Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas ISSN: 0717-7917 editor.blacpma@usach.cl Universidad de Santiago de Chile Chile Chaverri, Carlos; Cicció, José F. Composition of the essential oil from leaves of Smallanthus quichensis (Asteraceae) from Costa Rica Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, vol. 14, núm. 5, 2015, pp. 355-363 Universidad de Santiago de Chile Santiago, Chile Available in: http://www.redalyc.org/articulo.oa?id=85641105002 How to cite Complete issue More information about this article Journal's homepage in redalyc.org Scientific Information System Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Non-profit academic project, developed under the open access initiative © 2015 Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas 14 (4): 273 - 279 ISSN 0717 7917 www.blacpma.usach.cl Artículo Original | Original Article Arbuscular mycorrhizal symbiosis increases the content of volatile terpenes and plant performance in Satureja macrostema (Benth.) Briq. [La simbiosis micorrícica arbuscular aumenta el contenido de terpenos volátiles y el rendimiento vegetal en Satureja macrostema (Benth.) Briq.] Yazmín Carreón-Abud1, Rafael Torres-Martínez2, Brenda Farfán-Soto1, Alejandra Hernández-García2, Patricia Ríos-Chávez1, Miguel Ángel Bello-González3, Miguel Martínez-Trujillo1 & Rafael Salgado-Garciglia2 1 Laboratorio de Microbiología y Genética, Facultad de Biología, Laboratorio. de Biotecnología Vegetal, Instituto de Investigaciones Químico-Biológicas, 3 Facultad de Agrobiología Presidente Juárez, Universidad Michoacana de San Nicolás de Hidalgo, Michoacán, México Contactos | Contacts: Rafael SALGADO-GARCIGLIA - E-mail address: rsalgadogarciglia@gmail.com 2 Abstract: We studied the effect of Rhizophagus irregularis on plant performance and volatile terpenes content of the Mexican native medicinal plant Satureja macrostema (Benth.) Briq. (Lamiaceae) in greenhouse conditions. The growth parameters considered in this research and the composition of volatile components were quantified monthly in mycorrhizal and non-mycorrhizal plants. The essential oil was collected from aerial parts and analyzed by gas chromatography-mass spectrometry. Colonization by R. irregularis significantly increased biomass, shoot and root length, and the amount of volatile terpenes. The more concentrated volatile terpenes were limonene, βlinalool, menthone, pulegone, and verbenol acetate. It is concluded that the use of R. irregularis allows optimal growth of S. macrostema plants in low fertility soils and increased production of the main components of the essential oil. Keywords: Essential oils, medicinal plants, Rhizophagus irregularis, terpenes Resumen: El efecto de Rhizophagus irregularis sobre el rendimiento vegetal y la producción de los terpenos volátiles de Satureja macrostema (Benth.) Briq. (Lamiaceae), una planta medicinal nativa mexicana, fue estudiado en condiciones de invernadero. Los parámetros de crecimiento considerados en esta investigación y los componentes volátiles, fueron cuantificados mensualmente en plantas con y sin micorrizas. El aceite esencial fue colectado de la parte aérea y fue analizado por técnicas de cromatografía de gases-espectrometría de masas. La colonización de R. irregularis aumentó significativamente la biomasa, longitud de tallo y raíz, y la cantidad de terpenos volátiles. Los terpenos volátiles mayoritarios fueron limoneno, β-linalol, mentona, pulegona y acetato de verbenol. Se concluye que el uso de R. irregularis permitió un óptimo crecimiento de las plantas de S. macrostema en suelos de baja fertilidad, con un aumento de los componentes principales del aceite esencial. Palabras clave: Aceites esenciales, plantas medicinales, Rhizophagus irregularis, terpenos Recibido | Received: June 13, 2014 Aceptado | Accepted: December 6, 2014 Aceptado en versión corregida | Accepted in revised form: June 28, 2015 Publicado en línea | Published online: July 30, 2015 Declaración de intereses | Declaration of interests: This work was financed by COECYT and CIC/UMSNH (2.10rsg).. Este artículo puede ser citado como / This article must be cited as: Y Carreón-Abud, R Torres-Martínez, B Farfán-Soto, A Hernández-García, P Ríos-Chávez, MÁ BelloGonzález, M Martínez-Trujillo, R Salgado-Garciglia. 2015. Arbuscular mycorrhizal symbiosis increases the content of volatile terpenes and plant performance in Satureja macrostema (Benth.) Briq.. Bol Latinoam Caribe Plant Med Aromat 14 (4): 273 – 279. 273 Carreón-Abud et al. Mycorrhization increases terpenes and plant performance in S. macrostema INTRODUCTION Most of the medicinal plants cultivated in Mexico are not native to the country, such as chamomile (Matricaria recutita), lavender (Lavandula angustifolia), rosemary (Rosmarinus officinalis), thyme (Thymus vulgaris), marjoram (Origanum majorana) and spearmint (Mentha spicata), among others (Estrada et al., 1995). However, there is very little documented research of agronomic factors affecting native medicinal plant’s qualitative and quantitative characteristics. It is known that mineral fertilization increases biomass production, which is associated with increasing content and number of components of secondary metabolites of economic interest, such as volatile oils (Zelyazkovet et al., 2009). Furthermore, it has been reported that application of Arbuscular Mycorrhizal (AM) symbiosis contributes to the growth of the host plant and to the synthesis, accumulation, and quality of some secondary metabolites such as terpenoids, flavonoids, and phytoalexins (Akiyama & Hayashi, 2002; Larose et al., 2002; Yao et al., 2003). Arbuscular mycorrhizal fungi (AMF) affect secondary metabolism and the production of active compounds of medicinal plants and thus influence the quality of herbal medicines (Zeng et al., 2013). Mycorrhizal colonization increased the content and composition of essential oils produced by plants of Ocimum basilicum, Mentha arvensis, Santolina chamaecyparissus, Salvia officinalis, Lavandula angustifolia, Geranium dissectum, Origanum dictamnus, and Artemisia annua (Freiteas et al., 2004; Rapparini et al., 2008; Karagiannidis et al., 2012). Due to the high demand for native medicinal plants collected from the wild, investigations are needed for clarifying the factors determining the variation of volatile compounds in order to increase the yields of essential oils of plants that grown in wild and greenhouse (Estrada, 2002). Such is the case of Satureja macrostema (Benth.) Briq. (Lamiaceae) commonly known as “nurite” that is used in traditional medicine in central-western Mexico (Bello, 1993; Rzedowski & Rzedowski, 2001). S. macrostema contains a mixture of flavonoids that have been extensively studied for its antioxidant effects (Perez-Gutierrez & Gallardo-Navarro, 2010), but other volatile compounds are also reported, such as limonene, pulegone, and menthone, which are considered to be antimicrobials (Bello, 2006). Wild nurite plants are over-collected to satisfy the demand from regional and national markets (Bello, 2006), but there are no programs designed for its domestication involving propagation systems, cultivation practices in the greenhouse, and studies of volatile oil content variation. Therefore, the aim of our investigation was to determine the effect of Rhizophagus irregularis MUCL 41833 on the content of volatile compounds produced by S. macrostema plants grown in the greenhouse. Material and methods Rhizophagus irregularis MUCL 41833 Single cultures of Rhizophagus irregularis MUCL 41833 were subcultured and propagated in carrot hairy roots on low mineral media minimal (M medium) (Balaji et al., 1995). Several thousand spores were obtained in a period of 4 months, which were isolated by solubilization of the medium with citrate buffer (10 mM), and afterwards maintained in sterile deionized water (Cranenbrouck et al., 2005). Plant material and mycorrhizal inoculation Plants of Satureja macrostema (Benth.) Briq. (Lamiaceae) were propagated from seeds collected from plantations established in the experimental area of Nuevo San Juan Parangaricutiro, Michoacán, Mexico (19°25’23’’N, 102°07’47’’W). Seeds were sterilized for 10 min in NaClO solution (1.2%), and then rinsed three times for 5 min in sterile deionized water. Seeds were planted in germination trays containing soil:sand mix (v/v, 1:1, pH 5.5) in greenhouse conditions (16 h day length, temperature between 22-25° C, 70% relative humidity), and watered three times a week. Previously, the soil:sand mix was sterilized at 180° C for 2 h (Copetta et al., 2006). After 15 days of germination, seedlings (5 cm in height) with fully expanded cotyledonary leaves and well developed roots were individually transferred to pots containing soil:sand mix sterilized (1.5 kg, v/v, 3:1, pH 5.5). At 7 days after transplantation, soil at the base of S. macrostema plants was gently pushed aside to expose portions of the roots system, and then the inoculation was realized with the R. irregularis (50 spores/plant). Roots were covered with soil immediately after inoculum application. The S. macrostema plants with and without mycorrhiza were grown in greenhouse conditions and irrigated every 20 days, for up to twelve weeks, with Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/274 Carreón-Abud et al. Mycorrhization increases terpenes and plant performance in S. macrostema a low phosphate level (1 mM KH2PO4, modified Hoagland’s solution) (Hoagland & Arnon, 1950). Colonization percentage At 30 and 90 days of inoculation, the plants were taken out of the pots and washed carefully to remove substrate from roots. A simple sample (2 g) of roots of test plants was taken to determine mycorrhizal colonization, by staining with trypan blue in lactophenol (Phillips & Hayman, 1970). The percentage of colonization of the root system was quantified by the grid intersect method, which estimates the percentage of root colonized. Is a procedure whereby the presence or absence of colonization at each intersection of root and gridline is noted, after dispersing the roots above a grid of square drawn on a Petri dish and observing under a dissecting microscope at X40 magnification (McGonigle et al., 1990). Growth parameters Seven plants from each treatment were harvested at 30, 60 and 90 days after inoculation with R. irregularis for recording various morphological parameters: dry weight (dry wt) of shoots and roots (g/plant); shoot and root length (cm). Shoot and root dry weights were determined by weighing samples after being washed, dried with filter paper, and then drying for 24 h at 80° C. Plants were distributed in a randomized design and the data on morphometric parameters were compared by one-way ANOVA (P < 0.05). Standard errors were calculated for all data. GC-MS analysis Aerial parts (apical shoot with 3-4 leaves) from three specimens of plants with and without mycorrhiza were collected early in the morning. Plant material was extracted immediately by vigorous shaking with n-hexane (10 mL of n-hexane per gram of sample), the extract was macerated for 5 days (4° C), filtered (Whatman Nº 1 filter paper), and n-hexane was evaporated to dryness at 45° C in vacuum evaporator. The residues were dissolved in n-hexane at 1 mg/mL and were analyzed by gas chromatography-mass spectrometry (GC-MS). GC-MS analyses were performed on a GCHP6890-GCMS HP5973 fused silica analytical HP-5 MS capillary column (25 m x 0.25 mm x 0.25 µm film thickness). The temperature programmed for the gas chromatography was as follows: initial temperature of 60° C held for 5 min, linear gradient of 5° C/min to 300° C, a final hold time of 30 min. The injector temperature was 260° C, and injection was performed in a split radio 1/30. The carrier gas was helium (99.99% purity, 1.0 mL/min). Injection volume of each sample was 1 μL. Retention indices for all compounds were determined using n-alkanes (C8 to C20) as standards. Compounds were identified by comparison of their MS spectra with the NIST02 mass spectral library (National Institute of Standards and Technology), as well as by comparison of their retention indices with those described by Adams (2007). Quantitative determination was based on the total ion count detected by the GC-MS. Statistical analysis of the data was carried out by analysis of variance (P ≤ 0.05 significance level, n = 3). RESULTS AND DISCUSSION At 30 days of R. irregularis inoculation S. macrostema plants showed a percentage of colonization of 30% with observation of hyphae and arbuscules. The mycorrhizal colonization increased proportionally at 60 and 90 days, with 60 and 80%, respectively. Such values are considered acceptable for other medicinal plants to these times of cultivation (Freiteas et al., 2004; Binet et al., 2011; Karagiannidis et al., 2012). In Catharanthus roseus, mycorrhizal colonization with G. fasciculatum was 85% at 150 days (Karthikeyan et al., 2009). These results demonstrate that S. macrostema is a plant with high degree of mycotrophy. Plants of S. macrostema inoculated with R. irregularis showed improved growth and development when compared to control plants (nonmycorrhizal), being larger the root and shoot biomasses (dry weight) of inoculated plants. At 90 days, the length of shoots and roots, and the biomass was significantly higher in mycorrhizal plants (Table 1). The results are in agreement with the findings of earlier work by Gupta & Janardhanam (1991), who recorded a two-fold increase in growth and three-fold increase in biomass production as compared to plants without mycorrhiza in Cymbopogon martini on inoculation with Glomus aggregatum. A similar response was also observed in ten medicinal plants (Abrus precatorium, Cynodon dactylon, Euphorbia tirucalli, Gymnema elegans, Hemidesmus indicus, Ocimum basilicum, Plumbago zeylanica, Phyllanthus amarus, Sida acuta and S. rhombifolia) inoculated with three AMF species (G. mosseae, G. fasciculatum and G. monosporum), recording the highest AMF infection (86%) in Arbus precatorius, and the lowest in Phyllanthus amarus (36%) (Kumar & Murugesh, 2002). Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/275 Carreón-Abud et al. Mycorrhization increases terpenes and plant performance in S. macrostema In a study by Coppeta et al. (2007), inoculation of sweet basil (Ocimum basilicum var. Genovese) plants with Gigaspora rosea significantly increased shoot length (54.7 cm) and number of nodes (9.0) in comparison with control plants (46.9 cm, 7.7 nodes) and the other fungal treatments (Glomus mosseae or Gigaspora margarita) at 63 days of culture. The authors reporting that mycorrhizal inoculation was advantageous in terms of obtaining healthy vigorous seedlings, and a higher biomass of plants that grew better in the field. The reason for these effects may be the formation of external mycelium surrounding the roots by R. irregularis. The extramatrical hyphae produced by AMF act as extensions of roots increasing the surface area of the root system and making it more efficient for absorption of water, and for diffusion of limited nutrients. This effect is more pronounced in phosphorus deficient soils (Bagyaraj & Reddy, 2000). It has been demonstrated that plants inoculated with AMF utilize more soluble phosphate from soil than non-mycorrhizal plants (Antunes & Cardoso, 1991). Table 1 Effect of R. irregularis inoculation on the growth of Satureja macrostema plants in greenhouse Growth Non-mycorrhizal plants Mycorrhizal plants parameters 30d 60d 90d 30d 60d 90d Root dry wt 2.7 4.9 7.2 5.9 7.6 12* (g/plant) Shoot dry wt 5.8 8.8 11.2 9.2 18.4 24.6* (g/plant) Root length 8.82 17 24.2 14.2 20.8 28.1* (cm) Shoot length 18.2 29.7 34.6 22.1 42.1 48.2* (cm) *Significant difference (n=7, P < 0.05) The volatile compounds were identified and quantified during the 90 days of experimentation, the major terpenes found were limonene, β-linalool, menthone, pulegone, and verbenol acetate. However, the content of these terpenes varied according to plant development and mycorrhizal colonization. Table 2 summarizes the main volatile compounds identified by GC-MS in aerial parts of control plants of S. macrostema after 30 days under greenhouse conditions. Pulegone (58.3%) and β-linalool (30.4%) were the main compounds, followed by limonene (1.5%), menthone (2.8%) and verbenol acetate (7.0%). The volatile terpenes content did not change significantly during culture of S. macrostema nonmycorrhizal plants, with the exception of pulegone that dramatically increased at 60 days of culture (33.4%), diminishing again at 90 days (Table 3). However, volatile terpenes showed a tendency to increase in mycorrhizal plants, in which they were produced at larger amounts in non-mycorrhizal plants beginning at 30 days of culture. At 90 days, the production of limonene, β-linalool, menthone and verbenol acetate was highest in plants with mycorrhizal in comparison with non-mycorrhizal plants. However, pulegone content was most abundant at 60 days, diminishing at 90 days (Table 3). Table 2 Volatile compounds observed in aerial parts of Satureja macrostema at 30 days under greenhouse conditions. Compounda Rtb(min) IKlc GC aread (%) Limonene 9.08 1024 1.5 β-Linalool 11.14 1095 30.4 Menthone 13.05 1164 2.8 Pulegone 15.15 1233 58.3 Verbenol acetate 17.87 1340 7.0 a Identified by GC-MS; bRt: retention time; cExperimental Kovat´s Retention Index; dQuantified by GC. Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/276 Carreón-Abud et al. Mycorrhization increases terpenes and plant performance in S. macrostema Table 3 Content of volatile compounds from the aerial part of Satureja macrostema non-mycorrhizal and mycorrhizal plants. Content (µg/g fresh wt ± Sd) Non-mycorrhizal plants Mycorrhizal plants 30d 60d 90d 30d 60d 90d Limonene 1.05±0.07 1.07±1.13 1.21±0.19 2.13±0.11 2.19±0.19 4.49±0.37* β5.07±0.41 6.32±0.57 6.7±0.33 6.45±0.71 8.4±0.66 12.22±1.13* Linalool Menthone 0.667±0.09 0.991±0.11 1.17±0.18 1.03±0.97 1.47±0.13 2.97±0.21* Pulegone 11.5±1.18 16.71±1.81 12.0±1.31 18.1±1.19 34.4±2.99* 22.7 ± 2.19 Verbenol 1.11±0.09 1.14±0.14 1.16±0.17 1.16±0.18 1.28±0.13 2.42±0.27* acetate Sd: Standard deviation; *Significant difference (n=3, P ≤ 0.05) diuretic, stimulant and a carminative (Bailer et al., The content in mycorrhizal plants of β2001; Singh et al., 2005; Callan et al., 2007). These linalool, menthone, pulegone, and verbenol acetate results shown that mycorrhizal S. macrostema plants increased almost 100% respect to non-mycorrhizal produced in a greenhouse could be an alternative for plants at 90 days, but limonene incremented to 371%. production of plants with high contents of volatile The results of this experiment show that the compounds. application of R. irregularis on the roots of the plants of S. macrostema induces an increment of volatile terpenes content with respect to non-inoculated CONCLUSIONS The mycorrhizal colonization by Rhizophagus counterparts. The significant increase of the irregularis in Satureja macrostema had a positive concentration of volatile terpenes has also been effect on plant performance and increased contents of observed in Apiaceae species (Anethum graveolens, major volatile compounds. In all cases, pulegone was Coriandrum sativum and Foeniculum vulgare) the main volatile component, and limonene inoculated with Glomus spp (Kapoor et al., 2002; production was highest at 90 days of cultivation. Kapoor et al., 2004); and in Lamiaceae plants (Mentha arvensis, Ocimum basilicum and Origanum vulgare ssp. Hirtum) (Gupta et al., 2002; Copetta et ACKNOWLEDGEMENT This work was financed by COECYT and al., 2006; Toussaint et al., 2007; Morone-Fortunato CIC/UMSNH (2.10rsg). & Avato, 2008). The decrease in the content of pulegone at 90 REFERENCES days is related with the increase of limonene (Table Adams RP. 2007. Identification of essential oil 3). This behavior has been reported in plants of components by gas chromatography/mass Mentha piperita where pulegone content is highly spectrometry. 4th ed. Ed. Allured Publishing influenced by the stage of development of the plant Corporation, Carol Stream, Illinois, USA. and by the environmental conditions (Mahmoud & Akiyama K, Hayashi H. 2002. Arbuscular Croteau, 2003). mycorrhizal fungus-promoted accumulation Essential oils of different plants exert activity of two new triterpenoids in cucumber roots. antioxidant and anti-inflammatory, these effects are Biosci Biotechnol Biochem 66: 762 - 769. attributed to terpene components (Cardona & Mejia, Amanlou M, Dadkhah F, Salehnia A, Farsam H. 2009; Daniel et al., 2009). The antimicrobial, 2005. An anti-inflammatory and antiantioxidant or anti-inflammatory activity of essential nociceptive effects of hydroalcoholic extract oils of some species of Satureja have been of Satureja khuzistanica Jamzad extract. J demonstrated in vivo or in vitro models (Amanlou et Pharm Pharmaceut Sci 8: 102 - 106. al., 2005; Ozkan et al., 2007; Hajhashemi et al., Antunes V, Cardoso EJ. 1991. Growth and nutrient 2011). The high limonene content in mycorrhizal S. status of citrus plants as influenced by macrostema plants is very important by the properties attributed, it is used in pharmaceutical industry as a Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/277 Carreón-Abud et al. Mycorrhization increases terpenes and plant performance in S. macrostema mycorrhiza and phosphorus application. Plant Soil 131: 11 - 19. Bagyaraj DJ, Reddy BJD. 2000. Arbuscular mycorrhiza in sustainable agriculture. In Rajak RC: Microbial biotechnology for sustainable development and productivity. Ed. Scientific Pub., Jodhpur, India. Bailer J, Aichinger T, Hackl G, Hueber K, Dachler M. 2001. Essential oil content and composition in commercially available dill cultivars in comparison to caraway. Ind Crop Prod 14: 229 - 239. Balaji B, Poulin MJ, Vierheilig H, Piche Y. 1995. Responses of an arbuscular mycorrhizal fungus Gigaspora margarita, to exudates and volatiles from the Ri T‐DNA transformed roots of non‐mycorrhizal and mycorrhizal mutants of Pisum sativum L. Sparkle. Exp Mycol 19: 275 - 283. Bello GMA. 1993. Plantas útiles no maderables de la Sierra Purépecha, Michoacán, México, Campo Experimental Uruapan. CIRPAC, INIFAP, Michoacán, México. Folleto Técnico. Bello GMA. 2006. Catálogo de plantas medicinales de la comunidad Indígena Nuevo San Juan Parangaricutiro, Michoacán. México. Campo Experimental Uruapan. CIRPAC. INIFAP. Michoacán, México. Libro Técnico. Binet MN, van Tuinen D, Deprêtre N, Koszela N, Chambon C, Gianinazzi S. 2011. Arbuscular mycorrhizal fungi associated with Artemisia umbelliformis Lam., an endangered aromatic species in Southern French Alps, influence plant P and essential oil contents. Mycorrhiza 21: 523 - 535. Callan NW, Johnson DL, Westcott MP, Welty LE. 2007. Herb and oil composition of dill (Anethum graveolens L.): Effects of crop maturity and plant density. Ind Crops Prod 25: 282 - 287. Cardona LEH, Mejía LFG. 2009. Evaluación del efecto antioxidante de aceites esenciales y extractos de Eugenia caryophyllata, Origanum vulgare y Thymus vulgaris. Biosalud 8: 58 - 70. Copetta A, Lingua G, Berta G. 2006. Effects of three AM fungi on growth, distribution of glandular hairs, and essential oil production of Ocimum basilicum L. var. Genovese. Mycorrhiza 16: 485 - 494. Copetta A, Lingua G, Bardi L, Masoero G, Berta G. 2007. Influence of arbuscular mycorrhizal fungi on growth and essential oil composition in Ocimum basilicum var. Genovese. Caryologia 60: 110 - 116. Cranenbrouck S, Voets L, Bivort C, Renard L, Strullu DG, Declerck D. 2005. Methodologies for in vitro cultivation of arbuscular mycorrhizal fungi with root organs. In Declerck S, Strullu DG, Fortin JA: In vitro culture of mycorrhizas. Springer-Verlag, Heidelberg, Germany. Daniel AN, Sartoretto SM, Schmidt G, CaparrozAssef SM, Bersani-Amado CA, Cuman RKN. 2009. Anti-inflammatory and antinociceptive activities of eugenol essential oil in experimental animal models. Br J Pharmacol 19: 212 - 217. Estrada LE, Florencio MJ, Castellanos C. 1995. El método en etnobotánica: El enfoque transdisciplinario. In Estrada LE: Lecturas para el Diplomado internacional Plantas Medicinales de México. 2nd. Ed., Universidad Autónoma Chapingo, México. Estrada LE. 2002. Cultivo de plantas medicinales, una urgencia Latinoamericana. In Estrada LE: Lecturas para el Diplomado internacional Plantas Medicinales de México. 2nd. Ed., Universidad Autónoma Chapingo, México. Freiteas MSM, Martins MA, Curcino VIJ. 2004. Yield and quality of essential oils of Mentha arvensis in response to inoculation with arbuscular mycorrhizal fungi. Pesq Agropec Br 39: 887 - 894. Gupta ML, Janardhanan KK. 1991. Mycorrhizal association of Glomus aggregatum with palmarosa enhances growth and biomass. Plant and soil 131: 261 - 264. Gupta ML, Prasad A, Ram M, Kumar S. 2002. Effect of the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum on the essential oil yield related characters and nutrient acquisition in the crops of different cultivars of menthol mint (Mentha arvensis) under field conditions. Biores Tech 81: 77 79. Hajhashemi V, Zolfaghari B, Yousefi A. 2011. Antinociceptive and anti-inflammatory activities of Satureja hortensis seed essential oil, hydroalcoholic and polyphenolic extracts in animal models. Med Princ Pract 21: 178 182. Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/278 Carreón-Abud et al. Mycorrhization increases terpenes and plant performance in S. macrostema Hoagland DR, Arnon DI. 1950. The water-culture method for growing plants without soil. Calif Agric Exp Station 347: 1 - 32. Kapoor R, Giri B, Mukerji KG. 2002. Glomus macrocarpum, a potential bioinoculant to improve essential oil quality and concentration in dill (Anethum graveolens L.) and carum (Trachyspermum ammi L. Sprague). World J Microbiol Biotech 18: 459 - 463. Kapoor R, Giri B, Mukerji KG. 2004. Improved growth and essential oil yield and quality in Foeniculum vulgare Mill on mycorrhizal inoculation supplemented with P-fertilizer. Biores Tech 93: 300 - 311. Karagiannidis N, Thomidis T, Panou-Filotheou E. 2012. Effects of Glomus lamellosum on growth, essential oil production and nutrients uptake in selected medicinal plants. J Agric Sci 4: 137 - 144. Karthikeyan B, Joe MM, Jaleel CA. 2009. Response of some medicinal plants to vesicular arbuscular mycorrhizal inoculations. J Sci Res 1: 381 – 386. Kumar GS, Murugesh S. 2002. Studies on the VAM fungi improves growth of some medicinal plants. Adv Plant Sci 15: 43 - 46. Larose G, Chenevert R, Moutoglis P, Gagne S, Piché Y, Vierheilig H. 2002. Flavonoid levels in roots of Medicago sativa are modulated by the developmental stage of the symbiosis and the root colonizing arbuscular mycorrhizal fungus. J Plant Physiol 159: 1329 - 1339. Mahmoud SS, Croteau RB. 2003. Menthofuran regulates essential oil biosynthesis in peppermint by controlling a downstream monoterpene reductase. PNAS 100: 14481 14486. McGonigle TP, Miller MH, Evans DG, Fairchild GL, Swan JA. 1990. A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytol 115: 495 - 501. Morone-Fortunato I, Avato P. 2008. Plant development and synthesis of essential oils in micropropagated and mycorrhiza inoculated plants of Origanum vulgare L. ssp. Hirtum (Link) Ietswaart. Plant Cell Tiss Org Cult 93: 139 - 149. Ozkan G, Simsek B, Kuleasan H. 2007. Antioxidant activities of Satureja cilicica essential oil in butter and in vitro. J Food Eng 79: 13911396. Perez-Gutierrez RM, Gallardo-Navarro YT. 2010. Antioxidant and hepatoprotective effects of the methanol extract of the leaves of Satureja macrostema. Pharmacogn Mag 6: 125 - 131. Phillips JM, Hayman DS. 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans Br Mycol Soc 55: 157 - 160. Rapparini F, Llusià J, Peñuelas J. 2008. Effect of arbuscular mycorrhizal (AM) colonization on terpene emission and content of Artemisia annua L. Plant Biol 10: 108 - 122. Rzedowski GC, Rzedowski J. 2001. Flora Fanerogámica del Valle de México. 2nd Ed. INE-CNCUB, Michoacán, México. Singh G, Maurya S, De Lampasona MP, Catalan C. 2005. Chemical constituents, antimicrobial investigations, and antioxidative potentials of Anethum graveolens L. Essential oil and acetone extract: Part 52. J Food Sci 70: 208 215. Toussaint JP, Smith A, Smith E. 2007. Arbuscular mycorrhizal fungi can induce the production of phytochemicals in sweet basil irrespective of phosphorus nutrition. Mycorrhiza 17: 291 - 297. Yao MK, Desilets H, Charles MT, Boulanger R, Tweddell RJ. 2003. Effect of mycorrhization on the accumulation of rhishitin and solavetivone in potato plantlets challenged with Rhizoctonia solani. Mycorrhiza 13: 333 - 336. Zeng V, Ping Guo L, Chen BD, Hao ZP, Wang JW, Huang LQ, Yang G, Cuis XM, Yang L, Wu ZX, Chen, ML, Zhang Y. 2013. Arbuscular mycorrhizal symbiosis and active ingredients of medicinal plants: current research status and prospectives. Mycorrhiza 23: 253 - 265. Zheljazkov VD, Cerven V, Cantrell CL, Ebelhar WM, Horgan T. 2009. Effect of nitrogen, location and harvesting stage on peppermint productivity, oil content, and oil composition. Hort Sci 44: 1267 - 1270. Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas/279