Effect of regulated deficit irrigation and nitrogen levels

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Scholars Research Library Annals of Biological Research, 2012, 3 (6):2624-2630 (http://scholarsresearchlibrary.com/archive.html) ISSN 0976-1233 CODEN (USA): ABRNBW

Effect of regulated deficit irrigation and nitrogen levels on flavonoid content and extract performance of marigold (Calendula officinalis L.) Navid Rahmani, Tofigh Taherkhani, Peiman Zandi, and Amin Moradi Aghdam Department of Agronomy, Takestan Branch, Islamic Azad University, Takestan, Iran ______________________________________________________________________________ ABSTRACT Pot marigold (Calendula officinalis L.) is an annual medicinal plant which has been cultivated for herbal raw material (flower heads); its material has been benefiting the pharmaceutical and cosmetic industry since the ancient times. A field study was carried out in the years 2006-2007 in the Experimental Farm of the Islamic Azad University of Takestan in Iran. The study was conducted on a fine, mixed, thermic, and Typic Haplocambids soil with the granulometric composition of sandy-loam. The aim of the experiment was to determine the effect of different nitrogen rates (N0 = 0, N1 = 30, N2 = 60, N3 = 90 kg ha-1) and irrigation regimes (I1– irrigation after 40 mm, I2– irrigation after 80 mm, and I3– irrigation after 120 mm evaporation from class A pan) on some agronomic features of flower heads as well as on yield and quality of marigold raw material. Water deficit stress caused reduction in petal yield, extract yield, petal/flower weight ratio, and flower quality while it did not affect flavonoid content significantly. Extract content increased with increasing irrigation intervals based on 80 mm evaporation from class A pan. Extract yield of pot marigold was the lowest (112.8 kg ha-1) in the control treatment (without nitrogen) and the highest (133.7 kg ha-1) in the plots where nitrogen fertilization applied at the maximum rate (90 kg ha-1). It was understood that the application of 30 kg N ha-1to increased extract content drastically as compared to N2 and N3 treatments. Petal/flower weight ratio and flower quality did not differ markedly for fertilization rates from 0-90 kg N ha-1. The highest flavonoid content (1.59%) was achieved from plots which utilized a combination of I2×N1 treatment. However, the maximum petal yield was related to application of 90 kg N ha-1 and a normal irrigation (I1). Keywords: pot marigold, water deficit stress, nitrogen, yield, extract, flavonoid.

______________________________________________________________________________ INTRODUCTION Drought stress, caused by soil and atmospheric water deficiency, is one of the most significant environmental factors affecting plant growth and crop productivity in the majority of agricultural fields of the world [1]. Some studies have shown that stress caused by lack of water led to numerous morphological, physiological and biochemical plant changes [2, 3, 4]. Decreasing the growth trend of roots and shoots, leaf area, photosynthesis, transpiration, plant height, dry weight as well as stomatal closure, enzymatic interruption, destroys of proteins, structural changing in synthesized proteins/amino acids and also decreased chlorophyll accumulation are some the drought-induced losses reported by Jiang and Huang [5, 6] and Lei et al.[4]. Nitrogen is one of the elements which its deficiency is noticeable in arid/semi-arid regions; because the amounts of organic matter which are the main source of nitrogen storage are very low in this regions and in case of its existence, it decomposes immediately [7]. Nitrogen’s less or more availability to the plant causes some disorders in crop vital processes that may appear in the forms of abnormal growth and development, decrease in transpiration and even stunt in reproductive growth [8].

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Navid Rahmani et al Annals of Biological Research, 2012, 3 (6):2624-2630 _____________________________________________________________________________ Recognized as marigold, Calendula officinalis generally belongs to the family of Asteraceae (Compositae). Marigold is an aromatic, erect, annual herb with 60 cm height and angular and glandular stems; leaves 2.5-7.5 cm long; lower spathulate, entire, upper lanceolate with cordateamplexicaul base; flower-heads terminal, heterogamous, light yellow to deep orange; ray florets fertile; achenes 1.0-1.5 cm long, boat-shaped, faintly ribbed; indigenous to central, eastern and southern Europe, cultivated regularly for medicinal and ornamental purposes in North America, Balkans, Eastern Europe, Germany and India [9,10]. The plant has six annual herbaceous species in Iran including C. aurantica, C. alata, C. palaestina, C. persica, C. sancta, and C. tripterocarpa species [11].The flowers usually appears 40-50 DAP (days after planting) from early June to the cold season (70-120 days) in the optimum temperature of 30-40 °C [12]. Calendula officinalis has a long history of usage by the folk systems because of its rich medicinal values that have been reported to possess potent anti-inflammatory, antitumour, antioxidant, antibacterial, anti-HIV, anti-ulcer, antigenotoxic, chemoprotective and antiseptic properties [13]. The flowers of this plant are used to treat inflammatory conditions of internal organs, gastrointestinal ulcers, diuretic and diaphoretic convulsions [14]. Calendula extracts are also used in diverse preparations, mainly ointments for the treatment of some dermatological conditions, such as ulcers, eczema, burns and hemoroides [15]. Pharmacological studies of conventional marigold extracts (infusions, tincture, fluid extract) show that its most important constituents are saponines, glycosides of sesquiterpenes, flavonoids and triterpenes [16]. In spite of extensive undertaken studies on the impact of environmental stresses on growth and yield of medicinal plants, limited information is available on response to these stresses. No specific information on marigold irrigation is at hand and it is cultivated traditionally in Iran [17]. Based on a trial conducted by Yanive and Palevitch [18], it was revealed that the essential oil content in medicinal plants largely depends on genetic and environmental conditions. Shubhra et al. [19] found that seed yield, oil yield, plant height and number of flowers per plant in drought stress conditions severely reduced, while the oil percentage is increased under these conditions. In a research on Chamomile (Matricaria Chamomilla L.) with applied density of 33 plants m-2, the maximum essential yield (75%) gained from the plants irrigated with 100% of field capacity, but it was not significantly different from irrigation at 55% of field capacity [20]. Jangir and Sink [21] investigated the effect of 4, 5 and 6 times of irrigation on the yield of Cumin (Cuminum cyminum). Their results showed that irrigation regimes had a considerable effect on seed yield and its components. In addition, they found that five-time irrigations increased yield comparing with four-time irrigations, although more irrigation (six-time irrigations) did not increase the yield. Numerous studies [22, 23, 24] have shown that nitrogen fertilization results in a significant increase in quantity and quality of herbal plant yields. According to Rumińska et al. [25], the marigold does not require intensive mineral fertilization and high doses of nitrogen result in the decrease of yield of flower heads. Biesiada et al. [26] did not observe a clear correlation between nitrogen fertilization and the chemical composition of marigold flower heads. However, they noted a slight decrease in polyphenol content at higher nitrogen rates. Arganosa et al. [27], in their study on marigold with five levels of pure nitrogen (0, 20, 40, 60.80, and 100 kg ha-1), found that maximum biological yield, seed yield, oil yield and seed weight obtained from 80 kg N ha-1 and the highest oil percentage gained by using 40 kg N ha-1. Another study showed that the compounds percentage of garden thyme (Thymus vulgaris) was not significantly affected by applying nitrogen fertilizer in the range of 0-135 kg ha-1[28]. In the EU project entitled “Calendula as Agronomic Raw Material for Industrial Applications”, it was reported that Optimum Marigold N requirement for biological and economic yield was 100 kg N ha-1[29]. Clark and Menary [30], in a study on the peppermint (Mentha piperita L.), stated that irrigation and nitrogen fertilizer influenced essential oil yield, and the highest oil yield was achieved in the case of applying 300 kg N ha-1 coupled with 50 mm of irrigation weekly. This work was aimed to evaluate the effects of different irrigation frequencies and nitrogen fertilizer levels individually and in combination on flavonoid content and extract performance of marigold in the experimental field of Qazvin, Iran in 2006-2007. MATERIALS AND METHODS Site description and soil type. This study was taken up at the experimental farm of Islamic Azad University, Takestan, Iran (latitude 36° 04´N, longitude 49° 39´E, elevation 1283.4 m above mean sea level) during the periods of 2006–2007. This region has a semi-arid climate (252.6 mm annual rainfall). The soil analysis result of this region is demonstrated in Table 1. Table 1. The results of soil experiment in 2006. Soil depth (cm) 0-15

Sand (%) 60

Silt (%) 19

Clay (%) 21

K (mg ka-1) 287

P (mg ka-1) 6.6

N (%) 0.02

Organic carbon (%) 0.29

EC (dS m-1) 0.521

pH 7.7

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Navid Rahmani et al Annals of Biological Research, 2012, 3 (6):2624-2630 _____________________________________________________________________________ Agronomic practices. Individual experimental plot was consisted of 6 rows, 6 m long, 3.6 m width, 12 sowing lines, and 60 cm row spacing (Ridge’s interval). After preparing the land (plowing, disking, ridging), furrows and ridges were constructed by Furrower. The cluster sowing method was applied on 27 April 2006 (by placing 3 to 4 seeds into a hole of 2-3 cm depth). The planting date Selection was in association with the climate condition of the region (severe hail in early spring). For preventing any possibility of irrigation water transmission to the adjacent plots (treatments), 4 m space within the blocks was considered. After seedling emergence, for achieving desirable density (28 plants m-2), hand tinning was done. Hand weeding was performed at 4-5 leaves phase. No fungicides or insecticides were applied to experiment. Before applying the stress treatment, concerning the soil texture and meteorological situation, a particular case of furrow irrigation called Gholam-gardeshy irrigation in every 5 days was applied. The subsequent irrigations in each plot was performed based on experimental plan (irrigation treatments including I1, I2, and I3). The daily evaporation rate was recorded using a class A pan situated in meteorological station in the university. In general, weather condition during all trial period (during full season) was favorable. Proper management practices were adopted throughout the growing seasons to ensure good crop growth. Experimental design and treatments. The experiment was carried out as split plot based on a randomized complete block design (RCBD) in four replications in the research field of Takestan (I.A.U), Iran. Three irrigation levels consisting of I1–irrigation after 40 mm, I2–irrigation after 80 mm, and I3–irrigation after 120 mm evaporation from class A pan were applied in main plots. Subplots consisted of split application of four Nitrogen fertilizers from source of Urea (N0–without nitrogen application, N1–30, N2–60, and N3–90 kg N ha-1). The plots were irrigated based on irrigation treatments since the time of seedlings establishment (30 to 40 days after planting [DAP]) until the full maturity. Nitrogen fertilizer was used in two splits. The first application (1/2 of the total rate) was incorporated and added to soil as the starter at the time of seedlings emergence, and the second application was split equally at the end of stem elongation stage (prior to flowering) within the planting lines in a strip manner. Estimation of traits. At full blooming (100% flowering stage), ten random samples were hand harvested from each experimental unit (0.5 sq m of the central part of each plot) and the following parameters were determined: petal yield, extract content, extract yield, flavonoid content (based on rutin), petal/flower weight ratio, and flower quality. Flower heads were harvested gradually as the plants fully bloomed, at 4-day intervals (from July15th to August 20th). Immediately after the harvest, flower heads were dried in a well-ventilated drying room (under standard conditions, away from sunlight and moisture) at 35°C. The dried flowers were weighted using a precise scale (0.001 g). At the stage of highest dry flower's weight, after separating the petals of each flower, the weight of dry petals were calculated and expressed as kg ha-1 (petal yield). The petal/flower weight ratio was measured dividing petals weight to the flowers weight multiplied by 100.

Figure 1. Rutin standard curve.

Preparation of marigold extract and determination of Flavonoid percent. The dried flowers of Calendula officinalis L. (50 g per plot) were ground in a knife mill into fine particles (0.3 mm - mean diameter). The obtained powder (5 g of diced petal) was macerated twice with 150 ml of 50% methanolic solution (1:9, w/w) at 60 °C for 1 h. This mixture was submitted to mechanical stirring at 870 rpm (Fisatom, model 713 D) for 1 h at the beginning and end of the maceration period. Afterwards, the extract was filtered and dried at 40 °C in a stove with air circulation. Finally, the residue was dissolved into 50% hydroalcoholic solution (200 ml) and stored at -20 °C. Because of high concentration level in specimen and probability of overdose methanol, only 10 ml 50% methanolic solution was added to specimen as the final solution had 20 ml volume. Consequently, by applying this method, we were able to dilute it twice. The validation procedure was followed the methods described by Fonseca et al. [13]. Five ml of this sample was mixed with five ml of 5% aluminum chloride solution. After half an hour, the absorption was measured

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Navid Rahmani et al Annals of Biological Research, 2012, 3 (6):2624-2630 _____________________________________________________________________________ by blank without using the indicator. Based on rutin standard curve (combined with a 5% AlCl3 solution), flavonoid levels (rutin based) was determined (Fig.1). The flavonoid levels in marigold extract were determined using a Shimadzu (Kyoto, Japan) liquid chromatography system equipped with an LC-10 AT VP solvent pump unit and an SPD-10A VP UV-Visible detector operating at 430 nm. The test started with 430 nm wavelength and continued by the maximum range of 720 nm. The extract yield was estimated through multiplying weight of extract in petal yield. Flowers quality is an outstanding character for medicinal plants producers and indicating the percentage of flavonoid in the petals/flower weight ratio. Statistical analysis. Data were subjected to analysis of variance (ANOVA), the SAS software package [31]. A probability value of P < 0.05 was considered to denote a statistically significance difference to compare the means of treatments through least significant difference test (LSD). RESULTS The results in Table 2 revealed that various irrigation levels had a highly significant effect (P