
20
ab
mg
mg
ab
a
ab 10 a
100
0
0
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shoot fresh weight Control
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Fig. 1. Shoot fresh weight and root dry weight of M. piperita exposed to VOCs from three PGPR species. Letters above bars indicate significant differences according to Fisher’s LSD test (p < 0.05).
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Table 1 Effect of exposure to VOCs from three PGPR species on plant growth parameters of Mentha piperita. Data shown are mean SE. Values within a column followed by the same letter are not significantly different according to Fisher’s LSD test (p < 0.05). Treatment
Leaves (n )
Node (n )
Ramification (n )
Shoot length (cm)
Root length (cm)
Control P. fluorescens WCS417r B. subtilis GB03 A. brasilense SP7
23.48 2.23 33.81 3.61 34.91 5.63 28.22 4.17
7.26 0.29 8.04 0.67 7.23 0.32 8.05 0.37
1.14 0.13a 2.68 0.39b 2.92 0.46b 1.24 0.26a
4.58 0.35 5.05 0.45 5.70 0.54 4.50 0.61
6.32 0.37a 8.77 0.89b 8.61 0.28b 7.44 0.79ab
Little is known regarding the ability and efficiency of rhizobacteria to release VOCs. The present study showed that many growth parameters of peppermint (M. piperita) are increased upon exposure to VOCs from the bacteria P. fluorescens and B. subtilis, consistent with previous reports [12,15]. In our I-plate assays, only low molecular weight VOCs had such effect. In the study by Ryu et al. [15], two volatile components from B. subtilis were identified as strong promoters of growth in Arabidopsis thaliana (family Brassicaceae): 3-hydroxy-2-butanone (acetoin) and (2R,3R)butanediol. In the present study, root dry weight was increased 3.5- and 2.7fold, respectively, by exposure to B. subtilis and P. fluorescens. This may be attributed in part to root elongation (Table 1) induced by auxin. In a study of A. thaliana, Zhang et al. [16] presented transcriptional and histochemical data indicating that VOCs from B. subtilis promote growth by increasing auxin accumulation in roots. Shoot fresh weight was also significantly increased by exposure to bacterial VOCs. Since leaf number did not increase, this effect may be due to increases of ramification number (Table 1) and/or leaf area (Fig. 2). The above findings rule out the possibility that growth promotion resulted simply from increased plant hydration. Microarray data presented by Zhang et al. [17] suggest that exposure to B. subtilis VOCs enhanced photosynthetic efficiency, chlorophyll content, and cell expansion. Our previous study of sweet basil (Ocimum basilicum) showed that exposure to B. subtilis VOCs promoted shoot and root length, leaf number, leaf area, and shoot fresh weight [13]. The present results for M. piperita seem contradictory to previous findings by Vespermann et al. [5] and Kai et al. [6] that VOCs released by P. fluorescens strongly inhibited root and leaf growth of A. thaliana. However, use of different Pseudomonas species or isolates, and variations in growth conditions and testing systems, may account for the different results; bioactive compounds emitted by Pseudomonas species have not yet been identified. Regarding the effect of rhizobacterial VOCs on formation of plant secondary compounds, we observed an almost 2-fold increase in
accumulation of EOs in plants treated with P. fluorescens as compared to B. subtilis, A. brasilensis, or control. The major EO components of M. piperita (i.e., (þ)pulegone, ()menthone, () menthol, (þ)menthofuran) showed increased accumulation upon exposure to P. fluorescens, but not B. subtilis. In our previous study, in contrast, O. basilicum plants exposed to B. subtilis VOCs showed increased accumulation and content of major EOs [12]. Relative percentages (R%) of EOs were also changed by exposure to bacterial VOCs. In P. fluorescens-treated plants, R% of pulegone was 30% higher than in controls, and R% of menthofuran was reduced to 0.8%, as compared to 1.16% in controls or A. brasilensetreated plants. The enhanced levels of EOs were not due to increased biomass, suggesting that bacterial VOCs induce biosynthesis of secondary metabolites. The observed increase of monoterpenes in M. piperita may have resulted from induction of systemic resistance (ISR). ISR occurs upon stimulation of a plant’s defense mechanisms. Ryu et al. [15] observed that rhizobacterial VOCs caused ISR in Arabidopsis. ISR has been shown to increase plant cell wall strength, and to alter plant physiology and metabolic responses, leading to enhanced synthesis of defensive chemicals upon challenge by pathogens and/ or abiotic stress factors [18]. Terpenes play important roles in plant defenses, and help the plant’s photosynthetic apparatus recover from brief exposure to high temperature. Isoprene appears to physically stabilize thylakoid membranes at high temperature, and to quench reactive oxygen species, e.g., ozone, that cause membrane damage [19]. Several monoterpenes are synthesized de novo [20,21]. in aromatic and other types of plants [22,23]. in response to herbivory, apparently to prevent damage from further attacks [10]. Increased synthesis of EOs is a defensive response to colonization by microorganisms, since several EOs have antimicrobial properties [8]. Biosynthesis of terpenoids depends on primary metabolism, e.g., photosynthesis, on oxidative pathways for carbon, and on energy supply [24]. Bacterial VOCs are capable of improving photosynthetic efficiency [15,17]. In the present study, (þ)pulegone, the major EO of M. piperita, was enhanced >3-fold by exposure to
12
6 b
10
Total Essential Oil Yield (ug/mg fresh weight)
b
cm2
8 a 6
a
4 2
b
5
ab 4 3
a a
2 1
0 leaf area Control
P. fluorescens
B. subtilis
0 A. brasilensis
Fig. 2. Leaf area of 30-day old M. piperita plants exposed to VOCs from three PGPR species. Letters above bars indicate significant differences according to Fisher’s LSD test (p < 0.05).
Control
P. fluorescens
B. subtilis
A. brasilensis
Fig. 3. Essential oil (EO) yield in M. piperita exposed to VOCs from three PGPR species. Letters above bars indicate significant differences according to Fisher’s LSD test (p < 0.05).
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0,4
4
a
a
0,3
3 a
2 a a
fresh weight ug/mg
fresh weight ug/mg
b
a
0,2 bc c ab
0,1
1
c b
a
ab
bc
a
0
0
(-)-menthone
(+)-pulegone Control
P. fluorescens
(-)-menthol
B. subtilis
(+)-menthofuran
A. brasilensis
Fig. 4. Concentrations of major EO components in M. piperita exposed to VOCs from three PGPR species. Letters above bars indicate significant differences according to Fisher’s LSD test (p < 0.05).
rhizobacterial VOCs. Pulegone is a potent inhibitor of acetylcholinesterase (AchE) in invertebrates [10,25], and has been shown to cause a repellent effect [26], toxicity [10,27e30], or interference with development and reproduction [31] in various insect species. Pulegone can also provide a biochemical barrier to host plant utilization, by eliminating herbivore symbionts [10]. Menthone and pulegone compounds seem to be antagonistic, which may further enhance toxic effects [28]. (þ)Pulegone is a central intermediate in biosynthesis of ()menthol. Depending on environmental conditions, this branch point metabolite may be reduced to ()menthone en route to menthol by pulegone reductase, or oxidized to (þ) menthofuran by menthofuran synthase [32]. The observed changes in EO levels suggest that VOCs from soil bacteria, besides inducing biosynthesis of secondary metabolites in M. piperita, influence pathway flux or specific steps of monoterpene metabolism. Increased pulegone content in EOs may enhance pulegone reductase enzyme activity, and favor somehow reduction of pulegone to menthone, increasing cytochrome P450 (þ)MFS enzyme activity, rather than oxidation to menthofuran. Full interaction of bacteria with plants (inoculation) may induce secondary metabolite responses [13,33,34]. Inoculation of marjoram (Origanum majorana) with P. fluorescens increased yield and accumulation of major EOs. Plant growth can be promoted by bacterial effects including growth hormone production, VOC production, solubilization of phosphates, oxidation of sulfur, increased nitrate availability, increased root permeability, bacterial volatiles, and combinations of these effects. It is noteworthy that A. brasilense VOCs did not significantly affect on plant growth parameters or EO yields, and caused a decrease in menthol level. B. subtilis VOCs showed a growthpromoting effect in both O. basilicum and M. piperita, but caused increased yield of EOs only in the former species. These findings Table 2 Variation in relative percentage (R%) of major EOs in M. piperita treated with VOCs from three PGPR species. Data shown are mean SE. Treatment
(þ)Pulegone ()menthone ()Menthol
(þ)Menthofuran
Control P. fluorescens WCS417r B. subtilis GB03 A. brasilense SP7
45.29 9.90 59.95 7.71
1.65 0.68 2.23 0.78
9.53 2.80 6.11 1.89
1.16 0.09 1.44 0.31
50.07 9.93 56.48 9.51
2.59 1.13 2.92 1.05
11.3 2.89 5.91 2.01
1.39 0.35 0.88 0.25
suggest that effects of bacterial VOCs on plants are species-specific. I.e., VOCs from a given bacterial strain do not cause the same effects, or to the same degree, in all plant species; responses are characteristic of a specific plantebacteria combination. Possible explanations for this phenomenon are: (i) different plants respond to different component(s) of VOC mixtures; (ii) reactive sites are different; (iii) plant differ in their ability to metabolize VOCs. Various rhizobacteria show great variability in qualitative and quantitative complexity of their VOC profiles. The number of different compounds produced and emitted by a particular strain may be as high as 60 [4]. For most VOCs, the exact chemical structure remains to be elucidated [6]. Manifestation of characteristic volatile compound or profile is attributable to specific metabolic pathways active in the bacterium. Depending on the growth conditions or medium, the “bouquet” of released VOCs can vary for a particular species. Occurrence of VOC-mediated interactions between bacteria and plants are clearly demonstrated by results of this and previous studies. Such interactions are species-specific. Certain VOCs are capable of enhancing growth and productivity of plant species. Functional analyses in future studies will clarify roles of VOCs in plant interactions on the population or community level, and in maintaining ecological balance. VOCs from bacteria show a diversity and complexity comparable to those from plants and fungi, and are rich sources for new natural compounds. Biological functions of most bacterial VOCs are not yet well understood. In analogy to VOCs from plants and fungi, we expect that they may serve as: (a) “infochemicals” for communication within and among organisms; (b) cell-to-cell communication signals; (c) carbon release valves: (d) growth-promoting or -inhibiting agents. The present results indicate that VOCs from soil bacteria, released underground, play crucial roles in rhizosphere interactions. Structural elucidation and biological activities of these volatile compounds are subjects of ongoing and future studies. 4. Materials and methods 4.1. Bacterial strains, culture conditions, media, and treatments Three strains previously reported as PGPR (P. fluorescens WCS417r, B. subtilis GB03, A. brasilense SP7) [35] were assessed for their effects. These strains were grown respectively on media
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termed TSA (15 g/l tryptone, 5 g/l soy peptone, 5 g/l NaCl), and LB (10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl), for routine use, and maintained in nutrient broth with 20% glycerol at 80 C for longterm storage. For experiments, bacteria were grown on nutrient agar, single colonies were transferred to 100 ml flasks containing culture medium, and grown aerobically on a rotating shaker (200 rpm) for 48 h at 28 C. Bacterial suspension was diluted in sterile distilled water to final concentration 109 CFU/ml, and 20 ml of the resulting suspension was used for each treatment. 5e10 Plants were used for each treatment, and experiments were replicated 4. 4.2. Micropropagation of plants Young shoots from M. piperita plants grown in Traslasierra Valley (Córdoba province, Argentina) were surface disinfected by soaking 1 min in 17% sodium hypochlorite solution, and rinsed 3 in sterile distilled water. Disinfected shoots were cultured in 100 ml of MS culture medium containing 0.7% (w/v) agar and 1.5% (w/v) sucrose [36]. All culture media contained 30 g/l sucrose and 7.5 g/l agar. Stage I. Initial shoot-tip culture. After 30 days, apical meristems with foliar primordia, not showing contamination, were aseptically removed from terminal buds of shoots obtained in the previous step. Explants were cultured in test tubes, in 40 ml MS medium with 0.66 mg/l indolebutyric acid. Stage II. Growth and in vitro multiplication. Plantlets obtained from tips were multiplied by single node culture, and MS medium was adjusted to 5.6e5.8 pH prior to autoclaving (20 min, 121 C). Explants were placed in a growth chamber with controlled conditions of light (16/8-h light/dark cycle), temperature (22 2 C), and relative humidity (w70%). Stage III. Exposure to volatiles. One node, from aseptically cultured plantlet, was placed on one side of a specialized plastic Petri dish (90 15 mm) containing a center partition (I-plate, Fisher Scientific). Both sides of the dish contained 50% strength MS solid medium. 20 ml Suspension culture of various PGPR strains in sterile distilled water was applied drop-wise to the side of the dish opposite the plant node. By this method, plants were exposed to bacterial VOCs without physical contact. Dishes were sealed with parafilm, arranged in a completely randomized design, and placed in a growth chamber with controlled conditions of light (16/8-h light/dark cycle), temperature (22 2 C), and relative humidity (w70%). Plants were harvested after 30 days. 10 plants were used for each treatment, and experiments were replicated 4. 4.3. Plant growth measurement Plants were removed from the partitioned dish, roots were rinsed with water to remove MS medium, and growth-promoting effects of bacterial treatments were evaluated in terms of shoot and root lengths, numbers of nodes, leaves, and shoot ramifications, shoot fresh weight, root dry weight, and leaf area. Total leaf surface area was measured using integrated digital image analysis. Images (JPG file, 640 480 pixels) of plant trays from above were taken by digital camera at a distance of w80 cm. JPG files were adjusted using tools under the Image menu of Adobe PhotoshopÒ 3. Color was removed to obtain a grayscale-only image, and pixels were converted to cm as a unit of measurement. A “standard” dish was included within the image for calibrating the pixel conversion. All leaves in the image were outlined, and the “measure” option under “Analyze” menu was used to estimate surface area [37]. 4.4. Extraction of essential oils (EOs) Each shoot sample was weighed, subjected to hydrodistillation in a micro Clevenger-like apparatus for 30 min, and volatile fraction
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was collected in dichloromethane. Internal standard was added (0.1 ml dodecalactone in 50 ml ethanol). M. piperita plants contain w3% volatile oils, consisting of >50 different compounds. EOs, accounting for w60% of total oil volume, were (þ)pulegone, ()menthone, ()menthol, and (þ)menthofuran. These compounds were quantified in relation to dodecalactone, which was added during the distillation procedure. FID response factors for each compound generated essentially equivalent areas (difference < 5%). Chemical analyses were performed using a PerkineElmer Clarus 600 gas chromatograph (GC) equipped with CBP-1 capillary column (30 m 0.25 mm, film thickness 0.25 mm) and mass-selective detector. Analytical conditions: injector and detector temperatures 250 and 270 C, respectively; oven temperature programmed from 60 C (3 min) to 240 C at 4 /min; carrier gas ¼ helium at constant 0.9 ml/min flow; source 70 eV. Oil components were identified based on mass spectra and retention times, in comparison to standard [12]. GC analysis was performed using a PerkineElmer Clarus 500 GC, fitted with 30 m 0.25 mm fused silica capillary column coated with Supelcowax 10 (film thickness 0.25 mm). GC operating conditions: oven temperature programmed from 60 C (3 min) to 240 C at 4 /min, injector and detector temperatures 250 C; detector FID; carrier gas ¼ nitrogen at 0.9 ml/min constant flow. 4.5. Statistical analyses Data were subjected to analysis of variance (ANOVA) followed by comparison of multiple treatment levels with control, using post hoc Fisher’s LSD (least significant difference) test. Infostat software version 2.0 (Group Infostat, Universidad Nacional de Córdoba, Argentina) was used for all statistical analyses. 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). EB, JZ, and WG are Career Members of the CONICET. MS has a fellowship from the CONICET-MCyT Cba. The authors are grateful to Steve Anderson for English editing of the MS. References [1] O.O. Babalola, Beneficial bacteria of agricultural importance, Biotechnol. Lett. 32 (2010) 1559e1570. [2] L.C. Van Loon, Plant response to plant growth-promoting rhizobacteria, Eur. J. Plant Pathol. 119 (2007) 243e254. [3] R.S. Niranjan, H.S. Shetty, M.S. Reddy, Plant growth promoting rhizobacteria: potential green alternative for plant productivity. in: Z.A. Siddiqui (Ed.), PGPR: Biocontrol and Biofertilization. Springer, Netherlands, 2006, pp. 197e216. [4] M. Kai, U. Effmert, G. Berg, B. Piechulla, Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen Rhizoctonia solani, Arch. Microbiol. 187 (2007) 351e360. [5] Vespermann, M. Kai, B. Piechulla, Rhizobacterial volatiles affect the growth of fungi and Arabidopsis thaliana, Appl. Environ. Microbiol. 73 (2007) 5639e5641. [6] M. Kai, M. Haustein, F. Molina, A. Petri, B. Scholz, B. Piechulla, Bacterial volatiles and their action potential, Appl. Microbiol. Biotechnol. 81 (2009) 1001e1012. [7] R.E. Wheatley, The consequences of volatile organic compound mediated bacterial and fungal interactions, Antonie van Leeuwenhoek 81 (2002) 357e364. [8] N.S. Sangwan, A.H.A. Farooqi, F. Shabih, R.S. Sangwan, Regulation of essential oil production in plants, Plant Growth Regul. 34 (2001) 3e21. [9] D. Ram, M. Ram, R. Singh, Optimization of water and nitrogen application to menthol mint (Mentha arvensis L.) through sugarcane trash mulch in a sandy loam soil of semi-arid subtropical climate, Bioresour. Technol. 97 (2006) 886e893. [10] P. Harrewijn, A.M. Van Oosten, P.G. Piron, Natural Terpenoids as Messengers. A Multidisciplinary Study of Their Production, Biological Functions and Practical Applications. Kluwer Academic Publishers, London, U.K., 2001, p. 440. [11] K. Lind, G. Lafer, K. Schloffer, G. Innerhoffer, H. Meister, Organic Fruit Growing. CABI Publishing, Wallingford, UK, 2004, p. 281.
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