South African Journal of Botany

South African Journal of Botany 86 (2013) 68 72 Contents lists available at SciVerse ScienceDirect South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb Short communication Vase life of cut rose cultivars Avalanche and Fiesta as affected by Nano-Silver and S-carvone treatments Z. Nazemi Rafi, A. Ramezanian Department of Horticultural Science, Faculty of Agriculture, Shiraz University, Shiraz, Islamic Republic of Iran article info abstract Article history: Received 6 November 2012 Received in revised form 11 February 2013 Accepted 13 February 2013 Available online 16 March 2013 Edited by AK Cowa Keywords: Anti-microbial Cut flowers Essential oil Stomatal conductance Transpiration rate Water uptake Rosa hybrida L. is an important commercial cut flower. The vase life of this flower is usually short due to vascular occlusion. We assessed the effect of Nano-Silver (NS) and S-carvone in prolonging the vase life of R. hybrida L. cv. Avalanche and Fiesta. Hence, an experiment involving the treatment with NS at 0, 50, 100, and 200 mgl 1 and S-carvone at 0, 0.25, 0.5, and 0.75 mgl 1 with 10 replicates was conducted. Applying NS pulse treatments increased vase life, water uptake rate, and fresh weight and reduced the number of bacteria, water loss, stomatal conductance and transpiration rate. However, S-carvone treatments did not have a positive effect on the vase-life parameters of cut rose flowers. NS pulse treatments increased relative fresh weight (RFW), and water uptake rate (WUR) and decreased water loss (WL) (%) by 10, 89 and 31% for cultivar Avalanche, compared to the controls, respectively. Application of 200 mgl 1 NS led to the highest vase life (18 days) for roses. The results show that NS increased vase life by suppressing stomatal opening, decreasing transpiration from leaves and inhibiting bacterial proliferation. 2013 SAAB. Published by Elsevier B.V. All rights reserved. 1. Introduction Rose (Rosa sp.) is one of the highest demand cut flowers in the world and has a limited commercial value due to early dehydration (Van Doorn, 1997). Numerous investigations have demonstrated the helpful effects of various chemical additives on the postharvest water relations and extending the vase life of cut rose flowers (Ichimura and Shimizu- Yumoto, 2007). It is well established that one of the main causes for vase life reduction is the bacterial blockage of xylem vessel (Van Meetern et al., 2001). Stem occlusion in cut flowers is mostly due to the physiological wound-induced responses to cutting (Williamson et al., 2002), air embolism (Van Ieperen et al., 2002) and microorganisms that grow in a solution vase and their residue (Loubaud and Van Doorn, 2004; He et al., 2006). Many antimicrobial compounds, such as silver nitrate (Singh and Tiwari, 2002), aluminum sulfate (Rezvanypour and Osfoori, 2011), and 8-hydroxyquinoline sulfate (Ichimura et al., 1999) have been used to extend the vase life of cut flowers. The broad antimicrobial effect of Nano-Silver is well-known and, it has been used in different fields in medicine (healing wounds and anti-inflammatory) and water purgation (Jain and Pradeep, 2005; Chen and Schluesener, 2008). Corresponding author. Tel.: +98 9370740145. E-mail address: zahra_nazemi_rafi@yahoo.com (Z. Nazemi Rafi). Nanometer sized silver particles (NS) have a high surface area to volume ratio and because of that, they are considered to prevent bacteria and other microorganisms more intensely than the components of oxidation states of Ag (Furno et al., 2004). Recent studies have investigated the effectiveness of NS in extending the vase life of some cut flowers, including cut carnations, gerberas, acacias, and roses (Solgi et al., 2009; Lü et al., 2010; Hoseinzadeh Liavali and Zarchini, 2012; Liu et al., 2012; Moradi et al., 2012). S-carvone (chemical formula: C 10 H 14 O), a monoterpene, is an important oil extracted from caraway (Carum carvi) and dill (Anethum graveolens)seeds(de Carvalho and Da Fonseca, 2006). It has antibacterial, antifungal, and anti-wound healing properties (Oosterhaven et al., 1995a, 1995b). Oosterhaven et al. (1995a) reported that S-carvone delayed the induction of Phenylalanine ammonia lyase (PAL) activity and suberin formation. Previous workers reported the effect of S-carvone on the vase-life of Hakea francisiana (Williamson et al., 2002), Grevillea Crimson Yul-lo (He et al., 2006), Baeckea frutescens, Acacia holosericea, Chrysanthemum sp. cv. Dark Splendid Reagan and Chamelaucium uncinatum cv. Mullering Brook (Damunupola et al., 2010). There are no reports about the effects of S-carvone on the vase life of cut rose flowers. Also, genetic variations between cultivars of rose cause in different responses to various preservative solutions (Ichimura et al., 2002). Therefore, in our study, effect of NS was investigated on the vase life of cut rose cultivars (Rosa hybrida L. cv. Avalanche and Fiesta). In the present research, the efficacy of NS and S-carvone in extending the vase life of cut rose flowers Avalanche and Fiesta was evaluated. 0254-6299/$ see front matter 2013 SAAB. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sajb.2013.02.167

Z. Nazemi Rafi, A. Ramezanian / South African Journal of Botany 86 (2013) 68 72 69 Table 1 Effects of the different concentrations of Nano Silver (NS) and S-carvone (S-c) on measured traits and vase-life. Treatment Vase-life (d) RFW (% of initial) WUR (g g 1 ) WL (g stem 1 d 1 ) Avalanche Fiesta Avalanche Fiesta Avalanche Fiesta Avalanche Fiesta Control 9.6 c 12 c 113.8 b 109.74 b 10.69 c 17.66 17.38 a 16.08 NS 50 mgl 1 14.4 b 13 bc 116.84 ab 111.77 b 15.96 b 17.31 12.78 b 15.25 NS 100 mgl 1 14.6 b 14.20 b 121.17 ab 112.92 b 16.02 b 15.81 13.04 ab 14.99 NS 200 mgl 1 16 a 16 a 124.75 a 120.41 a 20.14 a 17.69 12.01 b 13.88 LSD 0.05 1.27 1.23 9.98 6.43 3.85 ns 4.60 ns Control 9.6 A 12 A 113.8 109.74 10.69 17.66 A 17.38 B 16.08 B S-c 0.25 mgl 1 6.2 B 6.4 B 108.57 107.47 7.08 12.1 AB 20.91 A 20.55 A S-c 0.5 mgl 1 5.4 B 6.2 B 110.01 105.4 7.33 9.22 B 20.32 AB 20.79 A S-c 0.75 mgl 1 5.8 B 5.4 B 104.34 106.85 7.07 9.85 AB 20.34 AB 22.75 A LSD 0.05 0.82 1.02 ns ns ns 8.12 3.10 2.78 Mean values of vase life (d), relative fresh weight (RFW), water uptake rate (WUR) and water loss (WL). Means followed by the same letter (s) are not significantly different at 5% level of significance. ns: LSD-test was not significant (P>0.05). 2. Materials and methods 2.1. Plant material Cut rose (R. hybrida L. cv. Avalanche and Fiesta) flowers were obtained from Lotus greenhouses in Shiraz, Iran. Flowers were harvested in the morning over summer (September) in 2011 and immediately stood upright in buckets partially filled with tap water. Harvested flowers were kept under shade in the field until being transported within 3 h to the laboratory. To minimize moisture loss, flowers were covered with a plastic film during transportation. At the laboratory, the stems were recut, removing 5 cm from end, and the stem length was then 35±2 cm. Deionized water (DI) was used to remove air emboli and the leaves from the lowermost 5 cm of the stems. 2.2. Experimental design and treatments Experiment was conducted at 20±2 C with 60 65% humidity (RH) and 12 μmol m 2 s 1 light intensity (cool white florescent tubes) under a daily light period of 12 h in a completely randomized block design with ten replicates. For pulsing, NS-treated stems were stood for 1 h in various concentrations of NS (Nanocid Co. Ltd., Iran) solutions at 50, 100 and 200 mgl 1 and were immediately stood in 1 L capacity plastic vases containing 800 ml of DI. Vase solution treatments of S-carvone (Sigma-Aldrich, St Louis, MO, USA) were carried out at 0.25, 0.5 and 0.75 mgl 1. Ethanol (80%) was used as a primary solvent to dissolve pure S-carvone. The final concentration of ethanol in treatment solutions was 0.05% (v/v) (Damunupola et al., 2010). Control treatment was DI. We used a sheet of aluminum foil to seal vases to minimize evaporation and inhibit contamination. 2.3. Measurements 2.3.1. Vase-life During the vase-life period, the visual quality of cut flowering stems was inspected daily. In our study, vase-life was defined as the period from the time of cutting to the time when 50% of floret petals wilted or abscised or floret necks bent. 2.3.2. Water uptake, water loss and fresh weight The cut flowers' fresh weight and the water uptake rate were measured daily. The weight of vases with and without cut flowers was recorded daily. Mean daily water uptake (g stem 1 day 1 ) was computed using the formula (S t 1 S t ), where, S t istheweightofvasesolution (g) at t=day 1, 2, 3,, and n. Mean daily water loss (g stem 1 day 1 ) was computed using the formula (C t 1 C t ), where, C t is the weight of vase solution (g) at t=day 1, 2, 3,, and n. Relative fresh weight (RFW) of stems was computed using the formula RFW (%)=(W t /W 0 ) 100, where, W t is the weight of stem (g) at t=day 0, 1, 2, and n, and W 0 is the weight of the same stem (g) at t=day 0 (Lü et al., 2010). 2.3.3. Stem end bacterial counts To determine the number of bacteria, 0.5 g (about 2 cm in length) segments were cut from the end of the stems. The explants were washed three times with sterile deionized water to decrease the surface load of microorganisms, and then dilution was made with a 0.9% normal salt solution. Liquid extract (0.1 ml) was spread on Plate Count Agar (PCA) plates. Before counting of bacteria, PCA plates were incubated at 37 C for 24 h (Balestra et al., 2005). 2.3.4. Stomatal conductance and transpiration rate Stomatal conductance and transpiration rate of the uppermost leaves were measured with a portable photosynthesis system (LCi-SD model, UK) every other day. 2.4. Statistical analysis Data were subjected to analysis of variance (one-way ANOVA) using the general linear model program of SPSS software. Means were compared by the LSD test at P=0.05. 3. Results 3.1. Vase-life S-carvone treatments at all concentrations caused damage to the leaves of cut roses and accelerated their abscission, resulting in a significantly shorter vase-life than the control. There were significant differences in vase life between NS-treated and control flowers. Increasing NS concentration markedly extended the vase-life of cut rose cv. Avalanche and Fiesta (Table 1). 3.2. Water uptake, water loss and fresh weight NS treatments reduced the declining rate of relative fresh weight and increased water uptake rate in both cultivars during vase life (Table 1). All NS treatments, particularly at 200 mgl 1 concentration delayed fresh weight loss, although the S-carvone treated flowers soon lost their fresh weight and reached to the minimum amount at day 7 (data not shown). Amount of water loss by the S-carvone treated and control flowers was greater than those pulses treated with NS, particularly at 200 mgl 1, although all treated and untreated plants decreased their water loss with progress in time. Changes in these factors showed similar trends for both cultivars, such that silver nanoparticles, particularly at 200 mgl 1 were the most effective treatments (Table 1).

70 Z. Nazemi Rafi, A. Ramezanian / South African Journal of Botany 86 (2013) 68 72

Z. Nazemi Rafi, A. Ramezanian / South African Journal of Botany 86 (2013) 68 72 71 3.3. Stem end bacteria Number of stem end bacteria was significantly controlled by NS treatments over the vase life period of both cultivars at 200 mgl 1 concentration (Fig. 1A). S-carvone showed no positive effect on controlling stem end bacterial proliferation (Fig. 1B). 3.4. Stomatal conductance and transpiration rate Stomatal conductance and transpiration rate were reduced markedly by NS treatments. These effects increased by increasing the NS concentration. Leaf stomatal conductance and the transpiration rate in 200 mgl 1 NS declined faster than in control. All S-carvone treatments increased stomatal conductance and transpiration rate in both cultivars during vase life (Fig. 1C, D, E, F). 4. Discussion Cut rose flowers are sensitive to water stress because after harvesting, the water balance of flower petals is easily disturbed. Also, rose flowers can often be affected by biotic stress, which causes microbiological vascular blockage of stem below the flower head (Van Doorn et al., 1989). Ethylene with vascular occlusion due to agglomerated bacteria in stem base or vase solution decreases the vase-life of cut flowers (Van Doorn et al., 1994). In this study, S-carvone at 0.25, 0.5 and 0.75 mgl 1 showed negative effects on relative fresh weight, water uptake, water loss, preventing the bacterial proliferation, vase life, and reducing the rate of stomatal conductance and transpiration rate of cut rose flowers compared to the positive effect of NS pulse treatment. He et al. (2006) did not report an antibacterial activity in their research, but they suggested that S-carvone could possibly have an antibacterial activity at the low concentrations (0, 0.032, 0.318 and 0.636 mm). However, concentrations over than 1 mm are effective against a range of plant pathogenic bacteria and fungi (Oosterhaven et al., 1995b). Damunupola et al. (2010) showed that S-carvone, in A. holosericea vase solutions, at low concentration (0.318 and 0.636 mm) did not decrease total bacterial populations. In this literature, we showed that unlike NS, S-carvone did not decrease bacterial populations. Kordali et al. (2008) reported that essential oils had a phytotoxic effect in various plant systems, e.g. treatment with essential oils from Origanum acutidens showed strong phytotoxicity in resistance to seed germination and seedling growth of Chenopodium, Amaranthus retroflexus, Rumex crispus and album. Probably, because carvone could damage cell membrane (Teper Bamnolker et al., 2010), in our experiment the S-carvone treatments caused visible damage to the leaves of cut roses and their early abscission, thereby resulting in a shorter vase-life than the control. Catechol oxidase and peroxidase enzyme activities after wounding have been connected with increase biosynthesis of lignin and suberin (Blee et al., 2001), which blocks the xylem vessels in cut flowers resulting in water imbalance. Loubaud and Van Doorn (2004) reported that in rose cv. Red One the reason for vascular occlusion was the accumulation of bacteria rather than the wound inducing. Also, the lack of S-carvone effect on extending the vase-life of cut flowers such as A. holosericea and Chrysanthemum sp. was reported (Damunupola et al., 2010). Similarly, unlike NS, S-carvone had no effect on the improvement of the vase life of cut rose flowers. Microbial proliferation is the main reason for vascular blockage (Van Doorn et al.,1989). Large surface to volume ratio of NS particles is increasing their contact with fungi or bacteria, and vastly improving their antifungal and antibacterial efficiencies (Shah and Belozerova, 2009). NS adheres to the bacterial cell wall surface, then directly gets inside the bacteria and quickly interacts with sulfhydryl (\SH) of oxygenic metabolic enzyme to deactivate them, to block inhalation and metabolism and suffocate the bacteria (Niemietz and Tyermann, 2002). In our study, NS treatments reduced the number of bacteria compared to the DI control. Microbiological vascular occlusion is an important factor in decreasing water uptake (Balestra et al., 2005). Also, it seems that besides the bactericidal property of NS, it has a positive effect on water uptake (Liu et al., 2009). In our research, pulse treatments with NS at 200 mgl 1 for 1 h significantly increased the water uptake rate of the cut rose cv. Avalanche (Table 1). The positive effect of NS treatment in decreasing the water loss was reported (Lü et al., 2010). In these experiments, pulse treatments with NS at 200 mgl 1 for 1 h decreased the water loss of the cut rose flowers (Table 1). Lü et al. (2010) reported that treatment with NS repressed stomatal aperture on cut rose leaves, and they ascribed this to the suppression of the primary channels of water transport across biological membranes (AQPs). Our results showed that NS treatments decreased stomatal conductance and transpiration rate, thereby reducing the water loss of the cut rose flowers (Fig. 1C, D, E, F and Table 1). In conclusion, the pulse treatment with NS at 200 mgl 1 improved the vase life of cut rose flowers cv. Avalanche and Fiesta. Furthermore, NS showed a great effect on inhibition of bacterial growth and increasing water uptake. However, S-carvone did not increase the vase life of cut rose flowers. Nevertheless, further research is necessary to identify the efficiency of S-carvone treatments on extending the vase life of cut flowers. References Balestra, G.M., Agostini, R., Bellincontro, A., Mencarelli, F., Varvaro, L., 2005. Bacterial populations related to Gerbera (Gerbera jamesonii L.) stem break. 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