Mycorrhizal fungi inoculation and phosphorus fertilizer on growth, essential oil production and nutrient uptake in peppermint (Mentha piperita L.)

This study evaluated the effects of inoculation with the arbuscular mycorrhizal fungi Glomus mosseae, Glomus intraradices A4 and Glomus intraradices B1 and two phosphorus levels (10 and 40 mg kg-1) on root colonization, plant growth, nutrient uptake and essential oil content in Mentha piperita L. The experiment was carried out in a greenhouse, in 4x2 factorial arrangement, in completely randomized design. At sixty days after transplanting, the mycorrhizal plants had significantly higher fresh matter, dry matter and leaf area compared to non-mycorrhizal plants. The inoculation increased P, K and Ca levels in the shoot which were higher under 40 mg P kg-1 of soil. Plants grown with 40 mg P kg-1 soil increased the essential oil yield per plant by about 4050% compared to those cultivated with 10 mg P kg-1, regardless of the mycorrhizal treatment. Among the studied fungal species, inoculation with G. intraradices A4 and a high level of P significantly increased plant growth and essential oil yield, compared to the other studied mycorrhizal fungal species. In conclusion, inoculation of arbuscular mycorrhizal fungi into peppermint plants is a feasible alternative to increase the essential oil production and reduce the use of fertilizers required to obtain economic production of peppermint under phosphorus-deficient soil condition.

Temperatures (ºC) insecticidal, antifungal and antibacterial activity of mint essential oils (Souza et al., 1991;Sokoviae et al., 2009;Derwich et al., 2010;Kumar et al., 2011).Arbuscular mycorrhizal fungi (AMF) are the most common type of mycorrhizal association (Hodge, 2000).This association allows plants to explore larger volumes of soil, expanding the area of root absorption, increasing the area of contact with the soil, favoring the absorption of water and nutrients, especially P, and stimulating plant growth (Bressan et al., 2001;Gupta et al., 2002).The responses of the mycorrhizal plants related to P concentration in the soil vary with the species and the genotypes (Clement & Habte, 1995).
Arbuscular mycorrhizal fungi are known to play an important role in the nutrition and in the growth of plants in many production-guided agricultural systems, but little is known about their potential effect on secondary metabolites in medicinal and aromatic plants (Copetta et al., 2006;Khaosaad et al., 2006;Kapoor et al., 2007).
Studies of Ocimum basilicum (Khaosaad et al., 2006) and Artemisia annua (Kapoor et al., 2007) have shown that root colonization by AMF increases essential oil content and quality.However, little is known about the effect of AMF, P availability and nutrient uptake on essential oil content in M. piperita.Sirohi & Singh (1983) reported better root infection, higher biomass, P uptake and essential oil yield in M. piperita when associated with Glomus fasciculatus, compared to non-inoculated plants.Moreover, Gupta et al. (2002) concluded that inoculation with the AMF G. fasciculatum significantly increased root colonization, growth, essential oil yield and nutrient uptake in M. arvensis.Freitas et al. (2004) also observed that inoculation with AMF provided increments of up to 89% in the essential oil content in mint plants (M.arvensis) in relation to noninoculated plants.Meanwhile, Maia (1998) determined that N and P deficiencies drastically reduced the production of fresh biomass and affected the essential oil composition in M. arvensis.From an agricultural point of view, mycorrhizal associations are important because they reduce the cost and the environmental contamination with chemical fertilizers (Azcón-Aguilar & Barea, 1997).
The aim of the present study was to evaluate the comparative effects of different species of mycorrhizal fungi and phosphate fertilization on growth, essential oil production and mineral composition in M. piperita plants.

MATERIAL AND METHOD
Plant material and growth condition: Experiments were carried out in a greenhouse at INFIVE, La Plata, Argentina (34º 52' S; 57º 58'W), between September 2007 and February 2008, under natural day length.Temperatures during the experiment were between the maximum 29.1ºC in January/08 and the minimum 10ºC in November/07 (Figure 1).
Apical shoots of M. piperita were obtained from the aromatic plant collection of the Biochemistry and Phytochemistry Laboratory (Faculty of Agricultural and Forest Science, University of La Plata, Argentina).The cuttings (10 cm long with two nodes and two pair of leaves) were surface sterilized in 0.5% sodium hypochlorite for 10 min, rinsed twice with sterile water and placed in plastic cells containing a sterile mix of perlite and vermiculite [1:1 (v/v)] and irrigated by sprinkling every day for rooting.
Inoculation period: Rooted cuttings were transplanted into 250 mL plastic pots (one per pot).
Growth period: The remaining plants (15 plants of each treatment) were then transferred to 3 kg plastic pots into a mixture of sand:soil (1:1) (the same as that for the inoculation period), tyndallized at 100ºC for 60 min on 3 consecutive days (Wolf et al., 1989;Barea et al., 1983).The plants were cultivated at two levels of P: 10 mg kg -1 (-P) or 40 mg kg -1 (+P) as (KH 2 PO 4 ).At flowering stage (60 days after transplanting), the plants were harvested and observations were recorded.
Estimation of mycorrhizal colonization: Fungal colonization was evaluated according to Trouvelot et al. (1986) and expressed as mycorrhization percent (M%).The roots were cleared with 10% (w/v) KOH and stained with trypan blue in lacto-phenol (Phillips & Hayman, 1970).Three replicates of 30 randomly chosen root fragments (1 cm long) were mounted on slides and examined microscopically.The M% was calculated as the proportion of infected roots over total number of root fragments.The efficiency of mycorrhizal colonization was estimated by mycorrhizal dependence (MD), which related the biomass of inoculated plants to that of non-inoculated plants grown under the same conditions; it was calculated as: Growth parameters: Harvesting of material for determination of shoot fresh (FW) and shoot dry weight (DW) of leaves and stems was conducted between 7h and 8h in the morning, according to Freitas et al. (2004).DW was obtained by drying the material in oven at 40ºC until constant weight.Leaf area (LA) per plant (Li 3000 leaf area meter, LICOR, Lincoln, NE, USA) was measured.
Mineral nutrient content: Nutrient content in shoot dry matter was determined at the end of the experiment.Total N was determined by micro Kjeldahl distillation.Phosphorus content was measured by the molybdovanadophosphate method (Kitson & Mellon, 1944).Potassium (K) and Calcium (Ca) levels were measured with a flame photometer as described by Haddad & Higginson (1990).
Extraction of essential oils: At flowering stage (60 days after transplanting), the plants were harvested and the essential oils (EO) in the shoot were determined by a hydro-distillation method on Clevenger-type apparatus for 3 h of each treatment (Maia, 1998).Essential oil content was calculated as mL of oil per 100 g DW and oil yield, as mL of oil per plant.For the extraction of essential oils, the plants were harvested between 7h and 8h in the morning according to Freitas et al. (2004).
Statistical analysis: The experiment was in 4 X 2 factorial arrangement, with four mycorrhizal levels (NI; Mmos; MintraA 4 ; MintraB 1 ) and two phosphorus levels (-P: 10 and +P: 40 mg kg -1 ) with 15 replicates (one plant/pot) per treatment in a completely randomized design.All data were analyzed using Analysis of Variance (ANOVA).The Tukey's test (p<0.05)was used to evaluate the differences between treatments and interaction means, using SigmaStat 3.5 software (Systat Sofware, Inc, USA).For statistical analysis, all percentage values were transformed to arcsine, since this transformation remarkably improves variance homogeneity.The number of replicates was n=3 replicates of 30 root fragments for mycorrhizal observations.

Inoculation period
None of the non-inoculated plants were colonized by Glomus species.At 40 days, mycorrhization was 36, 63 and 80% for Mmos, MintraA 4 and MintraB 1 , respectively, indicating that peppermint is a mycotrophic species.
The fresh and dry weigh and the leaf area of plants inoculated with G. intraradices A 4 and B 1 were higher than those of plants inoculated with G. mosseae and non-inoculated plants.MintraA 4 and MintraB 1 increased dry weight by 49% and 33%, respectively, compared to non-mycorrhizal ones; leaf area increased by 45% and 39%, respectively (Table 1

Growth period
At sixty days after transplanting, the percentage of root colonization varied between the fungi and the phosphorus fertilized soil.Mmos and MintraB 1 decreased M% by 14% under +P, compared to -P, while MintraA 4 did not show significant differences between phosphorus treatments (Figure 2).Other authors also reported the negative effect of the phosphorus increment on the percentage of colonized roots, and that effect depends not only on the plant species but also on the fungal species (Nogueira & Cardoso, 2000;Bressan et al., 2001;Kapoor et al., 2007).
AMF inoculation and/or phosphorus fertilization had a significant effect on all measured plant growth variables.However, the level at which plant growth was enhanced varied between the fungal inoculants.The fresh and dry weight of inoculated plants were significantly higher than that of noninoculated plants, regardless of P level, in agreement with Abdul-Khaliq et al. (2001) and Cabello et al. (2005).For +P treatment, the fresh and dry weight significantly increased in non-inoculated and inoculated plants, compared to -P treatment.In noninoculated plants, the dry weight increment was by 37%, whereas in Mmos it was by 39%, in MintraA 4 by 51% and inMintraB 1 by 43% (Table 2), in agreement with Jackson et al. (2002), who demonstrated the beneficial effects of AMF in soils with low nutrient conditions, especially P. The increment of the phosphorus level in the soil increased the leaf area in both non-mycorrhizal and inoculated plants.Nonmycorrhizal plants showed the lowest values, regardless of the P level (Table 2).Our data showed a significant interaction between fungal species and P levels on dry matter production and leaf area.) DW (g pl -1 ) LA (cm 2 pl -1 ) MD (%)  ) and Glomus intraradices B 1 (MintraB 1 ) with two P levels (-P: 10 mg P kg -1 soil, +P: 40 mg P kg -1 soil); 60 days after transplanting.
#Means followed by the same letter, uppercase letters on the line and lowercase letters in the column, for each evaluated parameter are not significantly different according to Tukey's test (5%).
Results showed that G. mosseae was less efficient in promoting the growth of plants compared to G. intraradices A 4 or G. intraradices B 1 .Some authors agree with our results and showed the beneficial effect of AMF on soils with low P levels (Martins et al., 2000;Gupta et al., 2002;Kapoor et al., 2007).
At the end of the growth period (60 days after transplanting), MintraA 4 had the highest mycorrhizal dependence (Table 2).This can be related to inoculum efficiency and/or inoculation percent.Freitas et al. (2006) observed in Mentha arvensis that the lowest values of mycorrhizal dependence occurred when the plants were inoculated with G. clarum and G. margarita and cultivated with 50 mg P kg -1 soil; the mycorrhizal dependence values increased with higher levels of P.
AMF symbiosis may improve nutrient uptake by improving the soil exploration and contributes to enhance the growth and the vigor of plants (Beltrano & Ronco, 2008).K content significantly increased in inoculated plants compared to non-inoculated ones, regardless of the P level.Potassium content in MintraA 4 was 45% greater than that in non-inoculated plants, whereas in Mmos and MintraB 1 this increase was about 25% (Table 3).The increase in K content was associated with a significant increase in shoot dry weight as previously observed by Freitas et al. (2006) and Siqueira et al. (2002).
Mycorrhizal plants consistently accumulated more quantities of phosphorus in their shoots than non-mycorrhizal plants, regardless of P availability.At low phosphorus levels, P content was enhanced by the inoculation (Mmos: 37%, MintraA 4 : 19% and MintraB 1 :13%), compared to non-inoculated plants.Meanwhile, at high P level, the phosphorus content was significantly higher than in -P, 44%, 17%, 37% and 39% in NI, Mmos, MintraA 4 and MintraB 1 , respectively (Table 3).Ca content was higher in inoculated plants compared to non-inoculated ones.Marschner & Dell (1994) determined that AMF could be responsible for mineral absorption of about 80% of P and 10% of K. Our findings indicated that AMF colonization had a beneficial effect on plant growth and P, Ca and K absorption, in concordance with Chen et al. ( 2010) and Freitas et al. (2006).
We found that N was not modified by mycorrhizal or by P fertilizer (Table 3).In contrast, our results did not agree with those reported by Chen et al. ( 2010) and Freitas et al. (2006), who showed that mycorrhizal plants increased N content in the shoot.
The increase in nutrient uptake and growth, by AMF colonization, was demonstrated especially under low P availability, although the extents of the colonization and the increase in growth parameters differ between host plants, species of AMF, and environmental conditions (Klironomos, 2003).Gupta et al. (2002) concluded that different cultivars of mint plants differ in nutrient uptake, and the mycorrhizal enhancement for the nutrient uptake was more pronounced in P than in N and K.Moreover, Marschner & Dell (1994) reported that the highest growth in mycorrhizal plants was caused by increased P absorption, particularly from sparingly soluble P sources.
Essential oils are a volatile lipophilic mixture of compounds from secondary plants, mostly consisting of monoterpenes, sesquiterpenes and phenylproponoids.These compounds derive from isopentenyl pyrophosphate (IPP) and dimethylally pyrophosphate (DMAPP).Hence, the biosynthesis of essential oils depends on the inorganic phosphorus content in the plant (Loomis & Corteau, 1972).Phosphorus is also known to have multifarious cellular functions in plants, including: signaling and transmembrane metabolic flux; therefore, the secondary metabolism is modulated by these mechanisms (Ram et al., 2003).

Treatments
EO content (mL 100 g -1 DW) EO yield (mL plant #Means followed by the same letter, uppercase letters on the line and lowercase letters in the column, for each evaluated parameter are not significantly different according to Tukey's test (5%).
#Means followed by the same letter, uppercase letters on the line and lowercase letters in the column, for each evaluated parameter are not significantly different according to Tukey's test (5%).or inoculated with Glomus mosseae (Mmos), Glomus intraradices A 4 (MintrA 4 ) and Glomus intraradices B 1 (MintraB 1 ) with two P levels (-P: 10 mg P kg -1 soil, +P: 40 mg P kg -1 soil); 60 days after transplanting.
The biosynthesis of secondary metabolites and, consequently, active ingredients in medicinal and aromatic plants depends on genetic, physiological and environmental factors.The concept of improving the levels of secondary plant metabolites through AMF is quite new; in recent years some research on this topic has been done.Studies focused on the accumulation of secondary compounds (essential oils) in the shoot of AMF-inoculated aromatic herbs.
Our results showed that the essential oil content (mL 100 g -1 DW) significantly increased in mycorrhizal plants, regardless of the P level and the fungal species (Table 4), in agreement with Sirohi & Singh (1983), Gupta et al. (2002) and Copetta et al. (2006).Toussaint et al. (2007) in sweet basil and Khaosaad et al. (2006) in oregano plants have shown that the increased accumulation of phytochemicals in mycorrhizal plants is mediated by direct effects of the AMF and is not solely the indirect results of improved P nutrition.
On the other hand, the results showed that essential oil yield per plant (mL plant -1 ) increased significantly with increased P level and inoculation.At +P treatment, the essential oil yield significantly increased in both non-inoculated and inoculated plants compared to -P treatment.In non-inoculated plants the essential oil yield increment was by 33%, whereas in Mmos, by 44%, in MintraA 4 , by 50% and MintraB 1 , by 47%.Plants inoculated with MintraA 4 showed a significant increase in essential oil yield in comparison with other treatments (e.g. the increment was 51% at low P level and 67% at high P level, compared to NI).The interaction AMF x P was positive.
Positive significant relationships were observed in inoculated plants between essential oil yield and colonization, as well as shoot dry weight.These results are in agreement with those reported by Praszna & Bernath (1993), who determined that plants grown under deficient nutrient conditions show less essential oil production, and with those reported by Maia (1998) who stated that the deficiencies in N and P drastically reduced the production of fresh matter.Piccaglia et al. (1993) also determined that the essential oil content in mint significantly increased with increasing rates of fertilizer probably due to the higher growth rate.Moreover, Freitas et al. (2004) reported that in M. arvensis, when phosphorus was not added to the substratum, the essential oil content increased by up to 89% in mycorrhizal plants, compared to noninoculated ones.No increment in essential oil content occurred when the levels of P increased.On the other hand, our results are in disagreement with those of David et al. (2006), who determined that plants of Mentha piperita subjected to 7.75/15.5mg L -1 of P showed a higher increase in essential oil content than those grown at 23.0/46.5 mg L -1 of P.
Alsafar & Al-Hassan ( 2009) stated that nitrogen and phosphorus fertilizers play a vital role in enhancing mint crop yield.Application of 75/50 kg N/ P 2 O 5 ha -1 significantly increased total dry weight and essential oil yield.
Our results showed that there was a significant increase in EO content and yield in mycorrhizal plants, which was related to an increase in shoot fresh and dry weight and P content.

CONCLUSION
Mycorrhizal colonization and P supply have a beneficial effect in Mentha piperita L, enhancing nutrient absorption (Ca, K and P), shoot dry weight and essential oil yield.Among the studied fungi, inoculation of M. piperita with Glomus intraradices A 4 resulted in a significant increase in shoot dry weight and mycorrhizal dependence essential oil yield compared to the G. mosseae and G. intraradices B 1 .Colonization of roots with Glomus intraradices A 4 was higher than with other AMF, which may show an efficient symbiotic potential of this fungus with M. piperita.AMF inoculation increases the productivity and reduces the fertilizer application required to obtain economic production of peppermint crop.

ACKNOWLEDGMENT
To O. Peluso and L. Wanhan (CONICET) for technical assistance; to C. Moreno (CIC BA) for the English revision; E. Soto and L. Coelho for his translation of the abstract to Portuguese and to the Universidad Nacional de La Plata and CIC-PBA for financial support.M. Ronco and J. Beltrano are researchers of CIC-PBA.

FIGURE 1 .
FIGURE 1. Monthly maximum, mean and minimum temperatures during the experimental period.

TABLE 4 .
Essential oil (EO) content and essential oil yield in shoots of Mentha piperita plants,non-inoculated (NI)