Effects of Cultivation Systems and Environmental Conditions on Peppermint ( Mentha × piperita L . ) Biomass Yield and Oil Content

Peppermint (Mentha × piperita L.) is perennial plant cultivated for essential oil production. In the present study, field experiments were conducted to determine the performance of peppermint under different cultivation systems for two consecutive growing periods in 2015 and 2016. The effects of environmental conditions on peppermint biomass yield and oil content was also evaluated. The experiments were carried out according to randomized complete block design with five replications per cultivation system. The results of the present study indicate that the total aboveground dry weight and dry leaves biomass were affected by cultivation system and year. For both growing seasons, the highest values were found under the conventional farming system. In contrast, oil content was approximately 14% higher in organic system than in conventional system. Moreover, oil content differed by year, with higher values being observed in 2015 comparing to 2016. Both oil content and biomass yield were positively affected by high temperatures. The results from this study demonstrated that both environmental conditions and the cultivation systems affect the biomass and oil content in peppermint crop.


Introduction
Peppermint (Mentha × piperita L.) is perennial plant cultivated around the world for essential oil production (Karkanis et al., 2017).According to Capuzzo and Mafei (2016), peppermint is a hybrid between water mint (Mentha aquatica L.) and spearmint (Mentha spicata L.).The last report of FAOSTAT on peppermint production (FAO, 2014), emphasize an increasing trend in the world peppermint production, which, in 2014 was 92,295 tons, over 3-fold bigger than world production in 1990 (28,672 tons).Moreover, in the last 24 years, an increase of about 70% in the cultivated area was registered.The major contributor to world peppermint production is Asia with 92.23% of total production and is followed by Americas, which contributes 7.61%.
Several studies have been conducted to evaluate the effects of various cultivation practices (i.e.intercropping, irrigation, herbicides, fertilization) on growth, yield and quality of peppermint (Singh et al., 1989;Jeliazkova et al., 1999;Zheljazkov et al., 1999;Verna et al., 2013;Karkanis et al., 2017;Németh-Zámbori et al., 2017).In contrast, a limited number of studies have been published about the effects of environmental conditions on growth and yield of peppermint crop (Clark and Menary, 1980;Piccaglia et al., peppermint cultivation in organic system was conducted according to principles of Council Regulations (EC) No 834/2007 of 28 June 2007 on organic production and labelling of organic products and repealing Regulation (EEC) No 2092/91.In addition, in conventional system, 210 kg ha -1 of an inorganic fertilizer (N:P2O5:K2O, ratio 15:15:15) was applied, while fluazifop-p-butyl (Fusilade Forte; Syngenta, Romania) was applied post-emergence at a rate of 0.195 kg a.i.ha -1 to control the grass weeds, in April.Broad-leaved weeds were controlled by hand weeding.

Sampling, measurements and methods
To determine the total aboveground dry weight and the dry leaves biomass, peppermint crop was harvested on the 15 July 2015 and the 15 July 2016.An area of 1 m 2 from each plot was hand harvested.Then, the dry weight was determined after drying at 60 • C until constant weight.For the extraction of the essential oil, plant samples were airdried at room temperature in the shade.The essential oil was extracted via the Hydro steam-distillation method using a Clevenger type apparatus, according to AOAC methodology (2005).The distillation was repeated until oil recovery stopped.The extraction was performed thrice for each plot.After the extraction, the oil was separated from the water phase.The oil content was expressed as percentage (v/w) of peppermint dry weight.Final, the oil yield was also measured using the equation: Oil yield (L ha -1 ) = (OC × DLB)/100, where DLB is the dry leaves biomass (kg ha -1 ) and OC is the oil content (ml/100g).
1993; Rita and Animesh, 2011).Therefore, this study has been carried out: (1) to determine the performance of peppermint under organic or conventional cultivation systems and (2) to evaluate the effects of environmental conditions (temperature and precipitation) on peppermint biomass yield and oil content in Southeastern Europe.

Study site and experimental design
A peppermint crop (Mentha × piperita L.) crop was established at an experimental field located in the Alba Iulia region in Romania (46˚04'17''N,and 23˚34'23''E).The soil was sandy clay loam (35% sand, 30% silt and, 45% clay), with pH 7.2.Peppermint was transplanted in the autumn (October) of 2014 in rows 20 cm apart and within the rows at 70 cm.The experiment was conducted for two consecutive growing periods (April-July), in 2015 and 2016.Meteorological data (temperature, wind velocity and precipitation) are presented in Table 1.
The experiment was set up over an area of 150 m 2 .Completely randomized block design was used in both years, with five replicates per treatment.The plot size was 10 x 5 m (50 m 2 ).The experimental treatments were as follows: Control, without agricultural inputs; ORG: organic cultivation system; and CON: conventional cultivation system.In organic cultural system, 2000 kg ha -1 of sheep manure was applied, while no herbicide application was made.The plots were kept weed free by hand weeding.

Statistical analysis
Analysis of variance (ANOVA) was conducted for all data to evaluate the effects of cultural systems, the year effects and the interactions between them.Data are presented in tables as means (n=5).Differences between means were separated by Fisher's Least Significant Difference (LSD) test at p ≤ 0.05 level.Statistical analysis was performed using the STATISTICA software package, version 8.0 (StatSoft Inc., Tulsa, USA).

Results and Discussion
The lowest mean monthly temperature of both growing periods are recorded in January (-0.71 °C in 2015, and -3.19 °C in 2016), and highest in July (21.77 °C in 2015, and 20.48 °C in 2016), months when we also report minimum, and maximum daily values (Table 1).By entire experimental period, the highest mean wind velocity is reported in April 2015 (10.43 m/s), while the lowest in December 2015 (4.94 m/s).A bigger sum of precipitations, 431.49mm, respectively, characterize the first growing period, compared to the second, when sum of precipitations was in amount of 411.44 mm (Table Concerning the dry leaves biomass there were significant differences between cultivation systems (Table both growing seasons, significant differences between cultivation systems were recorded.In the first growing season, the highest dry leaves biomass (917 kg ha -1 ) was found under the conventional farming system.For the second growing season, the lowest dry leaves biomass was recorded in organic plots (959 kg ha -1 ).
The total aboveground dry weight was also affected by cultivation system.In the first growing season, the highest values (2375 kg ha -1 ) were recorded in the conventional system and the lowest in organic cultural system (Table 2).Total biomass yield was comparable to those reported in the Mediterranean basin by Karkanis et al. (2017).Total aboveground dry weight of peppermint was approximately 25% higher than in conventional system than in organic system.Significant differences in total aboveground dry weight were also recorded in the second growing period.The highest total aboveground dry weight was found in conventional plots.Moreover, total aboveground dry weight differed by year, with higher values being observed in 2016 comparing to 2015.In contrast, Karkanis et al. (2017) have also reported that biomass yield of peppermint affected by year and the highest values were recorded during the first growing season in comparison to the second growing period.Similarly, Piccaglia et al. (1993) have also found lower biomass yield in the second growing season, compared with yield obtained in the first growing season.
Whatever cultivation system, both aboveground dry weight, and dry leaves biomass (Table 2)emphasize biggest values in the second cultivation period characterized by lower mean temperature (8.38 °C), and precipitations supply (411.44 mm), compared to the second growing period (Table 1).
The cultivation system affected the plant height in the second growing period, November 2014 -July 2016 (Table 3).Karkanis et al. (2017), report similar plant height values in control in 2015 (45.08 cm) and fluazifop-p-butyl herbicide treatment (42.83 cm), as we obtained in control (44.68 cm), and conventional cultivation system (41.12cm).
Regarding the oil content, there were significant differences between cultivation systems (Table 4).In the first growing season, the highest oil content (2.23%) was Table 2. Total aboveground dry weight (kg ha -1 ) and dry leaves biomass (kg ha -1 ) as affected by the cultivation system, in 2015 and 2016  259*** ORG-organic cultivation system, CON-conventional cultivation system.Means in each column followed by the some letter are not significant different according to LSD test, ns No significant differences at P>0.05, ***Significant differences at P<0.001.recorded in organic system and the lowest in conventional system.Oil content was approximately 14% higher in organic system than in conventional system.Karkanis et al. (2017) reported that essential oil content of peppermint varied from 1.87% to 2.11%.Significant differences in oil content were also recorded in the second growing period.The highest oil content was also found in organic system.Moreover, oil content differed by year, with higher values being observed in 2015 comparing to 2016.In contrast, Karkanis et al. (2017) have found that the oil content was not affected by year.
The oil yield was also affected by cultivation system.In the first growing season, the lowest values (17.39 L ha -1 ) were recorded in the conventional system and the highest in control plots (Table 5).The productivity of peppermint with respect to oil yields in this study was lower than those previously reported by Karkanis et al. (2017).Significant differences in oil yield were also recorded in the second growing period.The oil yield was also found in control plots.Moreover, oil yield differed by year, with higher values being observed in 2015 when biggest temperatures (8.57°C), and precipitations supply (431.49mm), are recorded comparing to 2016.In a previous study, Duriyaparan et al.
579 (1987) reported that the highest leaves dry weight was produced under 30 °C day temperatures, while Mitchell and Yang (1998) observed that both water stress and overirrigation resulting in reduced oil yield.Similarly, Khorasaninejad et al. (2011) also reported oil content, biomass and oil yield were significantly decreased by drought stress.
The results show similar intensities of correlations between oil content (%) and dry leaves biomass in both growing periods, 2015, and 2016, respectively (Table 6).They are moderate, with highest values in conventional cultivation system, and lowest in organic cultivation system.Reported to the same period, between oil yield (L ha -1 ) and dry leaves biomass moderate to strong correlations are identified, with highest values control, and lowest in organic cultivation system.Zheljazkov et al. (2010), report moderate correlation between oil production and fresh herbage yield in flower stage (R = 0.67), which are similar with those obtained in our experiment in 2016, but bigger, compared to those resulted in our experiment in 2015.
Analyse of variance shows biggest influence of year on dry leaves biomass, and oil content (Table 7).The meteorological characteristics of the year, precipitation and temperature, respectively influence in a different manner dry leaves biomass and oil content.If oil content is positively influenced by higher temperatures and higher precipitations supply, the dry leaves biomass increase in growing period with lower temperature and precipitations supply.Above ground weight is influenced by both cultural system and year.Our study shows that it is advantaged by lower temperature and precipitations supply, and conventional cultivation system.

Conclusions
The results of the present study indicate that the peppermint biomass and leaves yield, and oil production was affected by both the cultural system and the environmental conditions.Significant differences in total aboveground dry weight and dry leaves between the cultivation systems were recorded for both growing seasons.The highest values were recorded in the conventional system and the lowest in organic cultural system.In contrast, the highest oil content were found in organic system and the lowest in conventional system.Moreover, the oil content was affected by year, with higher values being observed in the second growing season comparing to the first growing season.Our study also reveals that oil content and biomass yield were positively affected by high temperatures.

The 577 Table 1 .
Mean monthly temperature (°C), wind velocity (m s -1 ), and precipitation (mm) at the experimental site during the two growing seasons (November 2014 to July 2015 and November 2015 to July 2016) CON-conventional cultivation system.Means in each column followed by the some letter are not significant different according to LSD test ***Significant differences at P<0.001

Table 3 .
The plant height (cm) of peppermint (Mentha × piperita) as affected by the cultivation system, in 2015 and 2016

Table 4 .
Oil content (% of dry weight) as affected by the cultivation system, in 2015 and 2016

Table 5 .
Oil yield from peppermint leaves (L ha -1 ) as affected by the cultivation system, in 2015 and 2016

Table 7 .
Analysis of variance for cultivation systems and year effects on total aboveground biomass, dry leaves biomass and oil content in peppermint crops values are shown.ns: not significant; *, **, *** denote significant differences at P< 0.05, P< 0.01, P< 0.001, respectively).