Yield and Oil Content of Mint under Different Nitrogen Fertilizer Treatments

Two species of mint Mentha piperitha (peppermint) and M. arvensis (Japanese mint) are widely cultivated in Iran, but their response to fertilizer regime has not been evaluated so far. A field experiment was conducted to investigate the effects of different organic and chemical fertilizer treatments [Control, 100% urea (95 kg N ha−1), 75% urea (71.25 kg N ha-1) + 25% vermicompost (3.3 t ha-1), 50% urea (47.5 kg N ha-1) + 50% vermicompost (6.75 t ha-1), 25% urea (23.75 kg N ha-1) + 75% vermicompost (10.1 t ha-1) and 100% vermicompost (13.5 ton ha-1)] on essential oil contents, yield and yield components of the two species of mint. Peppermint provided grater plant height, number of internodes, number of leaf and oil percentage compared with the Japanese mint under study. The results indicated that, irrespective of the mint species, plants treated with combined chemical and organic fertilizer presented taller plants, higher oil contents and oil yield compared with solo chemical or organic fertilizers. Oil percentage and essential oil yield of mint increased significantly under the treatment with 25% urea (23.75 kg N ha−1) + 75% vermicompost (10.1 t ha-1). Plant height and number of leaf increased along the replacement of organic fertilizer with chemical fertilizers. The results showed that there was a positive and significant correlation with leaf number and essential oil yield. Application of vermicompost in combination with chemical fertilizer increased plant height, oil percentage and essential oil in both species, suggesting that organic and chemical fertilizer combination improves performance and environmental sustainability.


Introduction
In recent decades, efforts have been made to reduce the usage of synthetic N fertilizers and improve the efficiency of them so to reduce their impact on the environment. Integrate nutrient management approaches the controlled use of nutrient fertilizers. Fertilizers' management refers to application of suitable amounts of organic and inorganic fertilizers, in order to improve the health and productivity of soil and persuade plant growth (Ram et al., 2012). Organic nutrients enhance the activity of soil organisms by providing organic matter for organisms such as fungal mycorrhiza which help plants in absorbing micro and macronutrients (Tilman et al., 2002). On the other hand, chemical nutrients may have long-term adverse effects on soil properties and living organisms (García et al., 2014). High application rate of nitrogen-based fertilizers can lead to increased leaching of nitrate into grand water (Yang et al., 2015). To increase nitrogen use efficiency and decrease negative effects of them on the environment, the fertilizer supply showed match during the growing season (Tilman et al., 2002). Ammonium ions absorbed preferentially over nitrate ions, which means excess nitrate ions dissolved by irrigation or rain into grand water. However, organic fertilizer improved the physical and biological properties of soil and reduced the effect of over fertilization.
For organic fertilizers, nutrient release rate are much lower than mineral (inorganic) fertilizers (García et al., 2014). Substitution of chemical fertilization with organic one or combining the two methods may be useful in many ecosystem processes such as nutrient cycling.
Vermicompost is one of the organic fertilizers, its nutrient profile being generally higher than other composts. Vermicompost can increase soil properties physically, chemically and biologically (Hussein et al., 2006). Soil treated with vermicompost has better porosity, aeration bulk density and water retention. Also, chemical properties such as EC and pH improved. Vermicompost can increase the size, biodiversity and activity of microbial population in the soil. Application of vermicompost promotes root growth and makes easier nutrient absorption. Plants grown with vermicompost contain higher levels of the main physicochemical properties of the organic compost are shown in Table 3.
The vermicompost (which was obtained from KODSA organic products company, Iran) and half of the total urea applied were broadcast by hand and incorporated immediately into the soil using a rototiller, three days before planting. The remaining half of the urea was applied as top dressing when the mint seedlings were at the four-leaf stage.
Plots were 2.5 m long and consisted of six rows, with 25 cm spacing between the rows (plot size 3.125 m 2 ). A two m alley was maintained between all plots to eliminate any influence of lateral water movement.
Mint seeds are available, but these species are natural hybrids (Tucker and Fairbrothers, 1990) and their future generation may produce seedling with different phenotype or oil percentage. Therefore, commercial mint species procreated only vegetatively (Lawrence, 2006) were used for the hereby study. To minimize disease and pest pressure within the research, certified virus free planting rhizomes were achieve from Medicine plant research, Inc, Karaj, Iran. The plants initiated from rhizome cuttings (7 cm long) and were planted directly by hand on 9 April 2016. The plants were thinned at the four leaf stage to achieve a density of approximately 20 plants m -2 . Weeds were controlled by hand-weeding using a hoe and/or a rototiller whenever necessary.
Because of the sandy soil, plots were irrigated frequently (at an interval of 1-2 days) with drip irrigation system, to avoid wilting at during the plant growth. The plants were harvested at lasting of August, at floral initiation, by cutting plants about 10 cm above the soil surface (Hussein et al., 2006).
Data of growth parameters were measured: plant height (cm), fresh and dry weights of herbage (g). Fresh weight of all plots was taken as fresh herbage yield. In term of dry herbage yield, the dry weight of 1 kg fresh herb dried and weighted was recorded after being air-dried for 24 h to avoid loss of essential oils (Topalov, 1989). The leaf content of samples was calculated after separating the leaf of dry herb samples. An all-glass Clevenger-type apparatus was used to conduct 2.5 h of hydro-distillation on dried aerial parts (which were collected at floral initiation) of the two species under study and expressed as ml 50 g -1 fresh herb. This method for the extraction of oils is recommended by the European Pharmacopoeia (European Pharmacopoeia, 1983).
Analysis of variance (ANOVA) of the data from each attribute was computed using the SAS package (SAS Institute, 2002). Significance of differences among species (S), various fertilizers (F) and their interaction (S×F) for variables were compared by LSD test (P≤0.05). Microsoft office Excel (2007) was used for figures drawing. The simple correlation among the parameters studied was also calculated to explore relationship between all characterizes.

Results and Discussion
Numerical data of yield for fresh and dry herb, essential oil and yield components with their statistical results are summarized in Table 2. Species treatment had significant nutrients required for plant growth, like nitrogen, compared to untreated plants (Ram et al., 2012). Increase in biological activity plant growth regulators or humic acid in the vermicompost are also responsible for enhancing plant growth. Roy et al. (2010) showed that plant height and shoot weight were the highest in the vermicompost treatment. The combination of vermicompost and chemical fertilizer also resulted in higher leaf area and shoot weight for common bean related to vermicompost alone (Suhane et al., 2008). Chand et al. (2004) observed that organic fertilizer had great effects on the growth of mint in terms of root and shoot dry weight, number of leaves per plant, leaf area and the mean plant height.
The genus Mentha is one of the important members of Lamiaceae family, containing 25 species such as M. piperita and M. arvensis. These species are fast growing and generally tolerant to a wide range of climate, so that can be cultivated in many countries. Their essential oil is considered industrially and widely used in pharmaceuticals, confectionery and cosmetic.
The current investigation aimed to study the yield and oil yield of two mint species affected by various fertilizers.

Materials and Methods
Field experiments were carried out in 2016 at the field research station of the Agricultural Research Center of Tarbiat Modares University in Karaj.
The climate in this region is semi-arid, with warm and dry summers, a mean annual rainfall of 286.6 mm and a mean annual temperature of 12 o C. The soil is classified as a sandy loam, with 38% clay, 22% silt and 40% sand. The soil was air-dried and crushed before its pH and electrical conductivity (EC) were evaluated. Next there were determined the total organic carbon (using the Walkley and Black method, which involves sulfuric acid), total nitrogen (using the Kjeldahl method), available phosphorus (using the Olsen procedure), available potassium after extraction with ammonium acetate, and levels of the micronutrients iron, zinc, copper, and manganese after extraction with diethylene triamine pentaacetic acid (Tandon, 1995). Details of the properties of field soil and properties of the organic compost are shown in Table 1.
The experiment was conducted as factorial in a randomized complete block design with three replications. According to the recommended N requirements (120 kg/ha) and soil N content for peppermint, 65 kg ha −1 N was supplied from fertilizer, using urea as N source, at two time points, before rhizome planting and remaining half of the urea was applied at fourth leaf stage respectively. Also, the crops were fertilized with 40 kg P2O5 ha -1 phosphorous as triple superphosphate (TSP). The fertilizers were incorporated into the soil before bed formation.

Plant height
Mean comparison showed that plant height were lower for Japanese mint. Among fertilizers' treatments, F3 showed the maximum plant height. However, F4 and F3 were not significantly different (Table 4). Actually, plant height was higher in all integrated fertilizer treatment compared with only chemical treatment.
Presumably, the application of vermicompost improved soil chemical, biological and physical properties and increased root activity, conducting to enhancement of plant absorption. Even more, unique mineral properties of vermicompost such as high association for NH4 (Alidadi et al., 2016) and high CEC, resulted in holding ammonium in treated plants with vermicompost. When vermicompost was applied to the soil, the ammonium ions were slowly exchanged and oxidized by nitrifying bacteria. In this way, the soil N concentration and N uptake by plants significantly increased (Yang et al., 2015).

Number of internodes and leaf number
Number of internodes and leaf number significantly responded to the two species under study. Results showed that peppermint significantly had more internodes and leafs per plant (Table 3). As show in Table 3, fertilizer treatments did not affect these two traits significantly, but numerically F5 had the highest number of internodes and leaf per plant. In fact, combination of chemical organic fertilizer treatment performed better than chemical or solo organic fertilizer in regard to plant internodes and leaf number. Vermicompost has high microbial activity due to the presence of bacteria, fungi and actinomycetes that can product plant growth regulators (PGRs) such as auxin, gibberellins, cytokinins and abscisic acid (Frankenberger et al., 1995). These results agreed with reports by other researchers who reported that co-application of organic fertilizers and chemical fertilizers improved crop production (Blaise et al., 2005;Hassanzadeh et al., 2011).  For a given mean within each column of each section, the same letters are not significantly different (p ≤ 0.05).

Oil percentage and essential oil yield
Oil percentage and essential oil yield in mint were remarkably impressed by species and fertilizer treatments. Although, most of yield components of mint significantly responded to species and integrated fertilizers, fresh and dry herbage were not affected by treatments (Table 2). Similar to oil percentage, essential oil yield was also improved when integrated fertilizers were applied. Regardless of the species, 75% vermicompost + 20% urea (F5) outperformed than other fertilizers (Table 3). None of the species and fertilization affected fresh and dry weight and harvest index. Further, peppermint showed remarkably greater number of leaf, oil percentage and oil yield than Japanese mint (Table 3).
In the current experiment, it was found a positive and significant correlation between the number of internodes and the number of leaves with oil percentage and essential oil yield (Table 4). These results showed that the production of leaves was higher for peppermint. In mint, leaves are the site where oil synthesis and accumulation is taken place. Therefore, it has special significance for improving the oil percentage and yield. This could also be the reason for the higher oil percentage and essential oil yield in peppermint (Table 4). Under the F5 treatment, plants had the oil percentage and essential oil yield of 0.64% and 23.4% kg/ha respectively, which were significantly higher than plants treated with solo chemical or vermicompost. The advantage of oil percentage and oil yield following integrated fertilization could have raised from modification in soil chemical, physical and microbial population, which all of them can affect yield and yield components. Vermicompost has large particulate surface area that is suitable for the strong retention of nutrients. Yang et al. (2015) reported that vermicompost application could increase N uptake by plants. Consequently, plants which received organic fertilizers provide greater nutrients than those which received chemical fertilizers. Aziz et al. (2009) have found a positive correlation between vermicompost and oil yield of chamomile. Base on previews results, most nutrient are easily available in vermicompost such as soluble potassium, nitrate and phosphate, which can be responsible for plant growth increase and high crop yield 95 (Yang et al., 2015). This result verifies again the advantage of integrated fertilizer over the other studies related essential oil yield production. Due to special properties of vermicompost, it provides good condition for microbial activity. Thus, it is not unexpected that soil treated with vermicompost is rich in plant growth regulation. Hormones like amylase, lipase, protease, pectinase and cellulose that cause decomposition of organic matter in the soil and provide the necessary nutrient for plant are present in higher concentrations. Improvement in soil hormones and available N, P and K due to integrated fertilizer have been reported in wheat by Suhane et al. (2008) and peppermint Ram et al. (2012).
Humic acid, and other plant regulator hormones, is another substance that can be found in vermicompost. It is demonstrated that soil bacteria can produce auxins, gibberellins and kinetin-like substances that had positive effect on plant growth directly (Barea et al., 1976). It is concluded that plant regulators in the vermicompost could also have had influence on the plant growth and increase yield of mint. Some of the effects of humic acid on yield and yield component of plants have been shown (García et al., 2014).

Conclusions
Yield of mint increased dramatically with the increase of vermicompost in fertilizer integrated treatments up to F5 scheme (10.1 t ha -1 ). However, at greater vermicompost rate (F6: solo vermicompost) the oil percentage and oil yield decreased. Such a result was probably because of the characteristic of organic fertilizers used in the study. Due to continuous, but slow, nutrient release of the organic fertilizer, vermicompost alone is not likely responsible for supplying the plant in the short term. From the above results, it is clear that vermicompost in suitable proportion can significantly enhance oil content and oil yield of mint, by improving various chemical, physical and biological characteristic of the soil.