Physiological performance and growth of Viburnum tinus L. on phytoremediated sediments for plant nursing purpose

Abstract

Sediments are fundamental resources for productive activities like plant nursing, which are also likely to be responsible of their loss. In contrast, other activities like the dredging of canals and waterways involve the extraction and continuous accumulation of sediments. Most dredged sediments are polluted, and need to be stocked and transported to landfills, with extremely high costs for transport and management. To address these problems, a low-cost remediation methodology was previously developed to decontaminate sediments which were tested for use in plant nursery field plantations located in Pistoia (Italy). The phytoremediated sediments were mixed in percentages of 33% and 50% with alluvial soil, which itself was used as control. We studied the characteristics of these mixtures, and the physiological response and growth of Viburnum tinus L. grown on each substrate, as well as its corresponding root ball. Substrates with sediments showed quick water infiltration and no waterlogging, in sharp contrast to what was observed in autumn in the control. Despite a rainy summer, V. tinus demonstrated a good acclimation to the different substrates, showing the lowest leaf water potentials in mixed substrates and no signs of stress. No differences in leaf carbon assimilation or transpiration were observed among substrates, while in late August plants grown on substrates with sediments showed a higher performance index for energy conservation from photons absorbed by PSII to the reduction of intersystem electron acceptors. In the 50% mixture, there was also an enhancement of electron transport from PSII to PSI. Moreover, no differences in growth and biomass were found. Plants in all substrates showed some thin-root mortality, likely due to the persistent rainfall, though a higher number of plants with dead roots was observed in control. Thanks to the dense and fibrous root apparatus of V. tinus, the mixture with 33% sediments produced satisfactory results even for the root ball, resulting in less deformation and a lower breakage percentage.

Introduction

Sediments are a dynamic and essential part of water basins, as well as fundamental resources for many productive activities, that can be also responsible of their depletion. For instance, the estimate of soil loss in plant nursing (especially due to the extraction of plants from field plantations) is about 5.2 millions cubic meters every year in Europe (Marzialetti P, personal communication). The net loss of soil at the basin level, however, may be moderated to the extent that this material is replenished using agricultural soil.

In contrast, other productive activities such as transport on waterways are so constrained by the accumulation of sediments that regular dredging is required to safeguard ship passage. According to another estimate, about 200 millions cubic meters of sediments are dredged every year in Europe, of which 65% are contaminated by heavy metals, hydrophobic organic compounds and hydrocarbons ([37]). Contaminated dredged sediments have to be stocked in confined disposal facilities, and then either transported at enormous cost to landfills according to the European Waste Framework Directive (Directive 2008/98/EC), or transformed ([33], [9]).

The European Life+ project CleanSed (“Innovative integrated methodology for the use of decontaminated river sediments in plant nursing and road building”) addresses these two issues by applying an effective methodology (developed in the Agriport project) for the decontamination and amendment of sediments ([23], [12]), and testing the remediated sediments for the plant nursery sector.

The Agriport methodology showed effective results regarding the decontamination of sediments dredged from the Navicelli canal (Pisa) and Livorno harbour. Heavy metals and petroleum hydrocarbons were removed from these soils by (a) modifying their physical and chemical properties with the addition of sandy soil (30% by volume) and a layer of compost, and (2) applying phytoremediation for two years with a combination of Mediterranean species. Afterwards, during the Cleansed project, the phytoremediated sediments were treated with land farming. The whole process was found effective in the removal of all contaminants, meeting the legal limits for industrial and commercial use ([12], [29]).

Based on this experience, we hypothesize that decontaminated sediments using the methodologies described above could replace the agricultural soil that is typically used to recover the soil lost in field-plantation nurseries. To test the suitability of such phytoremediated sediments, an experimental plan was set up and implemented in Pistoia, a province leader of plant nursing in Italy.

The aim of the study was to assess the physical, chemical and hydraulic properties of different substrates with varying percentages of phytoremediated sediments mixed with alluvial soil of the Pistoia plain, and to assess the growth performance of commonly used ornamental shrub species such as Viburnum tinus L.

Material and methods

Experimental design

The experiment was set up in the spring 2014 at the Center for Experimental Plant Nursing (Ce.Spe.Vi.) in Pistoia (Italy), which is a prominent center for plant nursing, especially for trees and shrubs in field plantations. The area is highly suitable to this sector because of its climate and soil characteristics. The climate is Mediterranean semi-continental, with maximum and minimum temperatures of 31.6 °C in August and 2.2 °C in January, respectively, and an average annual rainfall of about 1200 mm (climate series 1981-2010 - ⇒ http:/­/­www.­lamma.­rete.­toscana.­it/­clima-e-energia/­climatologia/­clima-pistoia).

The alluvial soil of the Pistoia plain, used in the experimental setup for control plots, is generally composed of loamy silt, with poor structural stability, high water retention and tendency to compaction.

Five cubic meters of sediments dredged from the Navicelli canal (Pisa, Italy) were decontaminated using the Agriport methodology, until heavy metal concentrations reached values below the legal limits for industrial and civil use (Dlgs. 152/2006, D.M. 161/2012 and Dlgs. 217/2006 - [12]). Land farming was then applied during the CleanSed project, to further lower the contaminant concentrations ([29]). The phytoremediated sediments were then mixed with the alluvial soil of the Pistoia plain that served in its original form as the control (CTL), in percentages of 33% (T33) and 50% (T50) by volume.

The three soil mixtures (CTL, T33 and T50) were arranged in six wooden-frame boxes (two plots each) of 3 m³ (3 m long, 2 m wide, 0.5 m deep) and insulated from the ground with non-permeable fabric. In each plot, 24 one-year-old plants (8 per species) of three ornamental evergreen shrub species (Photinia × fraseri var. Red Robin, Eleagnus macrophylla L., and Viburnum tinus L.) were planted in May 2014 at 50 cm distance from each other, according the traditional planting pattern. However, since Photinia × fraseri and Eleagnus macrophylla showed similar physiological response and final biomass across the three substrates, in this paper we present only the results for Viburnum tinus.

An irrigation system with drip emitters (one per plant) delivered from 0.5 to 1 L of water per day. Fertilization was done on July 20, using 50 g of Osmocote 8M (16N-11P-10K +2MgO) per plant.

Weather conditions

Weather parameters were recorded every 15 minutes at the Meteorological station in Ce.Spe.Vi. (long. 10° 54′ E, lat. 43° 56′ N, 60 m a.s.l.) equipped with a CR10 data logger (Campbell Scientific Campbell Park, Shepshed, Leicestershire, UK) and sensors for the main parameters. Summer 2014 recorded 50% more rainfall than the long-term climatic average, with very short dry periods; in particular, July was the wettest month with 187.6 mm of rain. The monthly mean temperature was around 23 °C from June to August whilst the maximum air temperatures were rarely above 35 °C, except in June (Fig. 1).

Fig. 1 - Weather conditions during the experimental period. (PP): Rainfall; (T Mean): Mean Air Temperature; (T Min): Minimum Air Temperature; (T Max): Maximum Air Temperature.

Results

Physical, chemical and hydrological characteristics of the substrates

The physical, chemical and hydrological characteristics of the three substrates are summarized in Tab. 1. The three substrates were characterized by a sandy loam texture (USDA classification) with CTL richer in silt compared to T33 and T50, which were richer in clay. In addition, T33 and T50 had a lower bulk density than CTL, which in contrast was more compact, and they were also more heterogeneous and looser in structure due to the presence of clay aggregates (reaching > 10 cm in diameter).

Tab. 1 - Physical and chemical characteristics of the three substrates. (T33): 33% phytoremediated sediments mixed with alluvial soil; (T50): 50% phytoremediated sediments mixed with alluvial soil; (CTL): 100% alluvial soil). Mean values and standard deviations are given. Different letters within the same row indicate significant differences between means of the three substrates after Tukey’s test (P < 0.05, N=3 for each substrate).

Parameter	T33	T50	CTL
Clay:Silt:Sand	6.6 : 39.4 : 54	10.5 : 34.4 : 55.1	10.3 : 34.9 : 54.8
pH	8.15 ± 0.05 b	8.06 ± 0.02 b	8.34 ± 0.05 a
Electric Conductivity (µS cm-1)	361.33 ± 35.22 b	597.33 ± 49.81 a	75.50 ± 3.16 c
Total C (g/100g)	2.70 ± 0.03 b	3.20 ± 0.22 a	1.90 ± 0.05 c
Total N (g/100g)	0.20 ± 0.03 a	0.20 ± 0.01 a	0.10 ± 0.01 b
C:N	17.60 ± 3.30	17.10 ± 0.40	14.30 ± 1.40
P (g/100g)	0.06 ± 0.00 a	0.06 ± 0.00 a	0.05 ± 0.00 b
K (g/100g)	1.00 ± 0.01	0.99 ± 0.06	0.96 ± 0.10
Ca (g/100g)	1.65 ± 0.01 b	2.29 ± 0.13 a	0.63 ± 0.05 c
Mg (g/100g)	1.06 ± 0.09	1.02 ± 0.09	1.00 ± 0.14
S (g/100g)	0.14 ± 0.00 b	0.23 ± 0.02 a	0.02 ± 0.00 c
NO3- (mg L-1)	9.10 ± 0.27 a	9.80 ± 0.29 a	7.10 ± 0.79 b
SO42- (mg L-1)	120.10 ± 7.98 b	264.70 ± 28.73 a	3.70 ± 0.94 c
PO43- (mg L-1)	0.10 ± 0.03 b	0.00 ± 0.00 c	0.30 ± 0.08 a
Bulk density (mg m-3)	1.10 ± 0.07 b	1.06 ± 0.05 b	1.15 ± 0.08 a
Water infiltration (mm hr-1)	5.50 ± 2.60 b	61.00 ± 27.00 a	0.14 ± 0.11 c

In terms of pH, all three substrates were alkaline, with the highest values in CTL, though it had the lowest concentration of Ca. EC showed higher values in the presence of phytoremediated sediments, especially in T50. Both treatments (T33 and T50) had a good C:N ratio, but were poor in macronutrients like P and K.

Regarding the hydraulic properties, water infiltration was very quick in T50 and T33, whereas CTL showed the lowest values. In summertime (after an initial inconsistency required changing the position of two sensors), the daily soil moisture at a depth of 20 cm (Fig. 2) was found to be slightly higher in CTL and T50 (ranging between 16.4% and 31.1 % in CTL and between 13.7% and 27 % in T50) than in T33 (range: 12.1% to 22.6%), with a pattern more linked to the rain occurrence. In the fall, soil moisture showed the highest values in all substrates, reaching saturation and waterlogging in CTL (data not shown). Both T50 and T33 showed a slight decreasing trend after the peak on November 18^th, and T33 had the lowest values.

Fig. 2 - Daily values of soil moisture (%) recorded by the Decagon 5 TE sensors placed at a depth of 20 cm. Data were recorded continuously from the 1st of August until the end of 2014. The sensors were removed temporarily during the extraction of plants (October-November).

Plant physiological performance

The lowest Ψp values were observed on July 18, with significant differences between CTL and the sediment treatments (T33 and T50). The latter recorded the lowest values (Fig. 3A), though on September 23rd after a period of rain, values increased. The lowest values of Ψmin were -2.3 MPa in T50 (measured on July 18) and -2.2 MPa in T33 on August 18, in accordance with the lower soil moisture value at the time of measurement (Fig. 3B, Tab. 2).

Fig. 3 - (A) Pre-dawn (Ψp) and (B) minimum (Ψmin) water potential in Viburnum tinus during the experiment. Mean values and standard deviations are shown for the three substrates (CTL, T33, T50). Significant differences resulted from the Tukey test at P[ less than ]0.05 are shown by letters in each date of measurement (N=18).

Tab. 2 - Soil moisture at the time of leaf water potential measurement, measured by Decagon 5 TE sensor (5TE), positioned at a depth of 20 cm in each plot. For each substrate the average value between two plots is given.

Measurement	CTL	T33	T50
18/08/2014 Hr. 4-5	20.1	16.3	18.7
18/08/2014 Hr. 13-14	29.5	21.4	26.4
23/09/2014 Hr. 4-5	20.2	16.2	18.4
23/09/2014 Hr. 13-14	28.2	20.1	25.7

In CTL V. tinus showed higher Ψp than in T33 and T50 and similar maximum values of ΔΨ (e.g., the difference between pre-dawn and minimum water potential) in the three substrates. The relationship between ΔΨ and Ψp given by the regression analysis revealed a linear dependence in CTL (ΔΨ = 2.65 Ψp + 2.3, R² = 0.45, P=0.002) and T33 (ΔΨ = 5.7 Ψp + 3.2, R² = 0.68, P=0.003), whilst in T50 the relationship was weak (ΔΨ = -2.26 Ψp + 1.25, R² = 0.0654). The intercept of the upper boundary of the points, represented by a straight line, ranged between 3.6 MPa in CTL to 5.6 MPa in T33 with higher slopes in T33 and T50 (Tab. 3).

Tab. 3 - Minimum absolute values of pre-dawn (Ψp, MPa) and minimum leaf water potential (Ψm, MPa), intercept value (Ψmax, MPa) and slope of the regression analysis between pre-dawn leaf water potential (Ψp) and the difference between predawn and minimum water potential for the three substrates (Ψ), according Rambal’s approach ([35]).

Substrate	Ψp	Ψm	Intercept	Slope
CTL	-0.21	-2.1	3.7	-9.6
T33	-0.25	-2.2	5.6	-13.0
T50	-0.26	-2.3	5.4	-22.0

On each date, V. tinus showed similar photosynthesis as well as leaf transpiration in all substrates. The highest rates of photosynthesis were observed on July 31, compared to the previous dates (P).

Fig. 4 - (A) Net photosynthesis (PN) and (B) transpiration (Tr) in Viburnum tinus during the experiment. Mean values and standard deviations are shown for the three substrates (CTL, T33, T50). Different small-capital letters indicate significant differences between means after Tukey’s test (p[ less than ]0.05, N=6 in each substrate). No differences were found between substrates at the same date, while significant differences were detected between similar substrates at different dates.

No significant difference was found between the substrates on any given date of measurement for F₀, Fm, Φ_PO, ΔV_IP and PI_ABS. However in T33, in late August, PI_ABS and ΔV_IP became higher on average than CTL (ca. 25% and 15%, respectively) although with high variability within the samples (Fig. 5).

Fig. 5 - “Spider plot” of the parameters of direct fluorescence during the experiment, expressed as percentage relative to the control (CTL=100, black polygon; N=16 in each substrate). (F0): initial fluorescence; (Fm): maximum fluorescence; ([phi]P0): maximum quantum yield of primary photochemistry in a dark-adapted leaf (i.e., probability of an absorbed photon to be trapped by the PSII reaction center); ([delta]VIP): probability of a PSII trapped electron to be transferred to PSI acceptors (i.e., efficiency of electron transport around the PSI to reduce the final acceptors of the electron transport chain); (PIABS): performance index of energy conservation from photons absorbed by PSII to the reduction of intersystem electron acceptors in the electron transport chain. See Tab. 3 for the significance of differences among substrates.

Discussion

Field-grown plant production presents many advantages, like low start-up costs and reduced maintenance during the growing period ([14]), but it is responsible for a consistent loss of topsoil when plants are transplanted from the nursery to their final location. For instance, in Europe the loss of soil due to plant nursery production is estimated at around 5.6 million cubic meters every year (Marzialetti P, personal communication). When new plantations are set up, suitable new soil is usually needed for augmenting the existing soil and improving its quality. In general, new soil is extracted from agricultural fields that undergo land use change.

In this experiment we tested the use of polluted river sediments, dredged from a waterway and remediated through low-cost and sustainable methodologies (Agriport and CleanSed), in nursery field-grown plant production. The Agriport methodology, followed by land-farming (during the Life+ Cleansed project), has proved to be effective in lowering the concentration of contaminants and producing a substrate suitable for agricultural purposes. In fact, these techniques stimulated the activation of microbial biomass (increase in biochemical activity) and reduced the concentration of heavy metals ([12], [13]), and they similarly reduced the concentration of residual organic contamination (by 15%), while increasing the homogenization of the substrate ([29]). The remediated sediments, with levels of contaminants below the limits set by Italian Law 152/06, were then tested for field-grown nursery production.

We looked at the properties of substrates containing remediated sediments in different percentages (0%, 33%, 50%), mixed with alluvial soil from the plain of Pistoia where the experiment was set up (a region known for its high-volume nursery production and for the outstanding growth performance of its cultivated ornamental species). Because the remediated sediments were characterized not only by a loose structure and sandy loam texture but also by a high degree of heterogeneity in terms of aggregate size, the position of soil moisture sensors in these mixed substrates had to be changed at the beginning of the experiment. However, later on in the fall, low evaporation and wet ground due to the persistent rains caused waterlogging in the control but not in substrates with the phytoremediated sediments. The improved drainage properties of these substrates were also demonstrated by very high water infiltration rates in the two sediment-containing mixtures, as opposed to minimal rates in the control.

Regarding the physiological performance of V. tinus, the analysis of the water status showed that in substrates with sediments plants had the lowest values of Ψp and Ψm along with the lower values of soil moisture. From the analysis of the relationship between ΔΨ and Ψp, we observed a strong correlation between the two parameters in T33 and CTL and higher values of the intercept (ΔΨ is the maximal daily amplitude when the soil water content is at field capacity and the evaporative demand high) and slopes of the regression in T33 and T50. These results suggest likely higher transpiration rates ([35], [3]), but also a greater control of leaf transpiration because at minimum changes in pre-dawn water potential the amplitude ΔΨ approaches zero. This is defined as “regulatory behavior”, which is typical of the xeric Mediterranean species ([22], [35]).

However, the plants were not exposed to water deficit, and the irrigation and the rainy season provided ample water. Leaf gas exchanges (carbon absorption and water transpiration) that were measured in the morning (at lower evaporative demand compared to midday) also did not show differences between the three substrates. Leaf transpiration recorded the lowest values on August 31, though without changes in water use efficiency (data not shown). The ecological conditions in which the plants grow play an important role in the performance of the photosystem ([32], [11]). Indeed, it is known that high temperatures associated with water stress stimulate the photosystem efficiency through a quicker electron transport between PSII and PSI ([5]). However, in this study, plants were watered, and in addition the season was particularly rainy. The photochemical efficiency of V. tinus was similar in the three substrates. The polyphasic chlorophyll a fluorescence transient with OJIP steps, which gives information about the efficiency of PSII photochemistry, in general did not evidence abiotic stress conditions. Φ_PO was on average around 0.75 in all substrates, similarly to other experiments ([15]), and it did not change throughout the summer. On August 31, plants grown in sediment mixtures also showed higher performance index values for energy conservation from photons absorbed by PSII to the reduction of intersystem electron acceptors, likely associated with a better energy transfer by open reaction centers at the PSII. In T50, there was also an enhancement of electron transport from PSII to PSI (ΔV_IP), connected to a quicker ability of ferredoxin reduction, used to reduce NADP+ into NADPH essential for the Calvin cycle.

On that date, air temperature at the time of measurement was 29 °C, likely below the critical value limiting photosynthesis in Mediterranean species ([6]), and CO₂ adsorption was similar to the previous dates. This, being associated with the enhanced electron transport as well as PI_ABS, would positively affect the CO₂ fixation ([27], [42]).

The long term effects of the physiological behavior are then noticeable on plant size and growth. The above-ground development is crucial for its growth and commercial purposes, as the below-ground apparatus and root ball guarantee plant survival at harvest and after transplanting ([10], [16]). V. tinus showed a good aboveground development, with fairly dense foliage and branching, with no difference between substrates. Moreover, V. tinus has a dense shallow root apparatus, likely with a large number of active root tips, which would favor regeneration and transplanting success ([10]). Despite this, we observed mortality of thin roots ().

Trees with fibrous root systems grow better in a looser structure, and afterwards are more successful in transplanting than those with coarse roots due to a higher regeneration potential ([40]). At the same time, a dense root apparatus reduces the level of root ball breakage. Root ball is important also to determine the depth level of the structural roots ([40]), though it may interfere with the time new roots need to explore the soil outside the root ball ([18]).

In this experiment, the substrates with sediments had a looser structure than the control, and showed a level of breakage of the root ball particularly high in T50 (with more than 60% of breakage). Root ball compaction is particularly important for preventing the loss of fine roots by mechanical injuries and desiccation in digging and preparing balled-and-burlapped plants, though the length of exposure to air between extraction and transplanting is also relevant ([17], [30], [31]). Indeed, soil removal from a root system can result in a loss of up to 90% of absorbing roots ([25], [19], [28]). Roots are extremely important because in close contact with soil, their loss and damage diminish the water uptake capability of plants ([43], [26]). However, the technique of planting bare root plants, together with accurately applying proper care, undoubtedly cheaper than transplanting a balled and burlapped plant or one in a container ([45]), has also reported positive results, with high rates of survival after transplanting ([21], [2], [4]), so that the sediments may be considered suitable for this kind of technique.

Finally, this study showed that a substrate with 33% remediated sediments mixed with alluvial soil characteristic of the Pistoia plain, resulted in conditions suitable for cultivating V. tinus. If we considered this percentage suitable also for other species ([41]), we could estimate for Pistoia province a soil recovery of 600-1200 cubic meters per year, depending on the species cultivated. In addition, due to the loamy silt texture of the plain soil ([1]), the remediated sediments would contribute to reducing the probability of anoxic conditions which impair the quality of the root systems.

Conclusions

This experiment focused on the use of phytoremediated sediments through the Agriport and Cleansed methodologies, in plant nursing for field-grown plantations. The remediated sediments, added in percentages of 33% and 50% by volume to a loam sandy soil with a higher content of silt (control), significantly improved water drainage. In the first year of growth, the species described here (V. tinus) showed physiological performance and final biomass similar to the control. In mixed substrates as well in the control, the plants showed some root mortality, although in the control the frequency was higher. However, thanks also to the dense and fibrous root apparatus, a sediment concentration up to 33% proved satisfactory even for the root ball, in terms of compaction and likely breakage.

Acknowledgements

The project was funded by the European Commission under the programme Life+ 2013-2016 (LIFE12ENV/IT/00652). We thank Dr. Paolo Marzialetti and Nicola Petrucciani (Centro Sperimentale per il Vivaismo, Pistoia, Italy) for their valuable suggestions, logistical support and assistance with plantation.

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