151 1. Introduction The genus Ocimum (Lamiaceae family), collectively called basil, comprises between 50 and 150 species of herbs and shrubs (Darrah, 1980). Basil is native to Asia (India, Pakistan, Iran, Thailand, and other countries) and can be observed growing wild in tropical and sub-tropical regions (Makri and Kintzios, 2008). The essential oil profiles of this group of plants are extremely variable, such that several aro- ma compounds can be found in chemotypes of basil such as citral, eugenol, linalool, methylchavicol, and methylcinna- mate that are traded in the international essential oil market (Simon et al., 1999). The diversity within basil species has been accentuated by centuries of cultivation and cross com- patibility, which has lead to great variation in morphology and chemical profile (Javanmardi et al., 2002). Basil species have antioxidant, antimicrobial and antitumor activities that are due to the presence of phenolic acids and aromatic com- pounds (Hussain et al., 2008). Lemon basil (Ocimum × citriodorum Vis.), a hybrid of sweet basil (Ocimum basilicum) and American basil (Oci- mum americanum), is a herb grown primarily in northeast- ern Africa and southern Asia (Fisher and Phillips, 2006). It is naturalized in Asia and cultivated for its lemon-scent- ed leaves due to the essential oils citral and neral as pre- dominant compounds (Grayer et al., 1996). Lemon basil is characterized by its small stature, early flowering, and small, narrow leaves. Application of organic sources of nutrients, with no or very little use of inorganic fertilizers, is rapidly gaining favor (Anwar et al., 2005). Compost tea is a highly con- centrated microbial solution produced by extracting ben- eficial microbes from compost. Compost tea is produced by mixing compost with water and incubating it for a de- fined period, either actively aerating (aerated compost tea, ACT) or not (non-aerated compost tea, NCT) and with or without additives that are intended to increase microbial population densities during production (Scheuerell and Mahaffee, 2002; Ingham, 2005). It is a source of foliar and soil nutrients, contains chelated micronutrients for easy plant absorption and the nutrients are in biologically avail- able forms for both plant and microbial uptake (Hendawy, 2008). Many researchers have pointed out the efficacy of organic manures, compost and compost teas in increas- ing vegetative growth, biomass and essential oil yield of sweet marjoram (Gharib et al., 2008), cumin (Safwat and Badran, 2002), fennel (Azzaz et al., 2009) and sweet basil (Khalid et al., 2006). Improvements in yield and quality following application of these organic-based substances has been attributed to an enhancement of the beneficial microbial communities in soil, an improvement of mineral absorption conditions for plants, and a stimulation of de- fense compounds, growth regulators or phytohormones in Effects of chicken manure and vermicompost teas on herb yield, secondary metabolites and antioxidant activity of lemon basil (Ocimum × citriodorum Vis.) J. Javanmardi(1), E. Ghorbani Department of Horticultural Sciences, Faculty of Agriculture, Shiraz University, Shiraz, Iran. Key words: essential oil, flavonoids, organic agriculture, sustainable agriculture, total phenolics. Abstract: Effects of chicken manure tea (CMT) and vermicompost tea (VCT) as soil drench on vegetative growth, herb yield, essential oil content, total phenolics, total flavonoids and antioxidant activity of lemon basil (Ocimum × citriodorum Vis.) was evaluated in a two-year field experiment. The greatest plant height, number of leaves and flowers, shoot fresh and dry weight and leaf chlorophyll content were obtained using CMT at either 1:5 or 1:10 dilutions with no significant differences. The highest number of lateral branches and flavonoid content were obtained when CMT at 1:5 dilution was applied. Essential oil content was at its highest level (0.618%) when CMT or VCT were used at 1:10 dilution, while the greatest total phenolic content and total antioxidant activity were obtained at 1:5 dilution of VCT. The results emphasize the possibility of using organic-based compost teas for enhancing herbal yield and important secondary metabolites in aromatic medicinal plants. Adv. Hort. Sci., 2012 26(3-4): 151-157 (1) Corresponding author: javann@shirazu.ac.ir. Received for publication 27 June 2012 Accepted for publication 31 January 2013 152 plants (Pant et al., 2009). Various liquid manures or their extracts are known to serve primarily as a source of soluble plant nutrients, growth stimulants and disease suppressors (Khalid et al., 2006). Several studies have reported the effects of compost tea on suppression of certain plant diseases such as damping– off caused by Phytium ultimum (Scheuerell and Mahaffee, 2004), gray mold (Botrytis cinerea) (Scheuerell and Ma- haffee, 2006), Alternaria solani and Phytophthora infestans (Koné et al., 2010). However, relatively little work has been done to investigate the effect of vermicompost or manure teas on yield, nutritional and quality factors, secondary me- tabolites and antioxidant activity of vegetable herbs. In the present work the effects of chicken manure and vermicompost teas as soil drench on growth characteris- tics, chlorophyll content, essential oil yield, total pheno- lics, total flavonoids and antioxidant activity of lemon ba- sil were evaluated. The significance of this study lies in the possibility of applying compost teas as soil amendments to improve yield components and secondary metabolites in organic agriculture. 2. Materials and Methods Site description, plant material and experimental design The experiment was carried out in two subsequent years, 2010 and 2011, in a field 1810 m above sea level in a silty-loam soil. Three soil samples from 0-30 cm depth were collected and sent to a certified local soil laboratory for analysis. The chemical properties of the soil are shown in Table 1. The local maximum-minimum mean tempera- tures and relative humidity during the growing period were 28.7-12.6°C and 47.3%, respectively. Seeds of lemon basil (Ocimum × citriodorum Vis.) were sown in double rows (20 cm apart) with 50 cm spac- ing. Seedlings were thinned three weeks after sowing to 15 cm between plants within the rows. Plants were harvested at ground level when they were at full bloom stage (90 days after sowing) for further analyses. Irrigation (using a drip-tube system), hand weeding, and other management practices were performed when required throughout the growing period. The experiment was carried out in a complete random- ized block design with three replicates per treatment, each of which consisted of 20 plants. Five fertilization treat- ments were applied as soil drench of de-ionized water (control), chicken manure tea (CMT) at 1:5 and 1:10 water dilution (v/v) and vermicompost tea (VCT) at 1:5 and 1:10 water dilution (v/v). Tea preparation and application method Vermicompost and chicken manure teas were pre- pared as described by Javanmardi (2010). Briefly, vermi- compost and chicken manure were separately mixed with tap water at ratios of 1:5 and 1:10 (v/v) in loosely cov- ered 14 l plastic containers. Water was allowed to stand for 24 h for passive chlorine removal before mixing. The mixtures were aerated using an aquarium pump for 72 h brewing time in a shaded area. Solutions were filtered through cheesecloth before application. Chemical prop- erties for teas are presented in Table 2. Treatments were started four weeks after sowing and applied five times as soil drench with 600 ml of solution per plant at weekly intervals. Fresh solutions were prepared for each applica- tion interval. Vegetative growth parameters At full bloom stage ten central plants from each repli- cate (to avoid marginal effect) were cut at ground level and plant height (cm), number of branches per plant, number of flowers, number of leaves, and fresh and dry weight of herb (g per plant) were recorded. Chemical analysis Chlorophyll content. Chlorophyll content was deter- mined as described by Saini et al. (2001). Randomly se- lected samples of fully expanded leaves (0.5 g) were used. Samples were homogenized with 5 ml of acetone (80% v/v) using a pestle and mortar and filtered through filter paper (Whatman No. 2). The process was conducted in the dark to avoid photo bleaching. Absorbance was measured with a UV-visible spectrophotometer (Camspec M108, Spectronic Instruments, Leeds, UK) at 652 nm and total chlorophyll content calculated using: Total chlorophyll (mg·g-1 FW) = [D 652 ×V]×V/W where: V is the total volume of acetone extract (ml) and W, the fresh sample weight (g). Essential oil content. Quantitative determination of the essential oil obtained from lemon basil subjected to the different treatments was achieved by placing the air-dried herbage in a 2 l flask with distilled water (1:15 w/v) and using a Clevenger apparatus, as described by Charles and Table 1 - Chemical properties of soil Organic matter (%) Total N (%) Available phosphorus as P (mg·kg-1) Bray method Available potassium as K (mg·kg-1) Fe (mg·kg-1) Cu (mg·kg-1) Mn (mg·kg-1) Zn (mg·kg-1) pH EC (ds·m-1) 1.21 0.05 13.50 540 4.88 1.19 0.39 0.23 7.73 1.50 153 Simon (1990). The average essential oil content of aerial parts is reported as percent of plant dry matter. Sample preparation for total phenolic and flavonoid content determination. Samples were prepared using the method described previously by Javanmardi et al. (2003). Briefly, 250 mg of dried plant material from each replicate were ground and dissolved in 10 ml of 80% acetone. Sam- ple extracts were rotated for 1 h in the dark and centrifuged at 5400 g for 10 min. One ml of supernatant was dried un- der vacuum at 45°C and kept at -18°C for further use. Each sample was dissolved in 1 ml acetone prior to analysis for total phenolic and flavonoid determination. Total phenolic compound analysis. The amount of total phenolics in extracts was determined with the Folin-Cio- calteau reagent using the method of Spanos and Wrolstad (1990), as described by Javanmardi et al. (2003). To 50 ml of each sample, 2.5 ml of 1/10 dilution of Folin-Ciocal- teau reagent and 2 ml of Na 2 CO 3 (7.5%, w/v) were added and incubated at 45°C for 15 min. The absorbance of all samples was measured at 765 nm using a UV-visible spec- trophotometer (Camspec M108, Spectronic Instruments, Leeds, UK). Gallic acid was used as standard and results are expressed as mg of gallic acid equivalent per g of dry weight (mg GAE/g dw). Total flavonoid analysis. The method described by Adom and Liu (2002) was adopted for total flavonoid con- tent analysis. To 0.5 ml of extract, 2.5 ml distilled water were added followed by 0.15 ml of 5% NaNO 2 solution. The mixture was left to stand for 6 min at room tempera- ture before adding 0.3 ml 10% AlCl 3 .6H 2 O solution. The mixture was left for an additional 5 min, then 1 ml of 1 M NaOH added and made up to 5 ml with distilled water. The solution was vortexed and the UV absorbance at 510 nm was recorded against catechin as reference. The result is expressed as µg/g DW. Total antioxidant activity assay. The antioxidant activ- ity of samples was determined by free radical scaveng- ing activity assay using 1,1-diphenyl-2-picryl-hydrazil (DDPH) reagent according to Brand-Williams et al. (1995). The ground leaves (1 g) were extracted with 50% methanol, 50% water. To 0.75 ml of the extract sample, 1.5 ml of freshly prepared methanolic DPPH solution (20 µg·ml-1) were added and stirred. The decolorizing process was recorded after 5 min of reaction at 517 nm and com- pared with a blank control. The total antioxidant activity is expressed in % calculated as (control absorbance - sample absorbance /control absorbance) × 100. Statistical analysis The experiment was carried out for two years in a random- ized complete block design (RCBD) with three replicates, each of which consisted of 10 plants. Data were analyzed using one-way analysis of variance (one-way ANOVA) and means of two years for each trait were compared with Least Significant Difference (LSD) at p≤0.05 by SPSS12 (SPSS Inc., Chicago, IL) computer software for Windows. The data presented in tables and figures are mean values ± standard errors of two years of data for three replicates. Pearson correlation analysis using SPSS12 (SPSS Inc., Chicago, IL) was performed to assess the relationship be- tween total phenolics, total flavonoids and essential oils with total antioxidant activity. 3. Results and Discussion The combined analysis of data from the experiment did not show any significant differences for all traits (data not shown). Growth parameters Analyses of variance showed that the growth param- eters of lemon basil including plant height, number of lateral branches, leaves and flowers, shoot fresh and dry Table 2 - Chemical properties of vermicompost tea (VCT) and chicken manure tea (CMT) vermicompost tea (1:5) vermicompost tea (1:10) chicken manure tea (1:5) chicken manure tea (1:10) Organic matter (%) - - - Total N (%) 6.30 3.14 2.21 1.10 Phosphorus as P (%) 1.01 0.50 1.31 0.65 Potassium as K (%) 6.18 3.08 5.61 2.81 Fe (mg·l-1) 9.20 4.51 9.00 4.48 Cu (mg·l-1) 0.07 0.03 5.00 2.48 Zn (mg·l-1) 25.0 12.0 3.20 1.60 Mn (mg·l-1) 0.18 0.89 0.08 0.04 pH 8.22 8.22 8.29 8.29 EC (ds·m-1) 4.41 2.20 2.82 1.41 154 weight at full bloom stage were significantly affected by VCT and CMT (Table 3). Plant height and number of leaves The greatest plant height and leaf number were ob- served at 1:5 CMT, which was not significantly different from 1:10 dilution. Other treatments did not show signifi- cant differences for plant height and leaf number (Table 3). The same result has been reported in Plantago plants under 300 ml·l-1 compost tea application (Hendawy, 2008). The increasing effects of organic manure and bio-fertilizers on plant height on fennel (Azzaz et al., 2009) and peppermint (Swaefy et al., 2007) have been previously reported. Number of lateral branches The highest lateral branch number was observed at 1:5 CMT dilution, however it was not significantly different from 1:10 CMT. The differences among other treatments (1:5 and 1:10 VMT and control) with regard to plant lat- eral branches were not significant (Table 3). A promoting effect of organic fertilizers on the number of branches was observed also by Azzaz et al. (2009) in fennel plants, es- pecially when organic fertilizers were used in combination with bio-fertilizers. Number of flowers The highest flower numbers were observed in lemon basil plants treated with CMT (1:5 and 1:10 dilutions) and VCT (1:5 dilution) with no significant differences. Con- trol plants and VCT-treated plants at 1:10 dilution showed lower flower numbers (Table 3). An increased number of flowers in Plantago plants under 300 ml·L-1 compost tea application has previously been reported (Hendawy, 2008). Shoot fresh and dry weight The highest shoot fresh and dry weights were observed at 1:5 CMT, which was not significantly different from 1:10 dilution. Other treatments did not show significant differences for these parameters (Table 3). The promoting effect of chicken manure tea on herb yield may be attrib- uted to the micronutrient content and to the action of living micro-organisms and microbial metabolites which stimu- late plant growth (Diver, 2002; Carpenter, 2005). Higher fresh and dry weights have also been attributed to the avail- ability of macronutrients, especially nitrogen, and/or to the improvement of soil water-holding capacity (El-Sherbeny et al., 2005). Furthermore, it has been stated that organic manure activates many species of living organisms which release phytohormones and may stimulate plant growth and absorption of nutrients (Naguib and Aziz, 2003). The same increased vegetative growth characters (including shoot fresh and dry weights) of basil plants under organic farming has been previously reported (Khalid et al., 2006). Chemical parameters Leaf chlorophyll content. The highest leaf chlorophyll content was observed at 1:5 CMT which was not different from 1:10 CMT dilution. Other treatments did not show sig- nificant differences for this parameter (Table 3). The pro- moting effect of highly N-containing chicken manure tea on chlo rophyll contents might be attributed to the fact that N is a constituent of chlorophyll molecule. Moreover, nitrogen is the main constituent of all amino acids and lipids that act as structural compounds of the chloroplast (Al-Tarwneh, 2005). In sweet basil, chlorophyll content was significantly higher when organic manure compost was applied than in non-fertilized control plants (Taie et al., 2010). Essential oil content. The essential oil content of lemon basil was affected by dilution levels of organic compost teas. The 1:10 dilution of VCT and CMT was higher in es- sential oil content compared to 1:5 dilutions (Fig. 1). The highest essential oil content was found in the 1:10 VCT treatment and it was about 3.12 times higher than in con- trol plants. Previously, the highest essential oil content in Ocimum basilicum was obtained following soil applica- tion of vermicompost at 10 t·ha-1 level compared to appli- cation of 10 t·ha-1 farmyard manure and control treatments (Anwar et al., 2005). In marjoram plants, aqueous extract of compost increased essential oil percentage and yield (Gharib et al., 2008). In basil as a source of essential oils and aroma compounds (Simon et al., 1990), the increase in essential oil yield has been attributed to increase in vegeta- tive growth or changes in leaf oil gland population (Gharib et al., 2008). Our data are in agreement with a previous work that found a higher essential oil percentage in ba- Table 3 - Effect of different dilutions of chicken manure tea (CMT) and vermicompost tea (VCT) on plant height, number of leaves, lateral branches and flowers, shoot fresh and dry weight and leaf chlorophyll content of lemon basil plants Treatment Plant height (cm) No. of leaves No. of lateral branches No. of flowers Shoot fresh weight (g) Shoot dry weight (g) Chlorophyll (mg·g-1 fw) Control 27.30±0.89 130.03±12.15 6.80±0.84 6.57±0.90 8.10±1.01 1.45±0.18 0.71±0.016 CMT (1:5) 30.47±1.27 225.93±22.80 10.23±0.92 11.80±1.62 17.63±2.44 2.64±0.36 0.82±0.012 CMT (1:10) 29.23±1.09 197.30±22.50 8.07±0.85 10.27±1.48 14.27±2.41 2.51±0.48 0.80±0.013 VMT (1:5) 27.57±0.64 136.70±13.58 6.77±0.93 10.47±1.80 8.37±0.92 1.60±0.18 0.74±0.016 VMT (1:10) 27.30±1.01 131.13±12.88 5.20±0.72 6.84±0.91 7.87±1.07 1.53±0.17 0.74±0.015 LSD value (p=0.05) 2.65 47.11 2.34 3.85 4.58 0.79 0.039 Data (per plant) are mean values of 3 replicates ± standard errors. 155 sil due to application of organic fertilizers as compared to control plants (Taie et al., 2010). Fig. 1 - Effect of different dilutions of chicken manure tea (CMT) and vermicompost tea (VCT) as soil drench on essential oil percent of lemon basil. Vertical bars show standard errors of the means (n=3). Total phenolic content. Application of VCT at 1:5 dilu- tion gave the highest total phenolic content. The differenc- es between CMT and 1:10 dilution of VCT were not sig- nificant (Fig. 2). Previously, Sousa et al. (2005) and Taie et al. (2010) reported that total phenolic contents achieved by organic culture were higher than those from conventional practice in tronchuda cabbage (Brassica oleracea L. var. costata DC) and sweet basil. Asami et al. (2003) and Wang and Lin (2002) also observed consistently higher levels of total phenolics in organically-grown crops compared with those produced by conventional agricultural practices. The amount of total phenolic content of lemon basil in this ex- periment is in the range of previously reported total pheno- lic content of different sweet basil accessions (Javanmardi et al., 2003). Plant phenolics constitute one of the major groups of compounds acting as primary antioxidants or free radical terminators (Lukmanul-Hakkim et al., 2008). Total flavonoids. The highest total flavonoid content was observed in 1:5 CMT-treated lemon basil plants and it was over 1.4 times higher than that produced in con- trol plants. The differences between the two dilutions of VCT were not significant (Fig. 3). In previous studies, the amounts of surface flavonoids in Ocimum x citriodorum specimens were reported in a range of 0.2 to 5.7 mg g-1 (Grayer et al., 2004). Our finding is in agreement with a previous work that reported a significant increase in fla- vonoid content of Ocimum basilicum due to compost or compost tea application in comparison with control plants (Khalid et al., 2006). It has been stated that the antioxidant activity in basil is largely due to the presence of phenolic components, including flavonoids and phenylpropanoids (Juliani and Simon, 2002). Fig. 3 - Effect of different dilutions of chicken manure tea (CMT) and ver- micompost tea (VCT) as soil drench on total flavonoids of lemon basil. Vertical bars show standard errors of the means (n=3). Total antioxidant activity. The antioxidant activity of lemon basil extracts is shown in figure 4. The total anti- oxidant activity (TAA) ranged from 48.28% (control) to 58.42% (VCT at 1:5 dilution). There were no statistically significant differences between CMT dilutions and 1:10 dilution of VCT. Pearson correlation analysis between secondary me- tabolites (total phenolics, total flavonoids and essential oils) Fig. 4 - Effect of different dilutions of chicken manure tea (CMT) and vermicompost tea (VCT) as soil drench on total antioxidant ac- tivity of lemon basil. Vertical bars show standard errors of the means (n=3). Fig. 2 - Effect of different dilutions of chicken manure tea (CMT) and vermicompost tea (VCT) as soil drench on total phenolic con- tent of lemon basil. Vertical bars show standard errors of the means (n=3). 156 with total antioxidant activity showed a significant positive correlation coefficient of R2=0.91 between the amount of total phenolics and total antioxidant activity (Table 4). This means that about 91% of total antioxidant activity in lemon basil plants was due to phenolic compounds. Other second- ary metabolites did not show significant contributions to the antioxidant activity. The same correlation was previously reported in sweet basil (Ocimum basilicum L.) accessions (Javanmardi et al., 2003). The antioxidant activity of phe- nolics is mainly due to their redox properties, which allow them to act as reducing agent, hydrogen donors, single oxy- gen quenchers and having possible metal chelating activity (Rice-Evans et al., 1995). Table 4 - Pearson’s correlation coefficients between total phenolics, to- tal antioxidant activity, total flavonoids and essential oil per- cent of treated lemon basil plants Total phenolics Total flavonoids Essential oil content Total antioxidant activity 0.919 0.071 ns 0.320 ns Data (per plant) are mean values of 3 replicates ± standard errors. 4. Conclusions The findings of this study indicate that fertilization with chicken manure and vermicompost organic teas im- proves herbal and essential oil yields as well as antioxida- tive agents such as total phenolics in lemon basil plants. The results point to the beneficial effects of compost tea as possible nutrition sources on growth characteristics and essential oil yields of basil. 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