EFFECT OF VERMICOMPOST AND VERMICOMPOST EXTRACT ON OIL YIELD AND QUALITY OF PEPPERMINT (Mentha piperita L.)

Organic fertilizers have beneficial effects on plants growth and quality. However, vermicompost increases electrical conductivity in soil due to increased salinity associated with continued usage. The experiment was conducted in a research field at the University of Guilan to determine effects of 7 Mt ha of cow manure vermicompost, vermiwash prepared from 7 Mt ha of vermicompost, leachate vermicompost + vermiwash, 50 Mt ha municipal solid waste compost (MSWC), chemical fertilizer (NPK 50–0–300) and no fertilization as a control on peppermint yield and quality. Fertilizer type affected all measured characteristics except number of nodes, stem fresh weight, total phenols and antioxidant capacity. Plants treated with vermicompost, vermiwash or leachate vermicompost + vermiwash were the tallest and had the highest levels of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids. Plants treated with vermicompost or vermiwash had the highest total plant fresh weight, leaf fresh weight and total fresh yield. The leachate vermicompost, vermiwash and vermicompost can be used as organic fertilizers for sustainable peppermint cultivation.


Introduction
Peppermint (Mentha piperita L., Lamiaceae) oil has a long history of safe use both in medicinal preparations and as a flavoring agent in foods and confectionery (Milovanović et al., 2009).Peppermint oil is used externally and internally.In Iran, peppermint is a perennial crop established with transplants in the spring and has an average stand life of about six years.In Guilan province, the crop is harvested only once a season.For the highest oil yields, growers apply enormous amounts of nitrogen (N) fertilizer to maximize oil yield because stress such as insufficient nitrogen reduces yield (Grigoleit and Grigoleit, 2005;Jelačić et al., 2005).
If appropriate amounts of fertilizers are not applied during production, physiological symptoms of deficiency can occur in plants (Takahashi, 1981;Olfati et al., 2012).Most producers use synthetic fertilizers because they are easy to transport, quickly available to plants, and produce high yields.However, with succeeding crops, quantities of chemical fertilizers must be increased because of low soil fertility (Thy and Buntha, 2005).Organic fertilizers have beneficial effects on soil structure and nutrient availability, help maintain yield and quality, and are less costly than synthetic fertilizers (Olfati et al., 2012;Thy and Buntha, 2005).
Large amounts of organic wastes, i.e., biosolids, animal manures and household wastes, are produced in Iran.These wastes may be used in production of herbs or to restore soil fertility (Benton and Wester, 1998) as they contain large quantities of nitrogen (N), phosphorous (P) and potassium (K) (Elliot and Dempsey, 1991).Compost is homogenous, retains most of its original nutrients and has reduced levels of organic contaminants because they are degraded before use (Ndegwa et al., 2000).It can be applied to increase soil organic matter and nutrients which can be released upon decomposition, to improve soil structure and increase cation exchange capacity.The use of composts in agricultural soils is a widespread practice and the positive effects on soil and vegetables are known from numerous studies (Gutierrez-Miceli et al., 2007;Peyvast et al., 2007Peyvast et al., , 2008aPeyvast et al., , 2008bPeyvast et al., , 2008cPeyvast et al., , 2008d;;Olfati et al., 2009;Shabani et al., 2011).However, thermophilic composting is generally time-consuming, requiring frequent mixing with possible loss of nutrients.
Certain species of earthworms can fragment organic material residuals into finer particles by passing them through a grinding gizzard (Ndegwa and Thompson, 2001).Additionally, earthworms reduce populations of human pathogens, an effect obtained in traditional composting by increasing temperature (Contreras-Ramos et al., 2004).There is evidence that earthworms produce plant hormones in their secretions (Suthar, 2010b).Earthworm processed materials 'casts' contain nutrients in forms easily available to plants (Suthar and Singh, 2008;Suthar, 2010a).
The final vermicomposting product has a high electrical conductivity (EC) which increases soil salinity with continued usage.To reduce EC, leachate vermicompost and vermiwash have been developed.Vermiwash may contain cytokinins, auxin, amino acid, vitamins, and enzymes possibly derived from microbes associated with earthworms (Suthar, 2010b).There is a demand for naturally derived agro-chemicals for sustainable farming systems, and organic production disallows the use of synthetic chemicals.There is no comprehensive study concerning the impact of organic fertilizer including vermiwash, leachate vermicompost or municipal solid waste compost on peppermint.
This study was undertaken to determine effects of these organic fertilizers in comparison with chemical fertilizer on oil yield and quality of peppermint.

Material and Methods
The experiment was conducted in a research field at the University of Guilan Campus, Agriculture Faculty, Rasht, Iran (altitude 7 m below mean sea level, 37°16′N, 51°3′E), from April to August 2012.The soil was a loam, pH 7.44, containing total N (1%), total C (1.08%), and there were 4,600, 1,700, and 4,000 mg•kg -1 of Ca, P, and K, respectively, in soil dry matter (DM), with an electrical conductivity (EC) of 0.1 dS cm -1 .The soil was prepared by plowing and disking.Local clones of peppermint were established by cutting on 15 April.Each plot area was 4 m 2 containing 80 plants.
Earthworms (Eisenia fetida) (25 g earthworms•kg -1 of cattle manure or 2.5 kg earthworms m -2 per bed) were added and vermicomposted for two months (Peyvast et al., 2008a(Peyvast et al., , 2008b)).The vermicompost had a water content of 380 g kg -1 , pH 6.82; total C content of 23.8% DM, and a total N content of 1.5% DM.The vermicompost (100 kg) was flushed with 50 L of water and leachate (vermiwash) was collected.Leachate vermicompost, after collecting vermiwash, was also stored for the next usage.
A completely randomized block design with three replications was used.Treatments included 7 Mt ha -1 of cow manure vermicompost, vermiwash prepared from 7 Mt ha -1 of vermicompost, leachate vermicompost + vermiwash, 50 Mt ha -1 municipal solid waste compost (MSWC), chemical fertilizer (NPK 50-0-300) and no fertilization as a control.Vermicompost, MSWC, and leachate vermicompost were spread over beds, and vermiwash was applied four times to plants at a sevenday interval with the first application one month after cutting.
At harvest, total leaf dry and fresh yield, oil yield, number of leaves, nodes and lateral branches per plant, leaves, stems and total plant fresh and dry weight, internode length, and plant height, leaf area index, content of essential oil per plant, total phenol, carotenoids and total chlorophyll, chlorophyll a, chlorophyll b, and antioxidant capacity were determined.Methanol extracts of sample (1 g of sample in 10 ml of methanol) were used for the determination of total phenolics.Total phenolic content was evaluated by colorimetric analyses using Folin-Ciocalteu's phenol reagent (Singleton and Rossi, 1965).The total phenolic content was expressed as mg galic acid equivalent/100 g of sample.The free radical-scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was evaluated with the methods of Leong and Shui (2002) and Miliauskas et al. (2004) with minor modification.In the presence of an antioxidant, the purple color intensity of DPPH solution decays and the changes of absorbance are followed spectrophotometrically at 517 nm.Total carotenoids (mg 100 g -1 ) were determined by a modified method of Ranganna (1997) using acetone and petroleum ether as extracting solvents and measuring absorbance at 450 nm.Chopped stems, leaves and plants were placed in a forced air drying oven at 75°C for 48 hours to determine dry matter.For the evaluation of the amount of essential oil a sample of 100 g of drying matter mixed with 800 ml of water was distilled for 3 hours using a Clevenger apparatus.

Results and Discussion
Fertilizer type affected all measured characteristics except number of nodes, stem fresh weight, total phenol and antioxidant capacity (Tables 1-4).The number of nodes, stem fresh weight, total phenol and antioxidant capacity averaged 11.48, 6.99 g, 5.35 mg galic acid equivalent/100 g sample and 60.78% respectively.Plants treated with vermicompost, vermiwash or leachate vermicompost + vermiwash were the tallest plants and had the highest levels of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids (Tables 5 and 8).Differences between plants treated with organic fertilizers were not significant for leaf area index (Table 7) where all plants treated with organic fertilizer had the higher leaf area index than the plants treated with chemical fertilizer and the control.There are no significant differences between different fertilizers for number of leaves, number of lateral branches, total plant dry weight, leaf dry weight, stem dry weight, total dry yield, oil yield and oil content per plant while the lowest values were related to plants cultivated without fertilization (Tables 5-7).Chemical fertilizer decreased plant internode length while there were no significant differences between other treatments.Plants treated with vermicompost or vermiwash had the highest total plant fresh weight, leaf fresh weight and total fresh yield (Tables 6-7).Organic fertilizers beneficially affect soil structure and nutrient availability, maintain quantity and quality of yield and can be less costly than synthetic fertilizers (Thy and Buntha, 2005;Olfati et al., 2012).The application of organic fertilizers may help alleviate soil erosion (Shahvali and Abedi, 2006) and saline and sodium problems as a result of excessive chemical fertilization and irrigation (Allahyari et al., 2008).The use of sustainable organic materials can increase fertility without negative effects on human health and environment.Vermicompost (Gutierrez-Miceli et al., 2007;Peyvast et al., 2008aPeyvast et al., , 2008b) ) and vermiwash (Suthar, 2010a) have been proposed as organic fertilizers previously.Most investigations confirmed that vermicomposts are beneficial to plant growth (Chan and Griffiths, 1988;Buckerfield and Webster, 1998;Atiyeh et al., 2000;Peyvast et al., 2008aPeyvast et al., , 2008d)).Leachate vermicompost contains slightly higher amount of sodium (Na), while vermiwash contains a high level of potassium (K), which as a primary nutrient, needed in high amounts for plant growth (Atiyeh et al., 1999;2000).
The main problem that can arise from excessive vermicompost application is plant toxicity due to high salt content.With leaching, the negative effects of vermicompost related to high EC (Gutierrez-Miceli et al., 2007) decreased and the continuous application of this material may be possible.The leachate vermicompost, vermiwash and vermicompost can be used as organic fertilizers for sustainable peppermint cultivation.

Table 1 .
ANOVA table effects of different fertilizers on vegetative characteristics., ** : non-significant or significant at P < 0.01 and P < 0.05, respectively. *

Table 2 .
ANOVA table effects of different fertilizers on total plant, leaf and stem fresh and dry weight.

Table 3 .
ANOVA table effects of different fertilizers on leaf area index (LAI), total fresh and dry yield, oil yield, and content of oil per plant.

Table 4 .
ANOVA table effects of different fertilizers on total phenol, antioxidant capacity, chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids content.

Table 5 .
Effects of different fertilizers on vegetative characteristics.

Table 6 .
Effects of different fertilizers on total plant, leaf and stem fresh and dry weight.

Table 7 .
Effects of different fertilizers on leaf area index (LAI), total fresh and dry yield, oil yield, and content of oil per plant.

Table 8 .
Effects of different fertilizers on total phenol, antioxidant capacity, chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids content.