Materials and Methods 1021 Microcosm Style Experiments 10211 Experimental Setup

The microcosm-scale experiment was carried out in a greenhouse facility of the Mediterranean University of Reggio Calabria. The microcosms consisted of 9.5 L PVC pipes (30 cm height x 20 cm diameter) filled with approximately 13 kg of a soil/perlite (80/20, v/v) mixture. The soil was a clay loam [sand 36.0%, silt 32.0%, clay 32.0%; bulk density 1.23 ± 0.04 kg dm-3; pH-H2O 7.2 ± 0.2; pHKa 6.4 ± 0.1; total organic C (TOC) 19.3 ± 0.4 g (kg dw [dry weight basis] soil)-1; total N (TN) 1.8 ± 0.2 g kg-1; C/N ratio 10.7; NH4+-N 17.1 ± 1.0 mg kg-1; NO3--N 13.0 ± 1.0 mg kg-1; Olsen P 18.3 ± 2.3 mg kg-1; total CaCO3 8.4 ± 1.0 g kg-1; active CaCO3 3.9 ± 0.2 g kg-1; cation exchange capacity (CEC) 17.1 ± 1.7 cmol(+) kg-1; electrical conductivity (EC at 25°C) 0.165 ± 0.004 dS m-1 (1 dS m-1 = 1,000 mS cm-1)] sampled from the surface (0-15 cm) Ap horizon of an agricultural field located in the Agricultural Experimental Station of the Mediterranean University of Reggio Calabria. After coarse sieving (4 mm mesh), the freshly collected soil was thoroughly mixed with commercial perlite (Agrilit®3, purchased from Perlite Italiana s.r.l., Milano, I) at 70/30 (v/v) soil/perlite ratio. Perlite properties were: particle size 2-5 mm, bulk density 100 ± 20 kg m-3, pH 6.5-7.5, CEC 0.79 cmol(+) kg-1, EC 0.020 dS m-1, water content at -1 kPa 26.30%, water availability 12.97%. Microcosms were closed at the bottom by a thin layer of nylon stocking material, separated from the soil/perlite mixture by a 1-cm drainage layer of water-washed quartz sand. Compost from agricultural by-products (pH-H2O 8.7, EC1:10 3.357 dS m-1, TOC 28.75%, TN 2.24%, C:N ratio 12.8) was provided by GESENU (Perugia, Italy). The water-soluble iron-porphyrin [meso-tetra(2,6-dichloro-3-sulfonatophenyl) porphyrinate of Fe (III)chloride], here referred to as the biomimetic catalyst (CAT), was synthesized according to the procedure of Traylor et al. (1984) as modified by Piccolo et al. (2005).

The microcosms were randomly set up in a 2 x 4 experimental block (6 times replicated) with two plant treatments (with and without maize plants) and four soil treatments, namely:

- Soil/perlite mixture without compost or synthetic iron-porphyrin, as control treatment (CONT).

- Soil/perlite mixture amended with compost at 2 kg m-2 rate (COM).

- Soil/perlite mixture added with 1 g m-2 synthetic iron-porphyrin (CAT); by assuming a porosity of 50% and a 15% volumetric water content at field capacity, the above CAT rate would have yielded an approximate concentration of 10 mM CAT in the soil solution.

- Soil/perlite mixture amended with compost (2 kg m-2) upon microcosms filling, and then surface sprayed (see below) with iron-porphyrin (1 g m-2) (COM + CAT) 10 days after microcosms filling.

The synthetic iron-porphyrin was surface sprayed upon CAT and COM + CAT treatments as a buffered solution (100 mM phosphate buffer, pH 7.0) soon after microcosm filling. De-ionized water was periodically added to soil microcosms to maintain the moisture between 13 and 15% of total soil volume.

Pre-germinated maize seeds (Zea mays L., var. Cecilia, kindly provided by Pioneer HI-Bred Italia s.r.l., Parma, Italy) were transplanted into soil microcosms 55 days after the beginning of the trial (one seedling per microcosm) and left growing for additional 21 days.

During the 76-day experimental period, greenhouse air temperature fluctuated between 12 and 25°C according to the day/night cycle and depending on external weather conditions, while soil temperature varied slightly within a range of 16-21°C. Plants Sampling and Root Morpho-Topological Analysis

At the end of experimental period, soil microcosms were destructively sampled and 21-days-old maize plants were gently separated into shoot and root, rinsed with de-ionized water, and their fresh weight was determined gravimetrically. Shoot dry weight (WS, g) was measured after drying in an oven at 70°C for 48 h. The root system was stained with 0.1% (w/v) toluidine blue O for 5 min and then scanned at a 600 dpi resolution (WinRhizo STD 1600, Instruments Regent Inc., Canada).

For topological analysis (Fitter 1986), the magnitude (m), the number of external links in the root system, and altitude (a), the number of links in the longest single path, were measured with WinRhizo Pro v. 4.0 software package (Instruments Regent Inc., Canada). These parameters allowed to calculate the topological index (TI = [(log a)/(log la)] (Glimskar 2000). TI values close to 1 indicate a "herringbone" root structure, where branching is largely confined to a main axis, whereas values close to 0.5 indicate a dichotomous root structure characterized by more random branching.

For morphological analysis, the length (LR, cm) and the volume (VR, cm3) of the whole root system and the total root length within diameter classes ("fine roots": 0-0.4, 0.4-0.8, and 0.8-1.2 mm; "coarse roots": 1.2-1.6,1.6-2.0, 2.0-2.4, 2.4-2.8, and >2.8 mm) were measured (WinRhizo software). Subsequently, root dry weight (WR, g) was determined after oven-drying (70° C) until a constant weight was reached. Total plant dry weight (WP, g) was obtained by summing WR and WS. Based on the measurements above, root fineness [(F = LR/VR), cm root length (cm3 root volume)-1] and tissue mass density [(TD = WR/VR), g root (cm3 root volume)-1], which represent structural root parameters, were calculated. These parameters are linked by the following relationship: LR = WR*(F/TD) (Ryser and Lambers 1995). Furthermore, root mass ratio [(RMR = WR/WP), g root (g plant)-1] was determined to evaluate the relative biomass allocated to roots. Soil Analysis

Soil respiration was periodically monitored by using a closed dynamic soil CO2-flux system (LI-8100 automated soil CO2 flux system, LI-COR Inc., Lincoln, Nebraska, USA) equipped with a 10-cm survey chamber for measuring soil CO2 efflux (mmol CO2 m-2 s1). Volumetric soil moisture (ECH2O EC-5 probe, Decagon Devices, Pullman, WA, USA) and soil temperature (Omega probe, Type E, LI-COR Inc.) were also monitored (at 5-cm depth) over time. Soil samples were collected at the beginning (day 0) and at the end of the experimental period (day 76). The pH, EC, TOC, and TN were determined using standard methods (Sparks 1996). Statistics

Soil variables, plant growth, and root morphological and topological parameters were firstly checked for deviations from normality (Kolmogorov-Smirnov test) and homogeneity of variances (Levene median test). Since the block effect was not significant (p > 0.05), the data were subjected to statistical analysis by using the Systat 12.0 software (Jandel Scientific, San Rafael, CA, USA). Tukey's post hoc test comparison (at p < 0.05) was applied for pairwise comparison of means. Data of Figs. 10.10 and 10.11 were statistically processed by two-way analysis of variance (ANOVA, CAT x COM) with repeated measures and three-way ANOVA, CAT x COM x plant with repeated measures, respectively, in order to give prominence to the main effect of the treatments (CAT, COM, and maize plant) alongside the variability given over time.

10.2.2 Laboratory Studies on Seed Germination and Post-germinative Processes

Direct in planta effects of CAT on seed germination and seedling establishment were evaluated on two fast growing and easily cultivable species amply used for laboratory studies, such as garden cress (Lepidium sativum L.) and carrot (Daucus carota L.). The used CAT concentrations were meant to be in the same range of those used for the microcosm experiments (see above); 0 (control), 3, 9, 27, 81, or 243 mM. These were obtained by diluting, in sterilized 10 mM phosphate buffer pH 6.0, the appropriate volumes taken from a sterilized stock of a 10 mM CAT solution in the same buffer. Previously sterilized seeds were germinated at 20°C in the dark in Petri dishes containing filter paper disks imbibed with CAT solutions at the concentrations stated above. Radicle protrusion was assumed to denote the completion of seed germination and the beginning of seedling establishment, i.e., the "zero time" for measuring root and shoot growth.

Chlorophyll a and b, as well as total carotenoids, were determined spectropho-tometrically on acetone/water (80/20, v/v) extracts of leaves, according to the method of Lichtenthaler et al. (1982).

Each reported result is the mean ± SE of at least ten measurements conducted for each of the 3-5 replicated independent experiments.

10.2.3 Laboratory Studies on Root Growth and Morphology

Previously sterilized seeds of A. thaliana ecotype Columbia (Col-0) were incubated in the dark at 4°C for 24 h in sterile distilled water. Seeds were then sown onto square Petri plates (10 cm x 10 cm) containing 40 mL of a sterile half-strength MS medium (Murashige and Skoog 1962), 3% (w/v) sucrose, 0.7% (w/v) plant agar, and final CAT concentrations of 0 (control), 3, 9, or 27 p.M obtained by diluting in sterilized 10 mM phosphate buffer pH 6.0 appropriate volumes taken from a sterilized stock 1 mM CAT solution in the same buffer. Petri dishes containing the germinating seeds were incubated at 25°C, with a photoperiod of 16 h light and 8 h darkness, in vertical position, to avoid root penetration in the medium. This allowed an easier measurement of the growth of the primary root and to visualize root hairs and lateral roots. Measurements were done after 5, 8, 11, and 14 days from seeds germination. Image analysis of the A. thaliana root apparatus was carried out as stated earlier. The root hair parameters, namely elongation zone, number, and length, were analysed with a stereomicroscope (Olympus MIC-D) at a 98 x magnification. The shoot analysis was performed by measuring the diameter and counting the number of leaf rosettes.

Each result is the mean ± SE of at least ten measurements conducted for each of the 3-5 replicated independent experiments.

10.3 Results and Discussion

10.3.1 Root Morpho-Topology in Microcosm-Scale Experiments

Roots can act as CO2 sink in soil by depositing both dead roots and photoassimilated C in the rhizosphere (Tresder et al. 2005). Conversely, roots can be considered biogenic sources of soil CO2 through root tissue respiration, and indirectly, stimulation of microbial activity. Beside this source/sink effect for CO2, roots also exert an indirect action on the rate and extent of SOM mineralization, often denoted as the rhizo-stimulated SOM-derived CO2 priming effects, through the release of easily decomposable C sources (Cheng and Kuzyakov 2005; Dijkstra and Cheng 2007; Cheng 2008).

Root morphology and topology represent primary factors for assessing the functional role of root system in the soil C dynamics. In fact, factors such as root length, surface area, diameter class, and topology, may contribute to CO2 source potentials, whereas other ones, such as root mass ratio, fineness, and tissue density, are related to CO2 sink capacity. In this respect, we analysed changes of these root parameters in response to CAT or COM amendments, as added alone or in combination.

The CAT treatment increased the total biomass of maize plants that, conversely, remained unchanged by the COM addition alone. However, the combination of the two treatments produced a synergic effect on total plant biomass (Fig. 10.1). The results mirrored the separate patterns observed for root and shoot dry weight (Fig. 10.2).


Fig. 10.1 Whole biomass (shoot + root) of maize plants (cv. Cecilia) growing in experimental microcosms filled with: control soil, CONT; compost-amended soil, COM; soil amended with iron-porphyrin, CAT; compost-amended and iron-porphyrin-treated soil, COM + CAT. Values are means (n = 6) ± standard deviation of the mean. Different letters denote statistically significant differences in respect to control (CONT; Tukey's test, at p < 0.05)


Fig. 10.1 Whole biomass (shoot + root) of maize plants (cv. Cecilia) growing in experimental microcosms filled with: control soil, CONT; compost-amended soil, COM; soil amended with iron-porphyrin, CAT; compost-amended and iron-porphyrin-treated soil, COM + CAT. Values are means (n = 6) ± standard deviation of the mean. Different letters denote statistically significant differences in respect to control (CONT; Tukey's test, at p < 0.05)

Plant root length was increased by COM amendment, though not significantly, while it was not influenced by CAT treatment. As already observed for biomass parameters (Figs. 10.1 and 10.2), a synergic effect was noticed when CAT and COM were added together to the microcosms (Fig. 10.3). Furthermore, COM amendment significantly increased the cumulative length of roots belonging to the fine roots classes (diameter range 0-0.4 mm), and even more so did the COM + CAT treatment (Fig. 10.4a). Conversely, CAT, either alone or in combination with COM, significantly increased the cumulative length of roots within the coarse class (diameter class, >1.2 mm), while larger diameter classes did not respond to COM addition alone (Fig. 10.4b).

Each of the two treatments alone, and even more their combination, tended to increase the root surface area (Fig. 10.5). By dissecting the treatments' effects for the surface area in respect to diameter classes, a pattern similar to that observed for root length was obtained (see above; compare Fig. 10.6 with Fig. 10.4). Nevertheless, no treatment modified the topological index of the maize root, whose architecture remained of the "herringbone" type (TI~1; data not shown).

Beside the morphological root features that potentially increase CO2 release from soil (soil as a CO2 source), we also evaluated those components of root morphology which, directly or indirectly, have the potential to enhance carbon storage in soil, thus promoting its role as CO2 sink. To such aim, we evaluated (1) the root mass



Fresh weight Dry weight



Fig. 10.2 Shoot (a) and root (b) biomass in maize plants in Fig. 10.1. Treatments acronyms and statistics as in Fig. 10.1



Fig. 10.2 Shoot (a) and root (b) biomass in maize plants in Fig. 10.1. Treatments acronyms and statistics as in Fig. 10.1

ratio (RMR), a component expressing the relative amount of biomass allocated to the root, and (2) the root fineness (F) and the root tissue density (TD), which are the root structural components. In particular, the RMR indicates the plant potential to allocate photosynthetically fixed carbon to soil, whereas the F and TD parameters provide indirect information on the "root biomass quality", that may have an impact on soil C stabilization after root degradation in soil. It has been observed that TD is negatively related to the root turnover rate (Ryser 1998), while it is positively related to the thickness of root cell walls, to the root sectional area (Wahl and Ryser 2000), and, finally, to the degree of exodermis lignification (Eissenstat and Achor,1999).

Fig. 10.3 Total root length in maize plants in Fig. 10.1. Treatments acronyms and statistics as in Fig. 10.1

Therefore, both a large TD and/or a low F indicate a high root lignification degree, whose alleged recalcitrance slows down root degradation in soil.

The results reported here suggest that the COM treatment, as compared to CAT, induced a greater biomass allocation to roots (larger RMR in Fig. 10.7). In term of structural components, the CAT treatment strongly reduced the fineness of the root system, implying the prevalence of root axes with large diameters, whereas this same parameter was significantly increased by COM, either alone or in combination with CAT (Fig. 10.8). Remarkably, both single treatments, as well as their combination, reduced root tissue density (Fig. 10.9). This, on the basis of previous considerations, would imply a decreased root recalcitrance to degradation, leading to an accelerated root turnover rate in soils amended with COM and/or treated with CAT.

All together, the above results appeared controversial, by showing a double role as source/sink of both CAT and COM amendments in soil C balance. On one hand, CAT increased the root surface area and length of coarse roots, possibly resulting in an enhanced surface contact between root and soil, that may stimulate CO2 emission from soil. On the other hand, the biomimetic catalyst reduced the fine roots length and, hence the release of root exudates, implying a decrease of supply to rhizospheric biota of promptly respirable substrates, and resulting in slowing down of CO2 emission rates from soil.

Likewise, compost amendment behaved similarly to CAT in increasing the root surface area and root length of fine roots, and both may have stimulated plant-derived CO2 release. However, COM concomitantly favored the increase of fine roots length, with consequent enhanced rhizodeposition, and larger biomass allocation to roots, thereby suggesting that compost may have promoted the role of roots as CO2 sink in soil. Consequently, the combined CAT + COM treatment exacerbated the contrasting role of the root system to act as either a source or a sink for the soil C balance.















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