Postharvest Biology and Technology

Postharvest Biology and Technology 66 () 8 5 Contents lists available at SciVerse ScienceDirect Postharvest Biology and Technology journa l h o me pa g e: www.elsevier.com/locate/postharvbio Effects of three different nano-silver formulations on cut Acacia holosericea vase life Jiping Liu a,b,d, Kamani Ratnayake b, Daryl C. Joyce b,c, Shenggen He a,, Zhaoqi Zhang d a College of Life Sciences, University of Zhongkai Agriculture and Engineering, Guangzhou 55, PR China b The University of Queensland, School of Agriculture and Food Sciences, Gatton, QLD 33, Australia c Agri-Science Queensland, Department of Employment, Economic Development and Innovation, P.O. Box 583, SCMC, Nambour, QLD 56, Australia d College of Horticulture, South China Agricultural University, Guangzhou 56, PR China a r t i c l e i n f o Article history: Received August Accepted November Keywords: Antimicrobial Cut foliage Hydraulic conductivity Nano-silver Water relations a b s t r a c t The relative efficacies of three chemically different nano-silver (NS) formulations were evaluated for their potential to extend the vase life of short-lived cut Acacia holosericea foliage. The novel proprietary formulations were neutral NS, acidic NS and ionic NS. They were characterised in terms of particle size, ph value, colour and odour. The NS treatments were applied as vase (lower concentrations) or pulse (higher concentrations) solutions. Among the treatments compared, neutral NS as a mg L vase solution or as a mg L h pulse treatment and acidic NS as a.5 mg L vase solution or as a 5 mg L h pulse treatment significantly (P.5) extended the vase life of A. holosericea. Vase life extensions over the deionised water (DI) controls were associated with better maintenance of relative fresh weight and vase water uptake, suppression of bacterial growth in the vase water and stem-end, and delaying stem blockage. In contrast, ionic-ns applied as a.5 or mg L vase solution treatment or as a 5 or mg L pulse treatment caused severe phytotoxicity to cut A. holosericea stems. The results suggest that NS treatments, especially neutral NS and acidic NS pulse treatments, could be a potential postharvest technology for commercial application to cut A. holosericea. Elsevier B.V. All rights reserved.. Introduction Acacia belongs to the family Mimosaceae and 35 Acacia species are found worldwide, with of these in Australia (Maslin, ). Acacia has long been sold as cut flowers and foliage in Europe under the name mimosa (Sedgley and Parletta, 993). In Australia, species such as Acacia baileyana, Acacia dealbata and Acacia retinodes are grown for cut flowers (Jones et al., 998; Horlock et al., ). A. holosericea is widespread across inland regions of northern Australia, where it shows strong survival and rapid early growth characteristics (Midgley and Turnbull, 3). A. holosericea bears large phyllodes densely covered with fine hairs, which give the foliage an attractive silvery-green colour. Therefore, A. holosericea has potential as a novel cut foliage (Damunupola, 9; Celikel et al., ). Cut Acacia stems tend to have a short vase life of just 6 d due mainly to rapid wilting (Williamson and Milburn, 995; Jones et al., 998). Reduced water uptake caused by xylem blockage is Corresponding author. Tel.: +86 8936; fax: +86 89368. E-mail addresses: liujipingpipi@63.com (J. Liu), k.ratnayake@uq.edu.au (K. Ratnayake), d.joyce@uq.edu.au (D.C. Joyce), heshenggen@yahoo.com.cn (S. He), zqzhang@scau.edu.cn (Z. Zhang). the typical reason for wilting (Williamson and Milburn, 995; van Doorn, 997; Jones et al., 998). Stem blockage in cut flowers is classified generally as microbial due to living bacteria and their decay products (van Doorn, 997; Teixeira-da-Silva, 3), physiological wound-induced (Williamson et al., ; Loubaud and van Doorn, ; He et al., 6), and physical due to air emboli (van Meeteren et al., 6). van Doorn (997) suggested that physiologically induced gum (gel) secretion into the xylem lumen may be a reason for early wilting of Acacia. Williamson and Milburn (995) observed that cavitation events in cut Acacia amoena stems were associated with their abbreviated vase life. Horlock et al. () reported that the use of a germicide and also lowering the ph of the holding solution can extend Acacia vase life. Supply of chemicals that inhibit suberin synthesis (apparently associated with physiological wound-induced blockage) increased the vase life of A. baileyana (Williamson et al., ). Silver ion (Ag + ) has long been recognised in medical applications and industrial processes as efficient in inhibiting growth of bacteria and other microorganisms (Feng et al., ; Rai et al., 9). As a novel antiseptic, nano-silver (NS) is used in the medical industry, in silver embedded fabrics and in water purification and vegetable disinfection (Davies and Etris, 997; Klaus et al., 999; Rai et al., 9). Owing to their high surface area to volume ratio, among other unique chemical and physical properties, NS 95-5/$ see front matter Elsevier B.V. All rights reserved. doi:.6/j.postharvbio...5

J. Liu et al. / Postharvest Biology and Technology 66 () 8 5 9 formulations provide good contact with microorganisms and are highly effective as germicides (Rai et al., 9). NS particles can attach to the cell membranes and penetrate into bacteria. Therein, NS can disrupt processes such as respiration and cell division, leading to cell death. NS releases silver ions (Ag + ) within bacterial cells, which enhance its bactericidal activity (Feng et al., ; Rai et al., 9). In cut flower applications, NS has been reported to be effective as an antimicrobial agent (Liu et al., 9a; Solgi et al., 9; Lv et al., a), an ethylene inhibitor (Kim et al., 5) and/or a regulator of stomatal aperture (Lv et al., b). In view of its potential as a novel cut foliage and as a representative of the largest family (species-wise) of attractive foliage and flowering plants native to Australia, it is important to screen treatments for extending the vase life of short-lived A. holosericea. Three physicochemically different formulations of NS were compared: neutral NS, ionic NS and acidic NS. The effects of these NS formulations on A. holosericea vase life were evaluated first with a view to determine optimum concentrations. The effects of pulse versus vase solution treatments with neutral NS were then investigated because pulse treatments are convenient for industry to apply prior to shipment. Lastly, the effects of neutral NS versus acidic NS were investigated because both pulse treatments had positive effects on vase life of A. holosericea.. Materials and methods.. Plant material Acacia holosericea A. Cunn. ex G. Don (Velvet Leaf Wattle) cut foliage stems were obtained from plants grown near Karalee, Queensland (5 56 E, 7 3 S). Stems were harvested with sharp secateurs at 6: am from late summer through to early winter (viz., February June). Harvested stems were immediately stood upright into buckets partially filled with deionised water (DI) and brought to the laboratory within h. Stems free of visual defects were used in experiments. Prior to imposition of treatments, the stems were recut under DI so as to remove the basal 5 cm in order to avoid stem-end air emboli. Recut stems were cm long... Chemicals Neutral NS, ionic NS and acidic NS from Shanghai Huzheng Nano Technology Co. Ltd. (Shanghai, China; http://www.hznano.com/: access date: -7-5) were used in this study..3. Experimental Experiments were conducted in a vase life evaluation room at the Postharvest Laboratory of The University of Queensland s Gatton campus. The room was operated at ± C, 6 ± % relative humidity (R.H.) and 8 mol m s light intensity (cool white florescent tubes) at foliage level under a daily h photoperiod. For vase solution treatments, cut A. holosericea stems were stood individually into plastic 3 ml capacity vases containing 5 ml of either DI or solutions of NS. For pulse treatments, stems were stood individually into 5 ml capacity glass tubes containing 3 ml pulsing solution for h, before transfer to DI in vases. Mouths of the plastic vases were covered with a sheet of plastic film in order to prevent contamination and minimise evaporation. All solutions were freshly prepared at the beginning of each experiment, and were not renewed in the course of the experiment. Experiment : Properties of three NS formulations. Appearance, size, colour, odour, ph value, stability, and reaction with acid (nitric), alkali (sodium hydroxide) or NaCl of three NS formulations were evaluated. Experiment : Effects of three NS formulations, as pulse and as vase solution treatments, on vase life. mg L NS pulse treatments and mg L NS vase treatments were applied. Neutral NS vase and pulse treatments were tested in February 9 and ionic NS and acidic NS vase and pulse treatments were applied in April 9. Pulsing was for h before the stems were put into vases containing DI. Vase life was assessed daily during the vase period. Experiment 3: Comparison of pulse and vase solution treatments. This experiment was carried out in May 9. Neutral NS pulse ( mg L h) and vase solution ( mg L ) treatments were compared to the control ( mg L ; DI). Vase life, relative fresh weight (RFW), relative water uptake, hydraulic conductance and antibacterial effects were assessed. RFW and relative water uptake of cut stems were recorded daily during the vase period. Measurements of tissue Ag contents were carried out in triplicate on stem-end, middle of the stem, top of the stem, lowermost mature phyllodes and uppermost unfolded phyllodes tissue samples on days (i.e., after the pulse), and 8. Sampling was carried out on additional stems maintained for each sample time. Also, on days,.5,,, and 8 during vase life evaluation, basal cm segments were excised under DI for hydraulic conductance assessment. To assess the antibacterial activity of NS, bacterial counts for pulse solutions were carried out on days and.5 and for vase solutions on days,, and 8. Vase solution treatment and stem-end ( cm) bacterial counts were carried out on days,.5,,, and 8. All destructive sampling was on additional separate stems maintained for each occasion. Experiment : Comparison of effective NS treatments. This experiment was carried out in June 9. With a view to NS treatment optimisation, neutral NS ( mg L ) and acidic NS (5 mg L ) pulses for h were compared to the DI control. Vase life, RFW, relative water uptake, hydraulic conductance and antibacterial effects were assessed as described above and below... Measurements Properties of three NS formulations: By way of partial characterisation of the proprietary NS products, the various formulations were examined by transmission electron microscopy (TEM). A L drop of NS was placed onto coated Cu grids ( mesh square; 3 mm diameter; ProSciTech, Thuringowa Central, QLD, Australia) and allowed to stand for min to settle the particles onto the grid. Liquid was then removed by absorbtion with the torn edge of a filter paper. Grids were held in air until completely dry and then observed with a JEM TEM [Japanese Electrical Optical Co. Ltd. (JEOL) Tokyo, Japan] at an accelerating voltage of 8 kv. Images were digitally recorded using item software (Olympus Soft Imaging Co., Münster, Germany). To wash out solvents and obtain clear images, the neutral NS grid was dipped in ultra pure water (Purelab UHQ, ELGA Lab Water Ltd., Lane End, Buckinghamshire, UK) while the acidic NS grid was dipped in % ethanol for s, and then allowed to dry completely. Images of neutral NS and acidic NS formulation (particles) were obtained at 8 kv and kv accelerating voltages, respectively. The solution ph values were detected using a ph meter (Hanna Instruments Co., Woonsocket, USA). The stability of NS formulations was assessed by observing the change in colour and clarity after long-time ( months) storage under ambient conditions and also after high temperature ( C, min) and after low temperature ( C, 8 h) evaluation conditions. Lastly, NS formulations were reacted with mm nitric acid, mm sodium hydroxide and mm NaCl, and the changes in colour and clarity were observed and recorded. Vase life, RFW and relative water uptake: Vase life of the cut foliage stems was assessed daily throughout the vase life evaluation periods. Vase life was considered to have ended when 5% of the foliages on a stem had wilted. Fresh weights (FW) of cut stems

J. Liu et al. / Postharvest Biology and Technology 66 () 8 5 were recorded daily during the vase period using an analytical balance. Relative fresh weight (RFW) was calculated as described by He et al. (6). The weights of vases without their cut stems were recorded daily. Average daily relative water uptake was calculated by: relative water uptake (g g initial FW day ) = (S t S t )/initial FW; where, S t is the weight of vase solution (g) at t = day,, 3, etc., and S t is the weight of vase solution (g) on the pervious day. Hydraulic conductance: Stems were removed from their vase solution and cm long segments were excised under DI using sterile scalpel blades. Hydraulic conductance measurements were carried out using the method described by Damunupola (9). To prevent solution leakage, Selleys Aqua Knead TM sealant was used after cutting to fill voids in the junction between stem segment ends and silicon tube. Hydraulic conductance was measured under a pressure head of cm DI for h as described by He et al. (6). Bacterial counts: To determine vase solution bacterial populations, aliquots were taken in triplicate from pulse or vase solutions. Samples were serially diluted with.9% sterile normal saline (NaCl) to achieve 3 3 bacterial colonies in any one Petri dish. The. ml aliquots were spread over each Plate Count Agar (PCA) plate (Liu et al., 9a), and they were incubated at 3 C for 8 h (de Witte and van Doorn, 988) before enumeration of bacteria. Colony counts were expressed as colony forming units per ml (CFU ml ). To assess bacterial population in stem-ends, cm long stem-end segments were cut with sterile sharp secateurs. These stem segments were surface sterilised with 7% ethanol and were further cut into cm lengths followed by splitting into 6 smaller pieces with sterile scalpel blades. These pieces were transferred into sterile tubes each containing ml of sterile.9% normal saline. Bacteria were dislodged by vortex mixing for min. Liquid extract (. ml) was then serially diluted with sterile.9% normal saline and spread onto PCA plates. Bacterial colonies were enumerated as described above. Ag content in tissues: Samples of the stem-end (5 cm), middle of the stem (5 cm), top of the stem (5 cm), two lowermost mature phyllodes, and three uppermost unfolded phyllodes were obtained from three replicate stems. They were dried at 6 ± 5 C for 8 h and then ground to < mm particle size. Sub samples weighing.3 ±.5 g were subjected to nitric perchloric acid (HNO 3 HClO ) wet digestion. Briefly, tissue samples were predigested overnight in concentrated nitric acid [(7% w/w; reagent grade), Chem-Supply, Gillman, SA, Australia]. Digestion was then completed in a mixture of nitric and perchloric acid [(7%, A.C.S. Reagent), Aldrich Chemical Co. Inc., Milwaukee, WI, USA] in a 5 position AIM digestion block (Analytical Instruments, Golden Valley, MN, USA). The digestion block was held at 8 C for 5 min, then at 6 C for min, and then ramped by C min to 85 C and held there for 3 min. Finally, the cooled digestion solutions were diluted to ml and analysed by inductively coupled plasma spectrometer (ICPS; Spectroflame Modula E, Spectro Analytical Instruments, GmbH, Kleve, Germany). Two blank samples and a reference standard (Ag Elemental Standard, Australian Chemical Reagents, Moorooka, QLD, Australia) were used in the digestion and subsequent analysis with each batch. The tissue Ag content was expressed as g g dry weight (DW) of tissue..5. Statistical analyses Treatments were arranged on benches in a completely randomised design (CRD) with 3 replicate stems for each treatment. Data were analysed using ANOVA in SAS version 9.3 (SAS Institute Inc., Cary, NC, USA) and means were compared by the LSD test at P =.5. For vase life experiments, data are presented as means ± standard errors (SE) and significant difference (P.5) was analysed using ANOVA. Fig.. TEM images of neutral NS (A), ionic NS (B) and acidic NS (C) formulations. Scale bar = nm. 3. Results 3.. Properties of three NS formulations (Experiment ) Clear images could be obtained for native (i.e., not rinsed) ionic NS, which had an average particle size of 9 nm (Fig. B). Only after the neutral NS grid was rinsed by dipping in ultra pure water

J. Liu et al. / Postharvest Biology and Technology 66 () 8 5 Table Properties of the three different formulations of NS (Experiment ). Property Neutral NS Acidic NS Ionic NS Colour Colourless Colourless Light brown Odour Odourless Acidic odour Odourless ph value 7.5.5 6.5 Size (nm) 9. ±.5 Not determinable 8.9 ± 6.8 Stability [after month storage or at high ( C, min) and low ( C, 8 h) temperature] Stable Stable Unstable Reaction with nitric acid (ph 5) Deposition No deposition Deposition Reaction with sodium hydroxide (ph 8) No deposition No deposition No deposition Reaction with NaCl (concentration 8.6 mm) No deposition No deposition Brown deposition Table Effects of different NS formulations applied as vase and pulse treatments on cut A. holosericea vase life (Experiment ). Data are means ± standard error (SE) and different small letters indicate significant difference from control at P.5, n = ). Treatment Concentration (mg L ) Vase life (days) A. Vase solution Deionised water 5.3 ±.5 a Neutral NS 7. ±.8 b Neutral NS 8.6 ±.8 b B. Pulse ( h) Deionised water 5. ±. a Neutral NS 9. ±.6 b Neutral NS.6 ±.8 b C. Vase solution Deionised water 7.9 ±.7 a Ionic NS.5 7.6 ±.5 a Ionic NS 8. ±.5 a Acidic NS.5 9.9 ±.9 b Acidic NS 9. ±.6 a D. Pulse ( h) Deionised water 7.5 ±.7 a Ionic NS 5 9. ±.6 a Ionic NS 7. ±.8 a Acidic NS 5. ±.9 b Acidic NS.9 ±. b and acidic NS grid was rinsed in % ethanol for s, could images of neutral NS (Fig. A) and acidic NS (Fig. C) be obtained at 8 kv and kv accelerating voltages, respectively. The average particle size of neutral NS was 9 nm. Attempts to obtain clear images of acidic NS were not successful. The three proprietary formulations of NS differed in size, ph value, colour, odour and other properties (Table ). Neutral NS is a colourless and odourless liquid with a ph of 7.5. It is stable at high or low temperature and during long term storage, and does not form precipitates with sodium hydroxide or NaCl, but forms white deposits with nitric acid. Acidic NS is a colourless liquid with an irritating acid odour and a ph of.5. Acidic NS is stable during storage and does not form precipitates under alkaline or acidic conditions, or with NaCl. Ionic NS is an odourless light brown liquid with a ph of 6.5. It forms brown deposits upon long storage and with NaCl, but does not form precipitates with acid or alkali. 3.. Effects of three NS formulations on vase life (Experiment ) Cut A. holosericea stems in DI had a vase life of 5 8 d depending on harvest time (Table ). The neutral NS treatments had positive effects in extending the vase life of A. holosericea cut stems as compared to the control (Table A and B). The mg L neutral NS pulse treatment gave a significant vase life extension over the corresponding control of 5. d (96.3% extension). The mg L neutral NS vase solution treatment was also beneficial, giving a significant vase life extension over the control of 3.3 d (6.3% extension). For ionic NS, vase lives for both vase (Table C) and pulse (Table D) treatments were not significantly different to the corresponding control. Ionic NS as a 5 mg L pulse or.5 mg L vase treatment caused phytotoxicity, with rapid wilting and drying of pyllodes and blackening of the veins (data not shown). Lower concentrations of.5 mg L as a vase solution and 5 mg L as a pulse for acidic NS enhanced the vase life of cut A. holosericea (Table C and D). The mg L vase and mg L acidic NS pulse treatments also extended vase life, but they were phytotoxic to the leaves, as was the case with ionic NS. That relatively high concentrations of neutral NS ( mg L pulse or mg L vase) were beneficial and relatively lower concentrations of ionic NS and acidic NS ( mg L pulse or mg L vase) were phytotoxic indicates that NS formulation chemistry influences efficacy. 3.3. Effects of neutral NS vase and pulse treatments (Experiment 3) In this experiment, neutral NS vase ( mg L ) and pulse ( mg L ) treatments both doubled the vase life of A. holosericea as compared to the control, and nearly doubled the vase life of A. holosericea (Fig. A). The RFW of the control was comparatively constant over the first 3 d and then rapidly declined from days to (Fig. B). After day 9, all control stems had wilted and dried, with RFWs of 5%. Both neutral NS vase and pulse treatments showed similar effects to one another. Although RFW declined during vase life evaluation period, there were significant positive differences as compared to the control from day 3 onwards. Even on the last day of measurement, both neutral NS treatments gave reduced RFW of <%. Relative water uptake (Fig. C) of the control stems over days and 3 was generally higher than that at day, and then declined rapidly from day. Both neutral NS pulse and vase treatments showed increasing relative water uptake until day 6, and thereafter maintained significantly higher relative water uptake as compared to the control throughout the experiment. There were no significant differences between the neutral NS vase and pulse treatments. The hydraulic conductance of stem-end segments dropped continuously in all treatments (Fig. D). stem-ends showed generally lower hydraulic conductance at days and 8 than did the two neutral NS treatments. There were no significant differences in hydraulic conductance between treatments until day. The neutral NS pulse treatment gave a higher hydraulic conductance than the vase treatment on day, but was not significantly different on the other days. The bacterial population in the vase solution for A. holosericea increased over the vase period (Fig. E). Numbers for the control had increased rapidly by.5 d and were relatively constant thereafter. They were consistently significantly higher than those in NS treatments during the early vase period. Bacterial numbers for the NS vase treatment were fewer than those for the NS pulse treatment from day onwards. However, the differences between these two NS treatments were not significant. In the stem-ends of A. holosericea, bacteria in the control treatment tended to increase over the first half of the evaluation period

J. Liu et al. / Postharvest Biology and Technology 66 () 8 5 Vase life (d) 6 8 6 A 6.5 a 3.9 b 3.8 b Neutral-NS vase Hydraulic conductance (ml cm -3 h - ) 5 3 D LSD.5 Neutral-NS vase 3 5 6 7 8 9 Relative fresh weight (% initial FW) Relative water uptake ( g/g initial FW) 9 8 7 6 5.6... B LSD.5 (n=) C Neutral-NS vase LSD.5 (n=) Number of bacteria (log CFU ml - ) 5 3 7 6 5 3 E F LSD.5 LSD.5 6 8 6 Vase period (days) 3 5 6 7 8 9 Vase period (days) Fig.. Vase life (A; values are means ± SE and different small letters indicate significant difference from control at P.5, n = ), and relative fresh weight (B; n = ), relative water uptake (C; n = ), changes in hydraulic conductance (D; n = 3), changes in bacterial population in vase solutions (E; n = 3) and stem-ends (F; n = 3) over time in cut A. holosericea stems with neutral NS vase ( mg L ) and pulse ( mg L ) treatments. The vertical bars indicate the LSD.5 for treatment comparisons. (Fig. F). Both NS treatments suppressed stem-end bacteria from day to the end of evaluation. Bacterial counts in both NS vase and pulse solution transiently declined over days.5 and, and thereafter trended to increase. The Ag contents in tissues from control stems were g g DW and essentially undetectable by the extraction and ICP-AES methods used (data not presented). The Ag contents in stem-end parts of stems provided with NS as a vase treatment rose over days 8 (Fig. 3). For the NS pulse treatment, the Ag contents in the stem-end and middle of the stem initially declined over time. Ag contents in the phyllodes were low and did not change markedly during the evaluation period. The Ag content in the stem-end of the NS vase treatment was lower than that of the NS pulse treatment on day, but subsequently higher on days and 8. Along the middle of the stem to top of the stem and lowermost mature phyllodes and uppermost unfolded phyllodes axis, Ag contents for both treatments were higher in basal (proximal) tissue than in upper (distal) tissue. After day in the neutral NS vase treatment, the inside of stem-end became black in colour (data not shown). 3.. Effects of neutral NS and acidic NS pulse treatments (Experiment ) Both neutral NS ( mg L ) and acidic-ns (5 mg L ) had positive effects in maintaining RFW and relative water uptake as compared to the control, and extended 79.7% and 76.6% the vase life of cut A. holosericea stems over the control respectively (Fig. A). There were no differences for RFW and relative water uptake between these two NS pulse treatments for A. holosericea stems over the d vase period (Fig. B and C). Both NS pulse treatments were significantly positively effective for RFW and relative water uptake after day. Similarly, NS pulse treatments maintained significantly higher hydraulic conductance as compared to the control throughout most of the 8 d evaluation period (Fig. D). Antibacterial effects of both neutral NS and acidic NS pulse treatments were discerned in vase solutions and stem-ends of cut A. holosericea (Fig. E and F). The number of bacteria in the control vase solution was at a relatively high level of 3 to CFU ml throughout the evaluation period (Fig. E). Acidic NS significantly

J. Liu et al. / Postharvest Biology and Technology 66 () 8 5 3 Ag content (µg g - DW) 5 5 8 6 Middle of the stem Neutral-NS vase A A A8 B B B8 6 Treatments on vase period 8 Top of the stem Lowermost mature phyllodes 3 Uppermost unfolded phyllodes 8 8 8 Stem-end Middle of the stem Top of the stem Lowermost mature phyllodes Uppermost unfolded phyllodes Fig. 3. Ag content in cut A. holosericea stem-end, stem middle, stem top, lower leaves (phyllodes) and upper leaves as determined by ICP-AES for mg L vase (A) and mg L pulse (B) treatments. Insets show data for stem middle, stem top, lower leaves and upper leaves after neutral NS vase and pulse treatments. On the x-axis, the number stands for the vase holding day. The values are means ± SE (n = 3). inhibited bacterial growth during the early pulse time (days and.5), but gave no significant difference with control from days to 8. Neutral NS treatments had fewer bacteria than for the control throughout the vase period, with significant differences on days,.5, and 8. The number of bacteria in the stem-ends of A. holosericea increased throughout the evaluation period (Fig. F). Both pulse treatments transiently lowered numbers of bacteria during the pulse period (day.5) as compared with day. They also significantly inhibited the number of bacteria thereafter compared to the control, except for the acidic NS at day 8. The neutral NS treatment appeared more effective after day as compared to the acidic NS treatment.. Discussion Recent research has demonstrated the efficacy of NS in extending the vase life of some cut flowers, including cut gerberas, carnations and roses (Liu et al., 9a,b; Solgi et al., 9; Lv et al., a,b). In the present study, the effectiveness of vase and pulse treatments with three different proprietary formulations of NS in extending the vase life of cut A. holosericea foliage was evaluated. The neutral NS pulse and vase treatments effectively extended the vase life of A. holosericea (Table A and B), with the neutral NS pulse treatment showing potential for commercial use. Acidic NS was also efficient in extending the vase life of A. holosericea, but the effective concentration range was relatively narrow as compared to neutral NS. When the treatment concentration reached mg L for vase or mg L for pulse, respectively, black discolouration appeared on leaves, and the stems wilted and became brittle (data not presented). Nonetheless, lower concentrations of.5 mg L for vase or 5 mg L for pulsing solutions did significantly prolong the vase life of A. holosericea (Table C and D). As compared to neutral NS and acid NS, ionic NS gave deposits upon reaction with NaCl. This precipitate may be indicative of free silver ion (Ag + ) in solution, which in turn may have been associated with the phytotoxicity observed with both vase and pulse treatments of ionic NS, even when the concentration was low (Table C and D). Although the 5 mg L ionic NS pulse treatment improved RFW and relative water uptake (objectively assessed; data not presented), it did not extend vase life (subjectively evaluated) because black staining in leaf veins constituted a loss of visual quality (data not shown). Phytotoxicity may have arisen because ionic NS might be rapidly taken up and accumulated by virtue of its small size ( 3 nm) and dispersibility in water (Fig. B). Neutral NS is comprised of relatively small sized particles (Fig. A) that seemingly prolong the vase life of A. holosericea predominantly by their antibacterial properties. Bacteria and their decay products are probably the most common cause of cut flower and foliage stem blockage leading to tissue water deficit (van Doorn, 997). Also, microorganisms potentially may secrete pectic enzymes, toxic compounds and/or produce ethylene, thereby accelerating senescence (Williamson et al., ). Loubaud and van Doorn () reported that stem blockage in Viburnum opulus Roseum and Rosa Red One was principally due to living bacteria and their decay products. van Doorn (997) also showed that bacterial suspensions at concentrations of 7 CFU ml or higher significantly reduced hydraulic conductance and vase life of the cut flowers. Silver nanoparticles are efficient antimicrobially (Rai et al., 9). For both pulse and vase treatments, the Ag contents at the stem-ends of A. holosericea were higher than in other parts of the stem (Fig. 3), suggesting that accumulation of neutral NS at the stem-end may inhibit bacteria. A strong antibacterial effect of neutral NS in both the vase solution and stem-ends of cut A. holosericea was recorded (Fig. E and F) in association with increased water uptake (Fig. C), maintained hydraulic conductance (Fig. D) and enhanced RFW (Fig. B) relative to the controls. In some experiments, the neutral NS vase treatment caused phytotoxicity to the foliage and at the stem-end, but only close to the end of the vase life evaluation period. In this context, it still maintained ornamental quality and extended A. holosericea vase life to around twice that of the control. An antibacterial effect of neutral NS is in agreement with the result of our previous study with gerbera (Liu et al., 9a). As a vase solution treatment, the ph of acidic NS was.5 in the beginning rising to 5. at day 3 (data not presented). As a pulse treatment, the ph of acidic NS was.5 during the pulse period. Acidic solutions are generally considered to have positive effects for most cut flowers in terms of enhancing vase solution flow rates in the stems and thereby prolonging vase life (Williamson and Milburn, 995; van Doorn and Cruz, ; Teixeira-da-Silva, 3). Pulsing with the acidic antioxidants l-ascorbic acid and benzoic acid (ph < ) could delay cut chrysanthemum leaf wilting (van Doorn and Cruz, ). Likewise, Williamson and Milburn (995) determined that provision of citric acid can extend the vase life of A. amoena through improved hydraulic conductance. As compared with neutral NS, acidic NS was less efficient in inhibiting bacteria, possibly due to its use at a lower concentration. Pulse treatment inhibited bacteria during the pulsing period, but thereafter gave no significant difference as compared to the control in either the vase solution or stem-ends (Fig. E and F). Nonetheless, acidic NS still had some effect in maintaining hydraulic conductance (Fig. D), RFW (Fig. B) and relative water uptake (Fig. C) and, in these regards, had no significant difference as compared to neutral NS pulse treatment. These observations suggest that its acidic property may have a positive effect for A. holosericea. Stem blockage usually occurs at the stem-end (van Doorn, 997; Williamson et al., ; van Doorn and Vaslier, ; He et al., 6). van Doorn and Vaslier () reported that stem blockage initially located in the basal cm of cut chrysanthemum flowers

J. Liu et al. / Postharvest Biology and Technology 66 () 8 5 Vase life (d) Relative fresh weight (% initial FW) Relative water uptake (g/g initial FW) 8 6 9 8 7 6 5.5..3.. A 6. a.5 b.3 b Neutral-NS vase B C Acidic-NS pulse LSD.5 (n=) LSD.5 (n=) 3 5 6 7 8 9 Vase period (days) Hydraulic conductance (ml cm -3 h - ) Number of bacteria (log CFU ml - ) 5 3 3 7 6 5 3 D E F LSD.5 LSD.5 LSD.5 Vase period (days) Acidic-NS pulse 3 5 6 7 8 9 Fig.. Vase life (A; values are means ± SE and different small letter indicate significant difference from control at P.5, n = ), and relative fresh weight (B; n = ), relative water uptake (C; n = ), changes in hydraulic conductance (D; n = 3), changes in bacterial population in vase solutions (E; n = 3) and stem-ends (F; n = 3) over time in cut A. holosericea with neutral NS ( mg L ) or acidic NS ( mg L ) pulse treatments. The vertical bars indicate the LSD.5 for treatment comparisons. (Dendranthema grandiflora Vyking ). He et al. (6) studied Grevillea Crimson Yul-lo and reported similar findings such that the hydraulic conductance of the segment immediately above the basal segment ( cm) was consistently higher throughout the vase life. Neutral NS treatments and acidic NS treatment helped to maintain hydraulic conductance at the stem-end and after day, and the difference was significant as compared to the DI control (Figs. D and D). Since the control stems began to lose quality after day, the differential results suggest that stem blockage is directly related to vase life of A. holosericea and that both the proposed neutral NS antibacterial effect and acidic NS acid effectly alleviate stem-end blockage and thereby prolong vase life. Besides antibacterial and acidic effects, NS could act as an antiethylene agent. Ag + is an effective ethylene action inhibitor (Beyer, 976; Veen, 979). Kim et al. (5) suggested that NS acted as anti-ethylene agent on cut Asiatic hybrid Lilium Dream Land and Oriental hybrid Lilium Sibera, although they did not study its effects in isolation from chitosan. However, unlike Lilium and based on our unpublished work, A. holosericea is relatively ethylene insensitive. In summary, pulse and vase solution treatments with suitable concentrations of neutral NS ( mg L for pulse and mg L for vase) or acidic NS (5 mg L for pulse and.5 mg L for vase) solutions evidently inhibit the growth of bacteria in the vase solution and thereby delay blockage (i.e., maintain hydraulic conductance) in the stem-end of cut A. holosericea foliage to prolong its vase life. Treatments with any of the three kinds of NS helped maintain RFW and relative water uptake. NS pulse treatments in particular could be a potential postharvest technology for commercial application to cut Acacia. The safe pulse solution concentrations for neutral and for acidic NS were < mg L and <5 mg L, respectively. Acknowledgements This research was supported by grants from the Natural Science Foundation of China (no. 37759 and no. 3789), the Natural Science Foundation of Guangdong Province (no. 855 and no. 5535), and the Science and Technology Foundations of Guangzhou, China (no. 8). Access for transmission electron microscopy was kindly provided by the Centre for Microscopy and Microanalysis (CMM) at The University of Queensland, Australia. The authors thank Richard Webb from CMM for assistance with the electron microscopy. Katherine Raymont and Allan Lisle of The University of Queensland are also gratefully acknowledged for their assistance with Ag analyses and with statistical analyses, respectively.

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