Impaginato 205 Adv. Hort. Sci., 2019 33(2): 205-214 DOI: 10.13128/ahs-23828 Yield and physiological response of Perilla (Perilla frutescens) under different soil fertility treatments M. Ghane 1, M. Mohammadi 2 (*), H. Pirdashti 1 1 Department of Agronomy, Sari Agricultural Sciences and Natural Resources University, Sari, Iran. 2 Department of Plant Sciences, University of Tennessee, USA. Key words: bio-fertilizer, chemical fertilizer, DPPH test, inoculation, organic fer- tilizer, Perilla, rosmarinic acid. Abstract: Medicinal plants are one of the main natural resources of Iran from ancient times. Perilla is one of the most important medicinal plants of the mint family Lamiaceae, since there is no study about adaptability of Perilla in Iran climate conditions and different fertilizer systems, this experiment was con- ducted in two experimental sites. The experiment was conducted as split-plot factorial based on a randomized complete block design with three replications at two experimental regions. The main factor was three chemical fertilizer lev- els (control, 50, 100, 200 kg/ha) and subplots were different kinds of organic fertilizer (control, humic acid, and compost application) and inoculation with Piriform osporaindica (inoculation and without). Among levels of chemical treatments, 50 and 100 kg/ha lead to a better result. Also, humic acid allows to achieve the highest amount of measured traits between different treatments of organic fertilizer. The highest plant yield (147.2 g/m2) and rosmarinic acid yield per area (3.432 g/m2) was achieved in 100 kg/ha at chemical fertilizer with humic acid and biological fertilizer application and the lowest plant yield (89.86 g/m2) and rosmarinic acid yield per area (1.253 g/m2) was observed in control. Also, the highest stomatal conductance was obtained with application of com- post fertilizer (67.67 mmol H2O m -2s-1). Integrated application of the studied fer- tilizers showed the more positive effect on yield and quality of Perilla than indi- vidual application of those fertilizers. 1. Introduction The Perilla is a medicinal plant belonging to the Labiatae family and is widely cultivated in Southeast Asian countries (Igarashi and Miyazaki, 2013). A number of studies have shown that advantages of Perilla are related to the metabolites contained therein (Ghimire et al., 2017). To date, very limited information exists regarding the adaptability and man- agement of chemical, organic and bio-fertilizer of Perilla in Iran. Organic matter effects on physicochemical properties and health of soil. It also affects the efficiency of fertilizer application, pesticides and herbicides. One of the most effective organic fertilizers on the growth of (*) Corresponding author: mmohamm9@utk.edu Citation: GHANE M., MOHAMMADI M., PIRDASHTI H., 2019 - Yield and physiological response of Perilla (Perilla frutescens) under different soil fertility treatments. - Adv. Hort. Sci., 33(2): 205-214 Copyright: © 2019 Ghane M., Mohammadi M., Pirdashti H. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 26 August 2018 Accepted for publication 18 February 2019 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2019 33(2): 205-214 206 plants and improvement of soil status is humic acid. Humic acid can be obtained from any material such as organic matter, coal, or well-decomposed com- post. This material plays an important role in increas- ing soil moisture, absorbing micronutrients and con- tributing to carbon sequestration (Spaccini et al., 2002). Another organic material that plays an important role in soil fertility is compost and it comes from p l a n t a n d f o o d r e s i d u e s . S o m e e x p e r i m e n t a l researches have shown the important role of com- post in crop production (Adugna, 2016). Compost retains moisture in the soil, slow release nutrients to crops and finally increase the crop yield. Application of compost obtained from plant remains leads to increased fertility, soil nutrients and increases water retention in the soil. Zemanek (2011) also confirmed that application of 50 t/ha and 100 t/ha compost has a positive effect on soil moisture retention. Moreover, biologic fertilizers assists well in miner- alization and channelization of nutrients leading to enhanced plant productivity (Fischer et al., 2007; Ansari et al., 2017). Biologic fertilizers adopt various possible ways to accelerate the rate of crop produc- tion (Rizvi et al., 2015; Ansari et al., 2017). They increase and improve plant growth by increasing access to nutrients in the root rhizosphere. These fer- tilizers provide nutrients through biological processes such as biological nitrogen fixation, phosphorus solu- bilization, and plant growth stimulation. Also, they help to natural nutrient cycle and build soil organic matter (Kapoor et al., 2015). Use of biologic fertilizers ensures healthy plants growth, while enhancing the sustainability and the vigor of the soil. These biologic fertilizers play a special role in increasing plant nutri- tion and fertility of soils (Vessey, 2003). A number of studies have shown that the chemical composition of secondary metabolites in the Perilla plant is influenced by various factors such as soil con- ditions, temperature, growth season (Kiazolu et al., 2016), geographic region (Ruberto et al., 2002), and phenological stages (Saeb and Gholamrezaee, 2012). Therefore, this study was carried out to evaluate the yield and physiological traits of Perilla cultivars under different fertilizer treatments in two regions. Based on the work, attempt was also made to provide ami- cable solutions to address the challenges of organic farming with the help of Perilla cultivation in two dif- ferent locations in dry region of Iran. 2. Materials and Methods The field experiments were conducted at two locations in Esfahan Province: 1-Mashhad Ardehal (latitude 34° North, longitude 51° East, Altitude 1800 m above mean sea level) 2- Sensen, Iran (latitude 33°, longitude 51°, Altitude 945 m above mean sea level) during 2015. Mashhad Ardehal is located in warm and dry condition and Sensen is located in the mountainous and dry region. The meteorological data recorded during the period of plant cultivation are given in Table 1. In order to determine the physical and chemical characteristics of experimental fields, two weeks b e f o r e p l a n t i n g , t h e s o i l s a m p l e s w e r e t a k e n . Physicochemical properties of experimental field soil are presented in Table 2. This experiment was conducted as split-plot facto- rial based on a randomized complete block design with three replications at two experimental sites in the year 2015. The main factor was three chemical fertilizer levels (50, 100, 200 kg/ha) plus control, and sub plots were different kinds of organic fertilizer (humic acid, and compost application), plus control; inoculation with Piriformospora indica (inoculation and without inoculation) was also evaluated. Table 1 - Weather characteristics in Mashhad Ardehal and Sensen Year Temperature mean (°C) Precipitation mean (mm) Humidity mean (%) Sensen Mashhad Ardehal Sensen Mashhad Ardehal Sensen Mashhad Ardehal 2015 19.8 18.4 89.3 90.8 42 49 Average of 5 years 20.86 19.88 143.36 133.42 40.6 46.6 Parameter Location Sensen Mashhad Ardehal Total Nitrogen (%) 0.09 0.28 Phosphorous availability (mg/kg) 15.56 12.18 Potassium availability (mg/kg) 245.6 209.7 pH 7.93 7.83 EC (dS.m-1) 2.82 0.89 Organic carbon (%) 0.53 1.63 Clay (%) 14.3 10.3 Silt (%) 33.3 43.4 Sandy (%) 52.4 46.3 Table 2 - Soil analysis of the experimental site Ghane et al. - Yield and physiological response of Perilla 207 F o l i a r - a p p l i e d h u m i c a c i d ( 9 5 % p u r i t y ) w a s obtained from Humic Strong company (70% w/w, pH 5.17, EC: 4.80 mS/cm) and added to the plots at 4 stages of the plant growth. Compost fertilizer was prepared from Barij Essence Company (including 1.5% nitrogen, 1.1% phosphorous, 0.9% potassium, 50% organic matter). Compost fertilizer was used as a strip one week after plantation. Perilla seeds were purchased from Barij essence Company, Isfahan, Iran. First of all, the seeds were germinated in the laboratory and then transplanted to the pots with 10 cm diameter and 15 cm height at t h e 4 - l e a f s t a g e . T h e m y c o r r h i z a l - l i k e f u n g u s Piriformospora indica was prepared in mycology lab- oratory of Sari University, Iran and then prepared biologic fertilizer was placed two weeks in incubator with 20-25˚C and 50 rpm under dark condition. Before planting of Perilla plants in the field, half of the plants in pots were inoculated with biologic fertil- izers and then translocated to the field. Each plot was 2×2 meter and had 4 furrows with 25 plant per plot. The recommended dose of chemical fertilizer (0- 50-100 and 200 kg/ha) in the form of urea, triple super phosphate and sulfate potassium was applied to grow the crop. Nitrogen was applied in three splits, the first along with phosphorus and potassium fertilizer at the time of soil preparation while the sec- ond part at the time of transplanting of plants and the third part at the flowering of crops. Date of trans- planting of both experimental sites was 20 of March. After plant growth, different traits were studied at the suitable stage. A furrow irrigation system was applied for both experimental sites. Hand weeding of the experimental area was performed as required. In both experimental sites, after flowering five plants from each plot were harvested from two central rows and then plant height, fresh weight and dry weight of plants were measured. Samples dried in an oven at 80°C for 24 hours and the mean of dry weight for each treatment at each replicate was determined. Rosmarinic acid determination by HPLC The dried seeds of Perilla were pulverized (60 mesh) for 3 min using an HR 2860 coffee grinder (Philips, Drachten, Netherlands), and each sample (1.0 g) extracted in 30 ml of 80% methanol for 6 h at room temperature in a shaking incubator. The super- natant was centrifuged at 3000g for 3 min and then filtered through a 0.45 lm syringe filter (Whatman Inc., Maidstone, UK) prior to HPLC analysis. For quan- tification, the peak areas of the isolated compounds were integrated from the HPLC chromatogram at 330 nm using the Dionex software. The stock solutions were prepared by dissolving in methanol to obtain a 1 mg/ml concentration. Calibration curves were obtained with methanol at eight different concentra- tions (0.5, 1, 2, 5, 10, 25, 50, and 100 g/ml). All cali- bration curves had coefficients of linear correlation r2>0.998. Stomatal conductance Twenty days after plantation stomatal conduc- tance was measured in the shade-enclosure with sat- urated light using Promoter (Model KR1301, KOREA TECH) Stomatal conductance (mmol H2O m-2 s-1) was measured at 12:00-14:00 hours on a clear, cloud-less day in fully expanded, healthy, turgid, three flat and uniform in color and size leaves of each samples (Barbieri et al., 2012). DPPH radical-scavenging activity This test was used for the determination of the free radical-scavenging activity of the extracts (Ebrahimzadeh et al., 2008). DPPH test was per- f o r m e d f o l l o w i n g t h e m e t h o d p r o p o s e d b y Ebrahimzadeh et al. (2008). Three young fully devel- oped leaves were selected from each replication. Polyphenoloxidase (PPO) Activity Crude extract was prepared by homogenization of frozen plant sample in buffer medium. Leaves of Perilla plant which were stored at -20°C was used for the enzyme extraction. 10 g of the sample were cut quickly into thin slices and homogenized in 50 mL of 100 mM sodium phosphate buffer (pH 7.0) containing 1 mM ascorbic acid and 0.5% (w/v) polyvinylpyrroli- done for 5 min at 4°C. The homogenate was filtered through three layers of cheesecloth and then the fil- trate was centrifuged at 5,000 x g for 15 min, and the supernatant was collected. Enzymatic activity was assayed by determining the rate of increase in absorbance at 420 nm and 25˚C in a Perkin-Elmer Lambda 15 UV/VIS spec- trophotometer (Shimadzu Corp., Tokyo, Japan). The reaction mixture contained 3.0 mL of catechol sub- strate, the solution freshly prepared in 0.05 M sodi- um phosphate buffer at pH 6.5 and a fixed quantity of PPO. The reference cuvette contained only the cat- echol substrate solution. The reaction was conducted at 25°C. The PPO activity was defined as a change of 0.001 in absorbance at the conditions of the assay (Pizzocaro et al., 1993). Catalase activity assay CAT activity was measured by monitoring the H2O2 decomposition at 240 nm in 3 mL of reaction Adv. Hort. Sci., 2019 33(2): 205-214 208 mixture containing 50 mmol/l phosphate buffer (pH 7.0), 15 mmol/L H2O2, 100 mL enzyme extract and 0.1% (v/v) Triton X-100 (Aebi, 1984). The activity was e x p r e s s e d i n t e r m s o f m m o l H 2O 2 r e d u c e d min/mg/protein. Statistical analysis The data were tested for homogeneity and nor- mality of residuals using the Bartlett tests and Kolmogorov-Smirnov, respectively. A combined ANOVA was used to compare treatments for 2 loca- tion using PROC GLM of SAS 9.1 software. Means were separated by application of LSD test when the F test proved significant at P≤0.05 and 0.01. 3. Results and Discussion Combined statistical analysis of two studied loca- tion is presented in Table 3. Also, the analysis of vari- ance of Mashhad Ardehal and Sensen locations are shown separately in Table 4 and 5. In Mashhad Ardehal location, the maximum plant yield (157.6 g/m2) was achieved in 100 kg/ha chemical fertilizer plus humic acid treatment and the minimum amount (90.68 g/m2) was produced in zero levels of chemical fertilizer and without application of organic treat- ment (Fig. 1, Table 3). While in Sensen location, the maximum plant yield (126.9 g/m2) was achieved with the application of 50 kg/ha chemical fertilizer plus humic acid (Fig. 1). Between treatments of chemical × organic × bio- logic fertilizer interaction, the maximum plant yield (147.2 g/m2) was produced in 100 kg/ha along with humic acid and application of biologic fertilizer (Table 6). Differences between plant yield (147.2 g/m2 and 89.86 g/m2) showed the importance of fertilizer application in improving yield in this plant. In all of the chemical fertilizer treatments, the plant yield was increased with application of organic and biologic fer- tilizer, especially with organic fertilizer. So, the inte- grated fertilizer application was very positive. In the present investigation, application of organic fertilizer especially humic acid significantly increased plant Fig. 1 - Effect of chemical and organic fertilizers on plant yield of Perilla in Mashhad Ardehal and Sensen regions. Different letters in each column denote a significant dif- ference at p≤0.05 (LSD test). *, ** significant at 5% and 1% probability levels, respectively. Table 3 - ANOVA of some morphologic, yield and essence traits of Perilla under different fertilizer systems in Sensen and Mashhad Ardehal in Kashan regions S.O.V. d.f. Means squares Plant yield Rosmarinic acid content Rosmarinic acid yield Stomatal conductance DPPH Catalase activity Polyphenol oxidase Location (L) 1 14012.64 ** 13.80 ** 8.87 ** 248.11 ** 224.60 ** 0.00357 * 0.000935 ** Replication (Location) 4 56.75 0.70 0.01 4.02 18.56 0.00047 0.000021 Chemical fertilizer (C) 3 10188.25 ** 244.44 ** 13.92** 1504.81 ** 99.75 ** 0.03396 ** 0.010834 ** L×C 3 1899.70 ** 27.75 ** 2.53** 184.57 ** 19.74 0.01396 ** 0.000478 ** Ea 12 28.48 1.18 0.04 5.74 44.51 0.00063 0.000072 Organic fertilizer (O) 2 1435.58 ** 27.93 ** 1.69** 76.66 ** 75.18 * 0.00178 0.000454** Biologic fertilizer (B) 1 308.17 ** 8.41 ** 0.53** 213.84** 0.24 0.00003 0.000146 * C × O 6 73.04 4.75 ** 0.06* 8.46 47.80 * 0.00170 * 0.000217 ** C × B 3 130.19 * 1.05 0.06 2.01 2.82 0.00257 ** 0.000022 O × B 2 236.97 ** 1.31 0.09* 2.47 19.55 0.00014 0.000008 L × O 2 71.26 4.79 ** 0.17** 13.49 2.83 0.00253 * 0.000275 ** L × B 1 88.64 4.42 * 0.22** 1.92 22.75 0.00026 0.000003 L × C × O 6 83.62 * 2.89 ** 0.03 10.55 5.98 0.00244 ** 0.000180 ** L × C × B 3 18.09 0.16 0.001 3.44 0.20 0.00178 * 0.000009 L × O × B 2 95.96 0.77 0.11* 9.61 0.13 0.00075 0.000010 C × O × B 6 191.38 ** 2.38 * 0.11 ** 2.76 18.59 0.00068 0.000004 L × C × O × B 6 66.33 1.42 0.04 3.05 6.50 0.00009 0.000006 Eb 80 37.44 0.79 0.02 5.84 19.15 0.00059 0.0000224 CV (%) - 5.11 4.69 6.66 3.64 2.42 5.48 3.06 Ghane et al. - Yield and physiological response of Perilla 209 tion in Perilla. The highest rosmarinic acid (23.48 mg.g DM) was acheved in 100 kg/ha chemical fertiliz- er plus humic acid and without application of biologic fertilizer (Table 6). Similarly, with plant yield, ros- marinic acid concentration was affected by fertilizer application. These results are in agreement with Hendawy et al. (2015) on Mentha piperita. They reported foliar application of humic acid increased growth character- istics and finally possessed the best oil percentage and yield in mint plant. Zaghloul et al. (2009) report- ed also the application of humic acid increased oil content of Thuja orientalis. At both of studied location and with/without application of biologic fertilizer, the maximum ros- marinic acid yield was achieved by using humic acid. The maximum rosmarinic acid yield (2.967 g/m2) was observed in Mashhad Ardehal and application of yield in all levels of chemical fertilizer. However, there was not clear trend about application of biolog- ic fertilizer. Previously, researchers (Ciarkowska et al., 2017) had highlighted the important role of humic acid, especially in root formation. Rosmarinic acid concentrations in Perilla were sig- nificantly affected by location, chemical and organic fertilization (Table 3) and, at both locations and all chemical fertilization levels, the humic acid applica- tion increased rosmarinic acid content in Perilla. This fact exhibited the importance of humic acid on ros- marinic acid content in Perilla. The maximum ros- m a r i n i c a c i d ( 2 5 . 0 1 m g . g D M ) w a s a c h i e v e d i n Mashhad Ardehal along with using 100 kg/ha chemi- cal fertilizer and application of humic acid (Fig. 2). Application of organic fertilizer especially humic acid with/without biologic fertilizer in all chemical fertilizer levels increased rosmarinic acid concentra- S.O.V. d.f. Means squares Plant yield Rosmarinic acid content Rosmarinic acid yield Stomatal Conductance DPPH Catalase activity Polyphenol oxidase Replication 2 61.95 0.46 0.01 5.24 24.06 0.0006 0.000040 Chemical fertilizer (C) 3 10082.10 ** 195.60 ** 13.42** 1256.35 ** 540.52 ** 0.0411 ** 0.00749 ** Ea 6 10.21 1.61 0.04 5.48 58.46 ** 0.0003 0.00013 ** Organic fertilizer(O) 2 1049.35 ** 27.60 ** 1.46** 42.86 ** 581.28 ** 0.0012 0.00071** Biologic fertilizer (B) 1 363.69 ** 12.52 ** 0.71** 87.60 ** 13.86 0.0002 0.00005 C × O 6 50.03 * 3.84 ** 0.07** 13.03 ** 11.17 0.0006 0.00037 ** C × B 3 42.09 0.63 0.03 2.73 1.39 0.0027 ** 0.00001 O × B 2 240.40 ** 1.60 0.18** 1.28 8.78 0.0006 0.00001 C × O × B 6 89.83 ** 1.67 0.08 ** 5.60 5.38 0.0009 * 0.000005 Eb 40 21.24 0.86 0.02 3.16 14.71 0.0004 0.00003 CV (%) - 3.56 4.79 5.65 2.63 2.32 4.48 3.64 S.O.V. d.f. Means squares Plant yield Rosmarinic acid content Rosmarinic acid yield Stomatal Conductance DPPH Catalase acti- vity Polyphenol oxidase Replication 2 51.56 0.96 0.01 2.80 13.07 0.00030 0.000003 Chemical fertilizer (C) 3 2005.86 ** 76.59 ** 3.03** 433.02 ** 326.18 ** 0.00677 ** 0.003819 ** Ea 6 46.75 0.76 0.04 5.99 30.56 0.00090 0.000009 Organic fertilizer(O) 2 457.49 ** 5.13 ** 0.39** 47.28 ** 521.70 ** 0.00307 * 0.000019 Biologic fertilizer (B) 1 33.13 0.31 0.03 128.16** 9.14 0.00005 0.000095 ** C × O 6 106.63 3.80 ** 0.01 5.98 36.55 0.00351 ** 0.000025 C × B 3 106.20 0.58 0.02 2.72 1.62 0.00156 0.000019 O × B 2 92.53 0.48 0.02 10.79 10.91 0.00030 0.000006 C × O × B 6 167.87 * 2.15 * 0.06 ** 0.21 19.72 0.00065 0.000006 Eb 40 53.64 0.74 0.02 8.51 23.59 0.00077 0.000012 CV (%) - 6.67 4.59 7.88 4.48 2.98 6.37 2.28 Table 4 - ANOVA of some morphologic, yield and essence traits of Perilla under different fertilizer systems in Mashhad Ardehal in Kashan regions *, ** significant at 5% and 1% probability levels, respectively. *, ** significant at 5% and 1% probability levels, respectively. Table 5 - ANOVA of some morphologic, yield and essence traits of Perilla under different fertilizer systems in Sensen in Kashan regions 210 Adv. Hort. Sci., 2019 33(2): 205-214 humic acid beyond biologic fertilizer (Fig. 3). Also, between chemical × organic × biologic fertilizer treat- ments, the maximum rosmarinic acid yield (3.432 g/m2) was observed in 100 kg/ha along with humic acid and biologic fertilizer application (Table 6). Integrated application of chemical, organic and bio- logical fertilizers improved soil physical and chemical properties and resulted in the increase in availability of nutrients and ultimately the yield and quality of plants. Darzi et al. (2007) reported the application of mycorrhiza and vermicompost and phosphate solubi- lizing biologic fertilizers determined increased essen- tial oil yield in fennel. Stomatal conductance Stomatal conductance is one of the most impor- t a n t f a c t o r i n d e t e r m i n i n g t h e p h o t o s y n t h e s i s amount. The study of stomatal conductance between location and chemical fertilizer treatments showed the highest stomatal conductance (75.88 mmol H2O m-2 s-1) in Mashhad Ardehal with application of 100 kg/ha chemical fertilizer (Fig. 4). At both of studied location especially in Mashhad Ardehal, application of chemical fertilizer increased the stomatal conduc- Table 6 - Means comparison of some morphologic, yield and essence traits of Perilla under different fertilizer systems in Sensen and Mashhad Ardehal in Kashan regions Chemical fertilizer Organic fertilizer Biologic fertilizer (Inoculation) Plant yield (g/m2) Rosmarinic acid content (mg/g D.M.) Rosmarinic acid yield (g/m2) 0 kg/ha Control (-) 93.12 lm 14.40 lm 1.34 l (+) 89.86 m 13.94 m 1.25 l Compost (-) 99.40 kl 15.31 l 1.52 k (+) 103.40 jk 17.29 jk 1.67 ij Humic acid (-) 99.91 kl 16.44 k 1.64 jk (+) 95.17 lm 17.31 jk 1.65 jk 50 kg/ha Control (-) 115.70 gh 19.52 def 2.25 gh (+) 128.40 de 19.38 defg 2.54 ef Compost (-) 129.50 de 19.56 def 2.52 ef (+) 126.90 de 20.11 ced 2.54 e Humic acid (-) 127.90 de 18.92 fgh 2.42 efg (+) 141.20 ab 20.37 cd 2.89 bc 100 kg/ha Control (-) 131.40 cd 21.05 bc 2.79 d (+) 126.70 de 21.58 b 2.75 d Compost (-) 138.00 bc 21.7 b 3.02 bc (+) 137.70 bc 21.63 b 3.00 bc Humic acid (-) 131.70 cd 23.48 a 3.11 b (+) 147.20 a 23.02 a 3.43 a 200 kg/ha Control (-) 107.20 ij 17.52 ij 1.88 i (+) 113.50 hi 18.4 ghi 2.09 h Compost (-) 126.20 def 18.2 hij 2.30 g (+) 119.40 fgh 19.13 egh 2.29 g Humic acid (-) 116.90 gh 19.33 efg 2.27 gh (+) 122.70 efg 19.07 fgh 2.35 fg Different letters in each column denote a significant differences at p≤0.05 (LSD test). Fig. 2 - Effect of chemical and organic fertilizer on rosmarinic acid content of Perilla in Mashhad Ardehal and Sensen regions. Different letters in each column denote a signi- ficant difference at p≤0.05 (LSD test). Fig. 3 - Effect of organic and biologic fertilizer on rosmarinic acid yield of Perilla in Mashhad Ardehal and Sensen regions. Different letters in each column denote a significant dif- ference at p≤0.05 (LSD test). Ghane et al. - Yield and physiological response of Perilla 211 tance of Perilla leaves. Increasing of stomatal con- ductance increases the photosynthesis activities and finally improved the plant yield (Atteya, 2003). The results of stomatal conductance are agreement with result of plant yield. This result showed the increas- ing of stomatal conductance can be useful to increas- ing of plant yield especially with 100 kg/ha applica- tion of chemical fertilizer. The highest stomatal conductance was obtained with application of compost fertilizer (67.67 mmol H2O m -2 s-1) (Table 7). Organic fertilizers can provide better moisture conditions by increasing the absorp- tion and preservation of water and the availability of water and food, and this can contribute to the favor- able conditions for photosynthesis in the plant. If there is enough stomatal conductance, CO2 gas will enter the stomata more easily and sufficient photo- synthesis will be performed. Also means comparison showed inoculation with mycorrhiza improved stom- atal conductance as compared to without inoculation (Table 7). The physiological effects of mycorrhizal fungi sym- biosis include aboveground modifications of water relations and physiological status in terms of leaf water potential, relative water content, stomatal conductance, CO2 assimilation, and efficiency of pho- tosystem II as compared to non-mycorrhizal plants (Barzana et al., 2012). Mycorrhizal fungi increase the contact surface with soil and moisture around the plant roots by 10 to 1,000 times, thus increasing the plant’s ability to use the resources in its surroundings (Sharma, 2002). DPPH test Many researchers have used DPPH test to express the antioxidant status of plants (Dasgupta and De, 2007; Sahu et al., 2013). The results showed the antioxidant activity of Perilla plant in Mashhad Ardehal (165.29 ug/ml) was higher than Sensen (162.79 ug/ml) location (Table 7). Perilla is very sensi- tive to free radicals productions. In a study in Korea on different species of Perilla, different species for DPPH test showed a significant difference (Choi et al., 2002). Increasing levels of chemical fertilizer reduced the plant’s efficiency in inhibiting free radicals. The high- est amount of DPPH test was obtained (169.47 μg/ml) under control condition and the lowest (55.85 μg/ml) was obtained from the highest level of fertiliz- er application (200 kg/ha) (Table 7). Omar et al., (2012) reported the antioxidant activity determined using the DPPH was high with the application of organic fertilizer compared to chemical fertilizer in cassava tubers. Organic fertilizers, especially compost (277.17 μg/ml), increased the antioxidant activity and inhibit- ed free radicals (Table 7). Uthairatanakij et al. (2017) reported an increase in antioxidant activity and inhi- bition of free radicals due to the application of organ- ic fertilizers. Organic fertilizers with plant sources activated defense mechanisms against pests, dis- eases and other tensions (Brandt and Molgaard, 2001). Catalase activity T h e h i g h e s t c a t a l a s e a c t i v i t y ( 0 . 5 3 3 2 n m o l H2O2/min) was observed in Mashhad Ardehal region with the application of 100 kg/ha fertilizer and with- Fig. 4 - Effect of chemical fertilizer on stomatal conductance of P e r i l l a i n M a s h h a d A r d e h a l a n d S e n s e n r e g i o n s . Different letters in each column denote a significant dif- ference at p≤0.05 (LSD test). Treatments Stomatal Conductance (mmol H 2 O m−2s−1) DPPH (ug/ml) Polyphenol oxidase (μmol/min) Location Mashhad Ardehal 67.61 a 165.29 a 0.156 a Sensen 64.98 b 162.79 b 0.152 b Chemical fertilizer 0 kg/ha 57. 36 d 169.48 a 0.130 c 50 kg/ha 65.99 c 161.73 c 0.152 b 100 kg/ha 72.05 a 166.41 b 0.167 a 200 kg/ha 69.80 b 158.55 d 0.168 a Organic fertilizer Control 66.03 b 158.54 b 0.152 b Compost 67.68 a 167.27 a 0.158 a Humic acid 65.19 b 166.31 a 0.153 b Biologic fertilizer (Inoculation) (-) 65.08 a 164.08 a 0.153 b (+) 67.52 b 164.00 a 0.155 a Table 1 - Weather characteristics in Mashhad Ardehal and Sensen Different letters in each column denote a significant differences at p≤0.05 (LSD test). Adv. Hort. Sci., 2019 33(2): 205-214 212 out application of biologic fertilizer. The lowest amount was obtained from Mashhad Ardeal area without using of chemical and biologic fertilizer. Also, in the Sansen location, the highest catalase activity was obtained from 200 kg/ha chemical fertilizer and biologic fertilizer application (Fig. 5). Among the chemical and organic fertilizer treat- ments in two studied locations, the highest catalase activity was obtained from 100 kg/ha of chemical fer- tilizer with compost application in Mashhad Ardehal region (0.5182 nmol H2O2/ min) and the lowest amount (0.338 nmol H2O2/ min) was obtained with- out using of chemical and organic fertilizer control in Mashhad Ardehal location (Fig. 6). Logan et al. (1999) stated that nitrogen had a significant effect on the activity of enzymes involved in photosynthesis, such as Ribulose-1,5-bisphosphate. In another study the researchers reported increased nitrogen levels caused to production of antioxidant enzymes like APX, SOD, CAT, and POD in the Populus yunnanensis plants (Lin et al., 2012). Our result indicated that sources of fertilizer had a significant influence on the level of catalase activity in field grown Perilla. Polyphenol oxidase activity Among the treatments of chemical and organic fertilizer in different locations, similar to catalase activity, the highest polyphenol oxidase activity was obtained from 100 kg/ha with compost application in Mashhad Ardeal location (0.193 μmol/min) and the lowest amount (0.129 μmol/min) was obtained from without application of chemical and organic fertilizer in Mashhad Ardehal area (Fig. 7). At all levels of chemical fertilizer in both locations, application of organic fertilizers increased the activity of polyphenol oxidase activity, and in most cases, organic matter compost was superior to humic acid. Also, the appli- cation of biologic fertilizer led to an increase in polyphenol oxidase activity (Table 7). Similar to our results, the application of biologic fertilizer increased the amount of polyphenol oxidase activity in triticale (Kheirizadeh-Arough et al., 2016). 4. Conclusions In the present experiment, the results showed that proper nutritional management of Perilla medic- inal plant has a special role in improving quantitative and qualitative traits. Among individual fertilizer treatments, 50 and 100 kg/ha of fertilizer and among organic fertilizers, humic acid played a more effective role in improving the studied indices, but the applica- tion of biologic fertilizer separately did not have a very significant effect on this plant. Integration with other fertilizers has been shown to be more effec- tive. Mashhad Ardehal area has better conditions for cultivating Perilla due to its soil characteristics and climatic characteristics. Also, the combined applica- tion of different fertilizer sources in comparison with the single application of each of them in both studied regions significantly improved the growth character- istics, as well as essential oil yield and biochemical Fig. 5 - Effect of chemical and biologic fertilizer on catalase acti- vity of Perilla in Mashhad Ardehal and Sensen regions. Different letters in each column denote a significant dif- ference at p≤0.05 (LSD test). Fig. 6 - Effect of chemical and organic fertilizer on catalase acti- vity of Perilla in Mashhad Ardehal and Sensen regions. Different letters in each column denote a significant dif- ference at p≤0.05 (LSD test). Fig. 7 - Effect of chemical and organic fertilizer on polyphenol oxidase activity of Perilla in Mashhad Ardehal and Sensen regions. Different letters in each column denote a significant difference at p≤0.05 (LSD test). Ghane et al. - Yield and physiological response of Perilla 213 indices of Perilla. 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