Impaginato 19 1. Introduction Milk thistle (Silybum marianum L.) is one of the most important medicinal plants in the pharmaceuti- cal industry worldwide, and is used in the production of flavonoids of the silymarin group (silybin, silidianin and silychristine) which are important in the modern pharmaceutical industry (Ghavami and Ramin, 2007). The origin of this plant has been reported to be the East Mediterranean region (Keville, 1991). Water availability may influence physiological and biochemi- cal properties and seed yield of this medicinal plant. Water stress severely limits growth and yield of plants by reducing ground green cover (Ghassemi- Golezani and Ghasssemi, 2013), chlorophyll content of leaves, photochemical efficiency of photosystem II (Ghassemi-Golezani and Lotfi, 2012) and photosyn- thesis (Munns et al., 2006). Water stress during vege- tative stages largely reduces plant height and bio- mass, while during reproductive stages it has the greatest negative impact on seed yield (Ghassemi- Golezani et al., 2008). Reports in oil crops indicated that water stress decreases oil and increases protein percentages of seeds. However, both oil and protein yields per unit area are decreased as a result of large reduction in seed yield per unit area due to water limitation (Ghassemi-Golezani and Lotfi, 2013; Ghassemi-Golezani et al., 2015 b). Some of the dele- terious effects of environmental stresses on plant performance could be alleviated by foliar application of growth regulators such as salicylic acid (SA) (Ghassemi-Golezani et al., 2015 a). It has been reported that the exogenous applica- tion of SA induces plant tolerance to several abiotic stresses including drought tolerance in wheat (Singh and Usha, 2003), salinity tolerance in safflower (Ghassemi-Golezani and Hosseinzadeh-Mahootchi, 2015) and mung bean (Ghassemi-Golezani et al., 2015 a), heat tolerance in mustard (Dat et al., 1998) and chilling tolerance in maize (Janda et al., 1999). These studies suggest that SA may enhance the mul- tiple types of stress tolerance in plants by interactive effects on several functional molecules. Hayat et al. (2008) found that there was a significant increase in photosynthetic parameters, chlorophyll and proline contents, and antioxidant enzyme activities in SA treated tomato plants. Loutfy et al. (2012) reported that SA induced drought tolerance and increased plant biomass, leaf relative water content, and the solute contents in four wheat cultivars. Moreover, Adv. Hort. Sci., 2017 31(1): 19-23 DOI: 10.13128/ahs-20721 Improving oil and flavonoid contents of milk thistle under water stress by salicylic acid K. Ghassemi-Golezani*, S. Ghassemi, I. Yaghoubian Department of Plant Eco-physiology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran. Key words: foliar application, plant biomass, seed yield, Silybum marianum L., water deficit. Abstract: Adverse environmental conditions such as water deficit can limit production. However, some of these adverse effects may be overcome by application of plant growth regulators including salicylic acid (SA). Thus, a field experiment was conducted in 2015 to evaluate the effects of SA (0 and 1 mM l-1) on yield components, seed yield and oil and flavonoid contents of milk thistle (Silybum marianum L.) under different irrigation treatments (I1, I2, I3 and I4: irrigation after 70, 110, 150 and 190 mm evaporation from class A pan, respectively). The experiment was arranged as split-plot based on randomized complete block (RCB) design in three replicates. Irrigation treatments and SA levels were located in the main and sub plots, respectively. The results indicated that plant biomass, seeds per plant, 1000 seed weight, seed yield per unit area and harvest index of milk thistle decreased as a consequence of water stress. Oil percentage and yield were also reduced, but flavonoid content enhanced with increasing water deficit. All these traits were considerably aug- mented by foliar application of SA under non-stress and stressful conditions. Therefore, it was conclude that SA can be used to improve field performance of milk thistle under different environmental conditions. (*) Corresponding author: golezani@gmail.com Received for publication 11 October 2016 Accepted for publication 20 December 2016 Copyright: © 2017 Author(s). This is an open access article 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. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2017 31(1): 19-23 20 foliar spray of SA decreased the inhibitory effects of drought on Phillyrea angustifolia (Munné-Bosch and Penuelas, 2003). Abd el-Lateef Gharib (2006) stated that oil content of Basil and Marjoram significantly increased with application of SA. However, the responses of some medicinal plants to SA treatment in stressful conditions were not documented so far. Thus, this research was undertaken to evaluate the effects of foliar application of SA on seed yield and oil and flavonoid contents of milk thistle under different irrigation intervals. 2. Materials and Methods Seeds of milk thistle (Silybum marianum L.) were obtained from Pakan bazr, Isfahan, Iran. The experi- ment was conducted in 2015 at the Research Farm of the Faculty of Agriculture, University of Tabriz, Iran (Latitude 38° 05′ N, Longitude 46° 17′ E, Altitude 1360 m above sea level). The climate is characterized by mean annual precipitation of 245.75 mm per year, mean annual maximum temperature of 16.6°C and mean annual minimum temperature of 4.2°C. The field experiment was arranged as split-plot based on randomized complete block design in three replica- tions, with irrigation intervals (I1, I2, I3, I4: irrigation after 70, 110, 150 and 190 mm evaporation from class A pan, respectively) in main plots and two levels of salicylic acid (SA; 0 and 1 mM l-1) in sub-plots. Seeds of milk thistle were treated with 3.3 g/kg Benomyl and then were sown by hand on 28 May 2015 in 3 cm depth of a sandy loam soil. Each plot consisted of 6 rows of 3 m length, spaced 25 cm apart. All plots were regularly irrigated up to seedling establishment, but thereafter irrigations were carried out according to treatments. Weeds were frequently controlled by hand during crop growth and develop- ment. Salicylic acid (SA; 0 and 1 mM) was sprayed at vegetative and flowering stages. Plant biomass and seed yield At maturity, plants in 1 m2 (8 plants) of the middle part of each plot were harvested and seeds per plant, 1000 seed weight and seed yield per unit area were determined. Then above ground biomass was oven- dried at 80°C for 48 hours and weighed and subse- quently harvest index was calculated. Oil extraction Oil was extracted from 3 g mature seeds of each plot in petroleum ether for 5 hours using a Soxhlet system according to the AOCS method (AOCS, 1993). Oil content was determined as a percentage for each sample and then oil yield per unit area was calculat- ed as: Oil yield = Seed yield × Oil percentage Flavonoid extraction Powdered air-dried mature seeds (1 g) were extracted in a Soxhelt extractor with 100 ml ethanol for an hour and the extract filtered. Three ml of the extract was placed in a 15 ml volumetric flask. Then 0.3 ml NaNO2 (1:20) and after 5 minutes 3 ml AlCl3 (1:10) and 6 minutes later 2 ml of 1 mol litre-1 NaOH were added and the total was made up to 10 ml with distilled water. The solution was mixed well again and the absorbance was measured against a blank at 510 nm with a HALO DB-20 spectrophotometer (Zhuang et al., 1992). The flavonoid content was cal- culated using the following linear equation: A = 0.01069C - 0.001163 Where A is the absorbance and C is the flavonoid content in µg/g. Analysis of variance Analysis of variance of the data appropriate to the experimental design and comparison of means at p≤0.05 were carried out, using GenStat 12 and MSTATC softwares. Excel software was used to draw figures. 3. Results Analysis of variance (Table 1) showed that plant biomass, seeds per plant, 1000 seeds weight, seed yield per unit area and harvest index were signifi- cantly affected by water limitation and SA, but the interaction of irrigation × SA was only significant for 1000 seed weight and harvest index. Source of vari- ation df Mean square Plant biomass Seeds per plant 1000 seeds weight Seed yield Harvest Index Replication 2 1198 2162 0.1163 61.7 0.0129 Irrigation (I) 3 38104 ** 56241 ** 4.6906 * 3121.7 ** 4.1715 ** Error 6 2795 4424 0.2785 140 0.1257 SA 1 837851 ** 1718420 ** 15.0417 ** 71195.6 ** 48.4504 ** I × SA 3 2633 NS 7585 NS 0.9128 ** 262 NS 3.3993 ** Error 8 3987 7553 0.1212 259.2 0.1058 CV (%) - 8.8 9.2 1.5 9.2 1.4 Table 1 - Analysis of variance of the data for plant biomass, yield components and seed yield of milk thistle affected by irrigation treatments and salicylic acid (SA) treatments NS, * and ** No significant and significant at p≤0.05 and p≤0.01, respectively. Ghassemi-Golezani et al. - Oil and flavonoid of milk thistle affected by watering and salicylic acid 21 Plant biomass, seeds per plant and seed yield per unit area decreased with decreasing water availabili- ty, but all these traits were considerably enhanced by foliar application of SA. Reduction in seeds per plant was only significant under severe water stress (I4), with no significant difference among I1, I2 and I3 treat- ments. Differences in plant biomass and seed yield between I1 and I2 and also between I2 and I3 were not statistically significant. Application of SA improved plant biomass, seeds per plant and seed yield by about 71%, 79% and 91%, respectively (Table 2). One thousand seeds weight (Fig. 1A) and harvest index (Fig. 1B) of untreated plants with SA gradually decreased as water stress increased. But, these reductions were only significant under severe water deficit. In contrast, SA treated plants did not show significant reduction in seed weight and harvest index due to water limitation. Oil percentage, oil yield and flavonoid content were significantly affected by irrigation and SA treat- ments, but the interaction of irrigation × SA was not significant for these traits (Table 3). Oil percentage and yield of milk thistle decreased, but flavonoid con- tent increased as a result of water stress. Seed oil percentage and yield and flavonoid content were sig- nificantly enhanced by foliar spray of SA. This superi- ority was more pronounced for oil yield per unit area (Table 4). 4. Discussion and Conclusions Reduction in plant biomass due to water stress (Table 2) was associated with diminishing leaf area expansion and plant growth during vegetative stages (Ghassemi-Golezani et al., 2009) and also with early leaf senescence (Hugh and Richard, 2003). Drought Fig. 1 - Mean seed weight (A) and harvest index (B) of milk this- tle affected by irrigation and SA treatments. I1, I2, I3, I4= Irrigation after 70, 110, 150 and 190 mm evaporation, respectively. SA0, SA1= 0 and 1 mM salicylic acid, respectively. Different letters in each column indicate significant difference at p≤0.05. Treatments Plant biomas (g/m2) Seeds per plant Seed yield (g/m2) Irrigation I1 797.2 a 1034.8 a 197.0 a I2 746.2 ab 984.0 a 185.2 ab I3 705.0 b 939.7 a 172.5 b I4 609.0 c 808.8 b 143.9 c Salicylic acid Irrigation 528 b 674 b 120.2 b SA1 901 a 1209 a 229.1 a Table 2 - Means of plant biomass, seeds per plant and seed yield of milk thistle for irrigation and salicylic acid (SA) treatments Different letters in each column indicate significant difference at p≤ 0.05. Source of Variation df Oil content Oil yield Flavonoid content Replication 2 2.2604 23.12 1.0208 Irrigation (I) 3 24.2749 ** 367.88 ** 20.0397 ** Error 6 0.6199 5.49 15046 Salicylic acid (SA) 1 5.9004 ** 3294.13 ** 26.8182 ** I × SA 3 0.0849 NS 0.59 NS 0.2903 NS Error 8 0.1625 10.8 0.3488 CV (%) - 2 9.2 1.2 Table 3 - Analysis of variance of oil percentage and yield and flavonoid content of milk thistle affected by irrigation and SA treatments NS, * and ** No significant and significant at p≤0.05 and p≤0.01, respectively. Adv. Hort. Sci., 2017 31(1): 19-23 22 stress decreases water potential of plant, leading to stomata closure and reduction in photosynthesis rate and leaf growth (Ozturk, 1999), which ultimately decreases plant biomass. This reduction in plant bio- mass resulted in decreasing the number of seeds per plant (Table 2), 1000 seeds weight (Fig. 1A) and con- sequently seed yield (Table 2) and harvest index (Fig. 1B). The losses in plant biomass and seed yield due to water deficit have also been reported for sesame (Kim et al., 2007), dill (Ghassemi-Golezani et al., 2008), maize (Ghassemi-Golezani and Dalil, 2011) and safflower (Ghassemi-Golezani et al., 2016). Application of SA largely improved seed yield of milk thistle by enhancing plant biomass, seeds per plant (Table 2), 1000 seeds weight and harvest index (Fig. 1). SA influences a wide variety of plant process- es, including stomatal regulation, chlorophyll content and photosynthesis (Yildirim et al., 2008). Ghassemi- Golezani and Lotfi (2015) found that exogenous application of SA enhances maximum quantum effi- ciency of PSII (Fv/Fm) and performance index (PI) in mung bean plants. In another report, Ghassemi- Golezani and Hosseinzadeh-Mahootchi (2015) stated that chlorophyll content index (CCI), photosystem II efficiency (Fv/Fm), relative water content (RWC), leaf area index (LAI) and finally seed yield of safflower were augmented by foliar application of SA. The low oil percentage due to water deficit (Table 4) may be resulted from the short seed filling dura- tion (Ghassemi-Golezani and Lotfi, 2013) and low seed weight (Fig. 1A). Adequate irrigation during plant growth and development can likely increase seed weight and oil storage. Decreasing oil yield per unit area as a consequence of water limitation (Table 4) strongly related with reduction in seed yield under stressful condition (Table 2). It was similarly reported that water limitation significantly decreases seed and oil yields of sunflower (Soleimanzadeh et al., 2010) a n d m a i z e ( G h a s s e m i - G o l e z a n i e t a l . , 2 0 1 5 b ) . Increasing seed yield (Table 2) and oil percentage by application of SA resulted in considerably higher oil yield of milk thistle (Table 4). Similar pattern of oil yield improvement by foliar spray of SA was observed in Ocimum basilicum and Origanium hortensis plants (Abd el-Lateef Gharib, 2006). With decreasing photosynthesis rate under water stress, carbons from the photosynthesis cycle shift to the shikimic acid pathway in order to produce higher flavonoid content (Table 4). It was found that pheno- lics and flavonoids are able to regulate plant growth and improve the physiological efficiency and can enhance effective partitioning of accumulates from the sources to the sinks in plants (Ghasemzadeh et al., 2010). Stimulation of flavonoid accumulation by SA treatment (Table 4) may protect plants from cer- tain biotic and abiotic stresses (Dučaiová et al., 2013). An increase in secondary metabolites content w a s a l s o d e t e c t e d i n c h a m o m i l e ( M a t r i c a r i a c h a m o m i l l a L . ) p l a n t s a s a r e s u l t o f S A s p r a y (Dučaiová et al., 2013). This result suggests that flavonoid accumulation is a biochemical response to water stress, and SA can induce flavonoid synthesis, providing an effective protection of milk thistle plants from stress. Water stress reduced plant biomass, seeds per plant, 1000 seeds weight, seed yield, harvest index, oil percentage and consequently oil yield of milk this- tle. However, flavonoid content of seeds increased with decreasing water availability. All these traits were considerably enhanced by foliar spray of SA under different irrigation intervals. This suggests that exogenous application of SA could be an effective way for improving field production of milk thistle under different environmental conditions. Acknowledgements We appreciate the financial support of this work by the University of Tabriz. References ABD EL-LATEEF GHARIB F., 2006 - Effect of salicylic acid on the growth, metabolic activities and oil content of basil and marjoram. - Int. J. Agric. Biol., 8: 485-492. AOCS, 1993 - Official methods and recommended practices of the American Oil Chemists’ Society. 4th edn. (Methods Ag 1-65 and Ce 1-62). - American Oil Chemists’ Society Press, Champaign, IL, USA. DAT J.F., LOPEZ-DELGADO H., FOYER C.H., SCOTT I.M., Table 4 - Means of oil percentage and yield and flavonoid con- tent of milk thistle for irrigation and SA treatments Different letters in each column indicate significant difference at p≤0.05. Treatments Oil content (%) Oil yield (g/m2) Flavonoid content (µg/g) Irrigation I1 22.13 a 43.60 a 48.05 c I2 21.33 a 39.50 b 49.12 bc I3 19.35 b 33.38 c 50.04 b I4 17.67 c 25.43 d 52.34 a Salicylic acid SA0 19.63 b 23.60 b 48.83 b SA1 20.62 a 47.24 a 50.95 a Ghassemi-Golezani et al. - Oil and flavonoid of milk thistle affected by watering and salicylic acid 23 1998 - Parallel changes in H2O2 and catalase during thermo-tolerance induced by salicylic acid or heat acclimation in mustard seedlings. - Amer. Soc. Plant Biol., 116: 1351-1357. DUČAIOVÁ Z., PETRUĽOVÁ V., REPČÁK M., 2013 - Salicylic acid regulates secondary metabolites content in leaves of Matricaria chamomilla. - Biologia, 68: 904- 909. GHASEMZADEH A., JAAFAR H.Z.E., RAHMAT A., 2010 - Synthesis of Phenolics and Flavonoids in Ginger (Zingiber officinale Roscoe) and their effects on photo- synthesis rate. - Int. J. Mol. Sci., 11: 4539-4555. GHASSEMI-GOLEZANI K., ANDALIBI B., ZEHTAB-SALMASI S., SABA J., 2008 - Effects of water stress during vegeta- tive and reproductive stages on seed yield and essen- tial oil content of dill (Anethum graveolens L.). - J. Food, Agric. Environ., 6: 282-284. GHASSEMI-GOLEZANI K., DALIL B., 2011 - Seed ageing and field performance of maize under water stress. - Afr. J. Biotechnol., 10: 18377-18380. GHASSEMI-GOLEZANI K., GHANEHPOOR S., DABBAGH MOHAMMADI-NASAB A., 2009 - Effects of water limi- tation on growth and grain filling of faba bean culti- vars. - J. Food, Agric. Environ., 7: 442-447. GHASSEMI-GOLEZANI K., GHASSEMI S., 2013 - Effects of water stress on some physiological traits and grain yield of chickpea (Cicer arietinum L.) cultivars. - Int. J. Biosci., 3: 62-70. GHASSEMI-GOLEZANI K., HOSSEINZADEH-MAHOOTCHI A., 2015 - Improving physiological performance of saf- flower under salt stress by application of salicylic acid and jasmonic acid. - WALIA J., 31: 104-109. GHASSEMI-GOLEZANI K., LOTFI R., 2012 - Responses of soy- bean leaves and grain yield to water stress at repro- ductive stages. - Int. J. plant, Animal Environ. Sci., 2: 63-68. GHASSEMI-GOLEZANI K., LOTFI R., 2013 - Influence of water stress and pod position on oil and protein accu- mulation in soybean grains. - Intl. J. Agron. Plant. Prod., 4: 2341-2345. GHASSEMI-GOLEZANI K., LOTFI R., 2015 - The impact of salicylic acid and silicon on chlorophyll a fluorescence in mung bean under salt stress. - Russ. J. Plant Physiol., 62: 611-616. GHASSEMI-GOLEZANI K., LOTFI R., NAJAFI N., 2015 a - Some physiological responses of mung bean to sali- cylic acid and silicon under salt stress. - Adv. Biores., 6: 7-13. G H A S S E M I - G O L E Z A N I K . , M A G H F E R A T I R . , Z E H T A B - SALMASI S., DASTBORHAN S., 2016 - Influence of water deficit and nitrogen supply on grain yield and yield components of safflower. - Adv. Biores., 7: 132- 136. GHASSEMI-GOLEZANI K., RAEI N., RAEI Y., 2015 b - Effects of water deficit and nitrogen levels on grain yield and oil and protein contents of Maize. - Azarian J. Agric., 2: 46-50. GHAVAMI N., RAMIN A.A., 2007 - Salinity and temperature effects on seed germination of milk thistle. - Commun. Soil Sci. Plant Anal., 38: 2681-2691. HAYAT S., HASAN S.A., FARIDUDDIN Q., AHMAD A., 2008 - Growth of tomato (Lycopersicon esculentum) in response to salicylic acid under water stress. - J. Plant Interact., 3: 297-304. HUGH J.E., RICHARD F.D., 2003 - Effect of drought stress on leaf and whole canopy radiation use efficiency and yield of maize. - Agron. J., 95: 688-696. JANDA T., SZALAI G., TARI I., PALDI E., 1999 - Hydroponic treatment with salicylic acid decreases the effects of chilling injury in maize (Zea mays L.) plants. - Planta, 208: 175-180. KEVILLE K., 1991 - Illustrated herb encyclopedia; A com- plete culinary, cosmetic, medicinal, and ornamental guide to hers. - Simon & Schuster, East Roseville, New South Wales, Australia, pp. 224. KIM K.S., PARK S.H., JENKS M.A., 2007 - Changes in leaf cuticular waxes of sesame (Sesamum indicum L.) plants exposed to water deficit. - J. Plant Physiol., 164: 1134-1143. LOUTFY N., EL-TAYEB M.A., HASSANEN A.M., MOUSTAFA M.F.M., SAKUMA Y., INOUHE M., 2012 - Changes in the water status and osmotic solute contents in response to drought and salicylic acid treatments in four different cultivars of wheat (Triticum aestivum L.). - J. Plant Res., 125: 173-184. MUNNÉ-BOSCH S., PENUELAS J., 2003 - Photo and antiox- idative protection, and a role for salicylic acid during drought and recovery in field grown Phillyrea angusti- folia plants. - Planta, 217: 758-766. MUNNS R., JAMES R.A., LÄUCHLI A., 2006 - Approaches to increasing the salt tolerance of wheat and other cere- als. - Journal of Experimental Botany, 57: 1025-1043. OZTURK A., 1999 - The effect of drought on the growth and yield of winter wheat. - Turk. J. Agric. For., 23: 531-540. SINGH B., USHA K., 2003 - Salicylic acid induced physiologi- cal and biochemical changes in wheat seedlings under water stress. - Plant Growth Regul., 39: 137-141. SOLEIMANZADEH H., HABIBI D., ARDAKANI M.R., PAKNE- JAD F., REJALI F., 2010 - Response of sunflower (Helianthus annuus L.) to drought stress under differ- ent potassium levels. - World Applied Sciences Journal, 8: 443-448. YILDIRIM E., TURAN M., GUVENC I., 2008 - Effect of foliar salicylic acid applications on growth, chlorophyll and mineral content of cucumber (Cucumis sativus L.) grown under salt stress. - J. Plant Nutrit., 31: 593-612. ZHUANG X.P., LU Y.Y., YANG G.S., 1992 - Extraction and determination of flavonoid in ginkgo. - Chinese Herbal Medicine, 23: 122-124.