Arboriculture & Urban Forestry 42(5): September 2016 MATERIALS AND METHODS Experiment 1: Carbon Uptake and Storage Under Optimal Water Avail- ability In autumn 2010, 84 uniform, two-year-old shrubs (12 per species) of Arbutus unedo, Elaeagnus × ebbingei, Laurus nobilis, Ligustrum japoni- cum, Photinia × fraseri, Viburnum lucidum, and Viburnum tinus, were potted in 3L containers. The substrate was a peat:pumice mixture 4:1, (v/v), added with 3 kg m-3 of a controlled release fertilizer (Osmocote Exact, 18-2,5-7, 8–9 months, Everris, Intl. B.B, Geldermalsen, The Netherlands). Shrubs were arranged in a randomized block design, with six blocks of two shrubs per species in each block, and grown outside in Sesto Fiorentino (43.816652 N, 11.199673 E), Florence, Italy, un- der full sunlight. Average maximum temperature of the area from 01 June to 01 September, calcu- lated over the last 30 years, is 30.2°C with peaks of 42°C. The average rainfall (June to September for the same period) is 149.8 mm (Wikipedia 2016). Shrubs were irrigated daily to container capacity to avoid water stress for the entire experimental period. Daily trends of net photosynthesis and transpira- assimilation and transpiration for all 12 days of mea- surement (Gebhardt el al. 2014). Mean daily assimi- lated CO2 mmol H2 per unit leaf area was obtained applying ter, applying an integral analysis to calculate the area underlying the curve. To obtain daily CO2 daily net CO2 assimilation versus time and, thereaf- assimila- leaf area of each species was multiplied per the total leaf area of shrubs, assuming that self-shading was not relevant because of the small size of the shrubs. Biomass measurements were carried out at the beginning and at the end of the experiment tion per plant, the mean CO2 assimilation per unit as reported by Fini et al. (2012). In detail, total assimilated CO2 integral analysis of the daily CO2 assimilation curve was estimated by plotting the mean ance on 18 current-season, fully expanded leaves per species. Water use efficiency (WUE, µmol CO2 tion were measured for 12 consecutive days during summer 2011, from 9:00 am to 6:30 pm, using an infrared gas analyzer (Ciras 2, PP-system, Ames- bury, Massachusetts, U.S.). Measurements were conducted at 380 ppm of CO2 and ambient irradi- / O) was calculated as the ratio between CO2 331 on four shrubs per species. In November 2011, shrubs were cut off at the root flare and roots were cleaned from the medium using an air flush. All leaves were separated from stems and imme- diately scanned with an A3 scanner. An image analysis software (Image Tool 1.3, UTHSCSA) was then used to measure leaf area. To determine dry weight, leaves, stems, and roots were oven dried at 70°C until constant weight was reached (≈72 hours). Root-to-shoot ratio was calculated as the ratio between root dry weight and leaves + stem dry weight (Fini et al. 2011). Biomass alloca- tion (%) was calculated as the change in dry mass of a specific plant organ (stems, roots, and leaves) over the change in dry mass of the whole plant (stems + leaves + roots) with the aim of identify- ing the species that allocated and stored more car- bon into more durable organs compared to leaves. Experiment 2: Carbon Uptake Under Drought Stress In October 2011, 144 three-year-old shrubs of the same species used in Experiment 1 (24 shrubs per species; Laurus nobilis was not used), were pot- ted in 5L containers. Experimental site, substrate, and fertilization were the same as in Experiment 1, but a novofol foil (see Fini et al. 2012) was used to protect shrubs from undesired rainfall. Shrubs were arranged in a randomized block design of three blocks with eight shrubs per species in each block. Container capacity, wilting point, and water- holding capacity of the substrate were determined using a gravimetric method (Sammons and Struve 2008) as described in other works (Fini et al. 2011; Fini et al. 2013). All shrubs were watered daily to container capacity (CC) until August, and then exposed to water stress according to the follow- ing treatments: 1) WW, 72 shrubs (four shrubs per species per block) were watered daily to container capacity throughout the experiment; and 2) WS, (72 shrubs), water withheld from four shrubs per species for two periods of five days each (drought phase 1 and 2), separated by 14 days of partial re- lief. During partial relief the substrate moisture was maintained at 30% of CC, corresponding to mild to moderate drought (Ruiz-Sanchez et al. 2000; Fini et al. 2011) (Figure 4A). Physiological parameters were measured six times during the 24 days of the water stress experiment according to the following plan: ©2016 International Society of Arboriculture
September 2016
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