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Open field experiment for the evaluation of Arundo donax ecotypes ecophysiology and yield as affected by soil water content
Industrial Crops & Products 140 (2019) 111630
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Open field experiment for the evaluation of Arundo donax ecotypes ecophysiology and yield as affected by soil water content
T
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Ezio Riggia, , Giovanni Avolaa, Giovanni Marinoa, Matthew Hawortha, Salvatore Luciano Cosentinob, Mauro Centrittoc a
National Research Council of Italy, Institute of BioEconomy, (CNR - IBE), via Paolo Gaifami 18, 95126, Catania, Italy Dipartimento di Agricoltura, Alimentazione e Ambiente (Di3A), Università degli Studi di Catania, via Valdisavoia 5, 95123, Catania, Italy c National Research Council of Italy, Institute for Sustainable Plant Protection (CNR - IPSP), Strada delle Cacce 73, 10135, Torino, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: Giant reed Available soil water content Physiological response Light response curve Biomass yield Ash content
The high photosynthetic efficiency and the drought tolerance of Arundo donax L. contribute to its potential use as a suitable biomass crop for drought prone environments. A modelling approach to study net assimilation and stomatal conductance as influenced by available soil water content (ASWC) can be used to obtain threshold values to support the management of irrigation practice. Physiological model parameters, morphological (stem height and max diameter, number of leaves per stem), and productive traits (yield and biomass ash content), can support ecotypes selection activity, crucial for genetic improvement in a crop, such as A. donax, widely distributed in natural habitats, but characterized by no viable seeds. To this end, an open field experiment has been conducted comparing three A. donax L. clones collected from habitats representing a gradient of increasing temperature and declining water availability (Morocco; South Italy, and North Italy). Two irrigation treatments (rain-fed, and well-watered) and two different harvest dates (December and March) were also imposed as experimental factors. Physiological model involved light response curve approach measuring net assimilation and stomatal conductance under different light intensity and different actual ASWC. The obtained physiological curve parameters in relation to ASWC (maximum net assimilation - AN-sat, photosynthetic quantum efficiency - Qapp, and maximum stomatal conductance - Gs-sat) were well fitted by a two-segmented piecewise regressions. The segment breakpoints were computed at 43%, 39% and 65% of ASWC, for AN-sat, Qapp, and Gs-sat, respectively. This means that between 65% and 43% of ASWC, only stomatal conductance appeared to be influenced by the decrease of ASWC, reducing stomata opening, whereas the efficiency of net assimilation resulted unaffected by water depletion. The capacity to sustain high net assimilation rates untill low ASCW and the capacity to full recover net photosynthesis after a severe drought stress, emerged as crop traits contributing to explain the drought resistant aptitude of A. donax. No differences have been reported for any of the studied physiological parameters within ecotypes. On the contrary, the three clones exhibited differences in terms of biometric characters, biomass production and its ash content. The results of the study emphasize the multipurpose value of Moroccan ecotype reporting best performances in terms of both quantity and quality (highest biomass yield - 26.4 t ha−1 - lowest ash content 4.8%).
1. Introduction Giant reed (Arundo donax L.) is a perennial grass attracting the interest of scientific and industrial community due to its multipurpose attitude related to high biomass yield, phytodepuration properties, strong adaptation capacity to environmental conditions, greenhouse gas (GHG) emissions mitigation, and lower tillage requirement than
⁎
traditional crop (Cosentino et al., 2006, 2008, 2014; Angelini et al., 2009; Mantineo et al., 2009; Fabbrini et al., 2019; Haworth et al., 2019). However, giant reed diffusion at a commercial scale is counteracted by many challenges recently reviewed by Ge et al. (2016). The main constrains include potential negative environmental impacts (invasive species attitude), negative biomass quality traits (limiting
Corresponding author. E-mail address: [email protected] (E. Riggi).
https://doi.org/10.1016/j.indcrop.2019.111630 Received 23 May 2019; Received in revised form 29 July 2019; Accepted 30 July 2019 Available online 07 August 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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precipitation per annum), in Catania, Sicily, South Italy (S - 26 °C mean summer temperature, approx. 400 mm precipitation) and in Florence, Tuscany, North Italy (T - mean summer temperature of 23 °C, approx. 800 mm precipitation), during March 2015. On April 2015 the rhizomes were planted in 4 x 3 m sized elementary unit at a distance of 50 cm from each other in row and 80 cm between rows (final density: 2.5 plants m−2) at the agricultural research station of the University of Catania (37°25′N 15°03′E; 10 m a.s.l.). To minimize edge effects, a 2.4 m thick border of three rows of A. donax, was planted around the experimental field. The soil was a typical clay-rich Xerofluvents soil (USDA, 1999) with 49.3%, 22.4% and 28.3% in sand, silt and clay, respectively and a bulk density of 1.1 g cm−3. In February 2016, the stems were cut at a height of 10 cm and new stems were allowed to develop for the second year growing season, representing the studied period in this paper. Irrigation was applied as main plot separating rain-fed and irrigated plot (3.0 m apart), and ecotypes were arranged in a randomized design within each main plot. During the year of establishment (2015), soil water availability was optimally kept in all treatments to allow root expansion and to achieve the desired plant density. During the trial (2016-17), water supplied to I100 was 722 mm. Irrigation was applied adopting a micro flow dispensing system with emitters at 0.5 m and 8 l h−1 flow rate. The irrigation schedule and calculation along the period between plant re-growth up to the last harvest (12 months) was determined according to Cosentino et al. (2014).
transformation processes), but also limited experiences in crop management strategies (starting from propagation high costs). Furthermore, being sterile, this species has a limited genetic variability (Balogh et al., 2012; Mariani et al., 2010), dramatically reducing the potential support of genetic improvement to its diffusion as a multipurpose crop. Nevertheless, high correlations between parent–progeny for some biometrical traits (culm height and diameter) have been assessed and they could be proposed as drivers for clonal selection (Pilu et al., 2014). Differences in biomass quality traits (ash content, biomass methane and biogas potential) have recently emerged in large collections of Arundo donax, from different geographical areas in Europe and China (Amaducci and Perego, 2015; Cosentino et al., 2016; Curt et al., 2018). The ranges of observed differences between clones, and the genetic control of ash and hemicellulose content, reported in some energy crop (Fabio et al., 2017), sustain further studies to ascertain the heritability of these characters. Moreover, photosynthetic capacity markedly characterizes giant reed with its high assimilation rate, uncommon for a C3 species, and for its tolerance to stressing environmental condition (Rossa et al., 1998; Nackley et al., 2014). However, the studies conducted on leaf level gas exchange, punctually measuring photosynthesis and transpiration rate, were not able to reveal relevant differences between clones (Haworth et al., 2017a, 2017b, 2017c). Nevertheless, knowledge on the relationship between soil water availability and physiological and productive traits could greatly contribute to describe plant responses to environmental drivers, but also to precisely define crop needs in term of irrigation. In this view, ecophysiological studies, within agricultural context, could support plant selection process to contribute to ideotype definition and mining (Haworth et al., 2018a). In a recent work, Fabbrini et al. (2019) proposed for semi-arid environment with dry and hot season, the stomatal resistance as a selection driver discriminating between ecotypes with different attitude to regulate water transpiration. The physiological responses to soil water status tend not to be linear (Jones, 2007), and species-specific thresholds could be ascertained for assimilation rate, leaf transpiration and stomatal conductance (Veihmeyer, 1956; Ritchie, 1974; Flexas et al., 2004; Cosentino et al., 2016; Romero-Munar et al., 2018; Haworth et al., 2018b). In this view, gas exchange response-curve to environmental variables could contribute to obtain a deeper interpretation of ecophysiological dynamic, escaping the effects of momentary ambient conditions. According to Rascher et al. (2000), the so-called cardinal points obtained in light response curve, resulting from a set of different light level measurements, provide highly useful data for the ecophysiological characterization of plant ecophysiology. We conducted an open field experiment on three clones of giant reed (collected from areas with different thermopluviometric characteristics) under contrasting irrigation treatments in order to test whether available soil water content (ASWC) could be used to determine threshold values to be emplaced in the irrigation management. Thus, the influence of ASWC on leaf level gas exchange was investigated adopting a modelling approach. Moreover, physiological model parameters, biometric, and productive traits were also studied to discriminate between ecotypes.
2.2. Leaf gas exchange measurements In this paper we report the physiological measurements conducted from the shoot emergence stage (occurred on early April 2016), to the early October. During this seven-month period, eight measuring sessions were performed at the beginning and end of April and May, at the end of June and July, and finally at the beginning of September and October, respectively. In each session, the measurements were made on three consecutive sunny days (one per ecotype on both irrigation levels), from 08:00 to 14:00 h solar time. Measurements of net assimilation rate and stomatal conductance were carried out using a LiCor Li6400XT (Li-Cor, Inc., Nebraska, USA) fitted with a 2 cm2 cuvette (LCF- 6400-40, Licor, U.S.A.). The leaf cuvette was clamped on the proximal segment of the lamina, just above the ligule, of the newest fully expanded leaf of three labelled stems per plot, monitored for the entire seven-month period. Leaves were acclimated to an incident photon flux of 2000 μmol m−2 s−1 prior to measurements and the CO2 concentration, temperature and relative air humidity in leaf chamber were kept at 400 μmol mol–1, 25 ± 0.5 °C and 60 ± 1%, respectively. The measurements were performed at photosynthetic photon flux density (PPFD) levels of 2000, 1500, 800, 400, 200, 100, 75, 50, 25, 0 μmol m−2 s-1 adjusted automatically by light-emitting diode (90% red light, 630 nm; 10% blue light, 470 nm) light source. To allow stomata acclimate to light level, intercellular CO2 concentration (Ci) was used as trigging value, and data were recorded when Ci reached ± 15% of the value obtained in the previous step. Determination of a single PPFD complete set (10 levels) took approximately 45 min including a dark adaptation period of 10 min.
2. Materials and method 2.1. Studied factors, experimental design and growth conditions
2.3. Light Response curve parameters calculation
The following factors were studied in a split-split-plot experimental design three time replicated: (i) Irrigation: I0 - rain-fed, I100 - 100% ETm restoration; (ii) Ecotypes: 3 clonal stands; (iii) Harvest dates: December 19th 2016 (early –EH) and March 9th 2017 (late –LH). Rhizomes were collected from single clonal stands in Marrakesh, Morocco (M - 30 °C mean summer temperature, approx. 200 mm
To describe the response of net assimilation rate to light intensity (AN:PPFD response curve), the best fit was found in the following three parameters modified rectangular hyperbolic model equation: y = y0 + a(1 – ebx) 2
(1)
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using GraphPad Prism 5 (GraphPad Software Inc., CA, USA). The same formula, expressed in a ‘physiological’ form, becomes
2.6. Statistical analysis
AN = Rd + AN-sat (1−e−(Qapp/AN−sat)PPFD)
For each of the physiological measurement sessions (8 dates), the AN:PPFD and Gs:PPFD curves were calculated for each of the 6 combinations Ecotypes x ASWC. Then, we performed a pairwise comparison of the curves for each date by means of the extra sum-of-square F test (Motulsky and Christopoulos, 2003). Fischer coefficient values as resulted from the Anova of linear regression relating ASWC in different soil layer (0–30, 30–60, 60–90, 0–60, 0–90, and 30–90 cm) vs. AN-sat, were, preliminarily, calculated to select the soil layer resulting in the most affecting the assimilation rate variations. To describe the effects of the explanatory variable ASWC on the calculated parameters of AN and Gs:PPFD response curves, a piecewise model and an iterative technique, based on two-segment, discontinuous piecewise-linear approximations (fixed design segmented regression) was applied by means of GraphPad Prism 5. The iterative technique, minimizing the residual sum of squares, leads to choose optimal breakpoints of the two segments expressing different behaviors in response to ASWC. Breakpoints are the values of ASWC where the slope of the linear function significantly changes, so representing threshold levels of available soil water content determining relevant differences in plant physiological behaviors. The statistic was applied on the entire data range and to each ecotype separately to verify any differences between clones. When significant breakpoints emerged, multiple regression analysis was conducted for each of the two segments generated with piecewiselinear analysis adopting ASWC and plant growth stage as independent variables. Irrigation, ecotypes and harvest period effects on biometrical traits, yield and ash content were tested by a three-way ANOVA using CoStat version 6.4 software (CoHost Software, Inc.) and means were separated using the Student-Newman-Keuls test (SNK).
(2)
where Rd is the dark respiration (μmol CO2 m−2 s-1), AN-sat is the maximum photosynthetic rate (μmol CO2 m−2 s-1) and Qapp is the ‘apparent’ light efficiency representing the maximum quantum yield achieved in the linear part of the curve and calculated from the first derivative of the equation ∂ AN= ∂ PPFD at PPFD = 0 (Tosserams et al., 2001). Similar approach has been applied to describe the response of stomatal conductance rate to light intensity (Gs:PPFD) and the best fit was found in the following two parameters modified rectangular hyperbolic model equation: Gs= Gs-sat (1 – eb
PPFD
)
(3) −2
-1
s ), and b a where Gs-sat is the maximum conductance rate (mol m constant value. The abovementioned parameters have been calculated on the entire data set of the replicates for each ecotype/available soil water content combination for each date.
2.4. Available soil water content (ASWC) and plant growth stage On the same date of physiological measurements, and on the same row where plants with measured leaves were located, soil samples were collected at three depths down to 0.90 m (0-0.30, 0.30-0.60 and 0.600.90 m). No deeper sampling were performed, assuming that, during the year after the crop establishment, Arundo crop was able to capture all the available water up to 100 cm soil depth (Monti and Zatta, 2009; Cosentino et al., 2014). The soil water content (SWC) was thermogravimetrically measured weighting sample from the three layers before and after oven drying at 105 °C until constant weight. Soil hydrological parameters were 11% (wilting point) and 27% (field capacity) of dry soil weight, and the available soil water content was calculated according to the following formula:
measured soil water content − wilting point x 100 ASWC = field capacity − wilting point
3. Results 3.1. Meteorological data During the trial, the average temperature raised from 10 °C of March to about 25 °C of July, where the maximum temperature peaked with more than 36 °C for few days (Fig. 1). Rainfalls during the active vegetative growth of giant reed reached a total amount equal to 117 mm from the shooting of March, when the 60% of the precipitation of the period was recorded, to August. Hereafter, from September to the first decade of October an amount of 146 mm was measured. The following autumn and winter seasons were unusually rainy, with more than 460 mm of precipitation.
(4)
According to the main scope of the experiment, available soil water content was calculated for each of the sampled layers separately and then aggregated in 0–90, 0–60 and 30–90 cm layers to focus on the layer showing the greatest influence on the most representative physiological parameter studied in the paper: the maximum photosynthetic rate obtained in the light response curve (AN-sat). On each gas exchange measurement day, number of nodes per stem has been registered and finally related to the number of nodes measured at final harvest (March 2017), to represent plant growth stage (%).
3.2. Available soil water content The available soil water content varied greatly in relation to soil depth and strongly increased with depth in absence of water restitution (Fig. 1). During spring, ASWC ranged from 70% (early April) to 38% (late May) in the average of the two deeper layers. On July, the rain-fed plots ranged between no available water content in the top layer, and 15.3% in the other layers. Whereas, the irrigated plots reported more than 75% of ASWC in the entire explored layer (0–90 cm). The rain events from late August to early October increased ASWC to values greater than 60%, even in rain-fed plots.
2.5. Biometric and biomass measurements At each harvest time, stem height and basal diameter, green leaves number per shoot and shoot number per m2, biomass fresh and dry weight was determined on a sample of 4.80 m2 in each plot (two consecutive rows), cutting the stems 5 cm above the ground level. Plant samples were partitioned into stems and leaves (lamina), in order to determine the proportion of the different components on the aboveground biomass. Dry matter content was obtained by oven drying at 70 °C until constant weight. Ash content in stems and lamina (separated) was determined placing samples in porcelain crucibles and combusted in a chamber furnace at temperature of 550 ± 10 °C according to the standard method UNI EN ISO 18122:2016.
3.3. Light response curves and related parameters vs ASWC The AN:PPFD response curves were well fitted by the modified rectangular hyperbolic model as indicated by R2 values (> 0.9), and the related physiological parameters were calculated. Then, to model the 3
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Fig. 1. Meteorological data during the trial, available soil water content (ASWC) in relation to soil depth, in irrigated (white circle) and rain-fed (black circle) plots.
The results showed fully superimposable trend for AN-sat and Qapp, and the intersection point for the two segments was computed at 43% and 39% ASWC30-90, respectively (Table 2). The causes of the divergent trends observed in the arounds of the breakpoints were mined applying a multiple regression involving soil water content and plant growth stage as leading factors. The Pearson correlation (Table 2) emphasized significant positive response to ASWC30-90 for both the assimilation parameters (AN-sat and Qapp) only when ASWC30-90 decline from breakpoint to extreme water stress. In the divergent segments (from breakpoints to maximum ASWC30-90) both parameters resulted not affected by ASWC30-90, whereas significant negative correlation emerged between AN-sat and plant growth stage (-0.0725 μmol CO2 m−2 s-1 for each 1% of the ratio actual vs. final nodes number). A higher breakpoint value resulted for the trend of Gs-sat variation in relation to soil water content, and stomata remained fully open until 65% of ASWC30-90, then, as water availability decreases, they begin to narrow until the almost stomatal closure (0.12 mol m–2 s–1) when the ASWC reached values around the 12%. The multivariate analysis showed no plant growth stage effects for both the divergent segments, whereas a strong ASWC30-90 effect emerged only for values lower than breakpoint when high correlation values have been calculated (r = 0.940; p < 0.001).
Table 1 Anova of linear regression (Fischer coefficient values) of the AN-sat vs. Available Soil Water content in relation to soil depth.
Probability level (*, **, *** significant at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively).
physiological response to the soil water content, we related the curve parameter AN-sat to the ASWC in different soil depth. The results of the regressions (Table 1) clearly showed that the top layer (0-0.30 m) tends to be less related [F (1, 42) = 4.23; p < 0.05)] to the physiological studied parameter whereas the narrowest relationship resulted for the 0.30-0.90 m layer. Consequently, all the calculated light response curves parameters were regressed to the selected layer (ASWC30-90). In Fig. 2, we plotted the AN:PPFD response curves of four representative days (early May, July, September and October). The pairwise comparison of curves by the extra sum-of-square F test led to emphasize significant differences exclusively for July and September. Precipitation that preceded the October measuring session lead to an ASWC always higher than 59%. In these conditions, no differences emerged between curves within each ecotypes in relation to soil water content. The light response curves parameters were then regressed in relation to ASWC30-90 (Fig. 3) combining all ecotypes, and the data were well interpolated by a two-segmented piecewise regressions for all the parameters, except dark respiration. Extra sum of square F-test of regressions ascertained no significant differences related to ecotype for all the two-segmented piecewise regressions parameters (breakpoints, slope 1 segment, slope 2 segment).
3.4. Biometric characters, yield and ash content Contrary to what has just been reported for physiological parameters, clones distinguished themselves when biometric variables and biomass yield and ash content were taken into account. On late harvest (Table 3), higher plants were measured (322.6 and 300.8 cm at LH and EH, respectively) confirming the continuous growing of Arundo, in Mediterranean environment, also during winter 4
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Fig. 2. AN:PPFD response curves for all the studied treatments in four representative dates. Average of the available soil water content in the 0.30-0.90 m soil depth (ASWC30-90) in irrigated (white circle) and rain-fed (black circle) plots.
larger biomass amount was collected on early harvest (25.4 t ha−1) than on late one (19.2 t ha−1). Furthermore, slight but significant higher values were registered in watered (23.6 t ha−1) than in rain-fed plots (21 t ha−1). A relevant reduction in terms of leaves could be observed comparing early and late harvests. In this case, significant interactions “Harvest Date x Ecotype” and “Harvest Date x Irrigation”, emphasized low leaves yield at LH in the average of all the other treatments (0.9 t ha−1), but also a lower amount in Morocco (4.9 t ha−1) when compared to the other ecotypes (7.4 and 6.2 t ha−1 for S and T, respectively) at EH. Moreover, consistently with the previous reported reduction of green leaves number, the leaves yield resulted larger in rain-fed treatment only for late harvest. The dry matter content at harvest showed that only the irrigation treatments resulted in low but significant differences between rain-fed and irrigated (52% and 51%, respectively). The ash content in the whole above-ground dry biomass was significantly affected by Harvest Date and Ecotypes and, to a lesser extent, by Irrigation, and no interaction emerged. In particular, on LH was
time. Stems resulted 50 cm taller in irrigated than in rain-fed plots (336 and 288 cm, respectively). The largest differences emerged for ecotypes and the tallest one, Morocco, resulted 19% higher than the lowest (Tuscany). Paralleling what already stated for plant height, Moroccan ecotype (16.9 mm) and late harvest (15.9 mm) reported the highest values of stem basal diameter. Ecotypes was the only factor affecting the density of stems, and the Tuscany exhibited the highest value (28.7 stem m−2), whereas 22 stem m−2 were counted for Sicilian and Moroccan clones, averaging the remaining factors. For all the studied ecotypes five green leaves were measured at LH, averaging irrigation levels, but the significant effect of interaction “Harvest Date x Irrigation”, put into evidence that rain-fed plant obtained 4-fold green leaves number when compared to irrigated (8.9 and 2 respectively). Relevant differences emerged in biomass yield in response to ecotypes, to harvesting periods and, in a lower extent, to irrigation, without interaction effects. Moroccan ecotype resulted the most productive and Tuscan the less (26.4 and 18.6 t ha−1, respectively), and 5
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observed a 40% reduction when compared to EH (4.37 and 7.27%, respectively), and Sicily and Tuscany ecotype (undifferentiated) contained 32% more of ash content when compared to Morocco (6.34 and 4.8%, respectively).
4. Discussion In this open field experiment, the light response curves approach was adopted to obtain a deeper interpretation of gas exchange dynamic, escaping the effects of momentary ambient conditions. According to Rascher et al. (2000), the so-called cardinal points obtained, resulting from a set of different light level measurements (10 in our study), provide highly useful data for the ecophysiological characterization of the gas exchange behavior of the studied crop. The large amount of light response recordings (more than 1400 during the plant growing season), converged to a range of calculated parameters comparable to the values described by different authors. In particular, in well-watered conditions, the range of AN-sat and Gs-sat (18–32 μmol CO2 m–2 s–1, and 0.5-0.99 mol m−2 s-1, respectively) resulted in agreement with the maximum rates (27–30 μmol CO2 m–2 s–1 and 0.6-1.1 mol m−2 s-1 for AN and Gs, respectively) measured in open field studies (Cosentino et al., 2016; Webster et al., 2016; Nackley et al., 2014; Erickson et al., 2012). Relationship concerning gas exchange at leaf level and actual ASWC were studied by means of the picewise-2segmented regressions that provided quantitative estimate of assimilation and transpiration capacity, and led to a quantification of ASWC thresholds, supporting the management of irrigation practice. The breakpoint estimated by the piecewise regression analysis for assimilation activity (43% and 39% of ASWC for AN-sat and Qapp, respectively) resulted in accordance with those obtained by Fracasso et al. (2017) on sorghum, a C4 species, grown in a controlled environment. In the abovementioned experiment, two genotypes with different sensitivity to drought stress showed significant reduction in net assimilation when ASWC decreased under 45%. However, contrary to our findings, Cosentino et al. (2106) reported a higher threshold in an open field experiment on giant reed, as a decrease in net assimilation was registered when ASWC dropped below 60%. The calculated Gs-sat breakpoint quantified the soil water content range (from maximum value to 65%) corresponding to a condition of fully stomata opening similar to what obtained by Cosentino et al. (2016). Therefore, between 65% and 43% of ASWC30-90, AN-sat and Gs trends diverged and only conductance appeared to be influenced by the decrease of ASWC, reducing stomata opening, whereas the efficiency of photosynthesis appeared unaffected by water depletion. At AN-sat breakpoint (43%) and until lowest ASWC recorded values, the fully
Fig. 3. Two-segmented piecewise regressions for AN-sat, maximum photosynthetic rate (μmol CO2 m−2 s-1), Qapp, ‘apparent’ light efficiency, Gs-sat, maximum conductance rate (mol m−2 s-1), Rd dark respiration (μmol CO2 m−2 s-1), in response to ASWC.
Table 2 Results of piecewise 2-segmented and multiple regressions on the main physiological light response curve parameters (ecotypes combined). piecewise 2-segmented regression
Multiple regression Plant Growth Stage1
ASWC breakpoint ASWC (%)
P
ASWC (%)
r
P
b
r
P
b
AN-sat
43
< 0.001
Rd Qapp
– 39
0.827 < 0.0001
Gs-sat
65
< 0.0001
min-43 43-Max – min-39 39-Max min-65 65-Max
0.863 −0.364 – 0.535 0.040 0.940 −0.301
< 0.001 0.059 – 0.027 0.837 < 0.001 0.468
36.4*** −8.19 ns – 0.04 * 0.01 ns 1.63 *** −0.43 ns
−0.844 −0.644 – −0.551 −0.171 −0.118 −0.070
< 0.001 0.001 – 0.022 0.375 0.566 0.869
−18.8*** −7.25*** – −0.03 * −0.01 ns 0.15 ns 0.01 ns
1
Plant growth stage = actual/final number of nodes at harvest (March 2017) per stem. r Pearson correlation coefficient of the multiple regression analysis. P Probability level of the Pearson correlation coefficient. b Regression coefficient of the multiple regression (ns = not significant; *, **, *** significant at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively). 6
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Table 3 Anova results (probability level) and the average values for studied biometric, productive parameters and ash content at harvests. Height cm
Stem ∅ mm
Stems N m−2
Green Leaves N stem−1
Biomass Dry Matter Whole plant
Main effects
Ash Stem
Leaves
Whole
Stem
Leaves
t ha−1
% DM
t ha−1
t ha−1
%
%
%
Harvest Date Irrigation Ecotypes Interaction HD x I HD x E IxE HD x I x E
* *** ***
*** ns ***
ns ns ***
*** *** *
*** * ***
ns ** ns
ns ** ***
*** ns **
*** * ***
** *** ***
*** ** ***
ns ns ns ns
ns ns ns ns
ns ns ns ns
*** * ns ns
ns ns ns ns
ns ns ns ns
ns ns ns ns
** ** ns ns
ns ns ns ns
ns ns ns ns
** * ns ns
EH LH
300.8b 322.6a
13.9b 15.9a
23.9x 24.9x
– –
25.4a 19.2b
51.6a 51.2a
19.3x 18.3x
– –
7.27a 4.37b
4.32a 3.98b
– –
I0 I100
287.5b 336.0a
15.2 14.5
23.7x 25.1x
– –
21.0b 23.6a
52.2a 50.6b
17.5b 20.1a
– –
5.60b 6.05a
3.91a 4.40b
– –
M S T
356.5a 302.3b 276.4c
16.9a 14.4b 13.1c
21.1b 23.4b 28.7a
– – –
26.4a 22.0b 18.6c
51.9a 51.5a 50.9a
23.6a 18.0b 14.8c
– – –
4.80b 6.22a 6.45a
3.78b 4.30a 4.39a
– – –
M S T M S T
– – – – – –
– – – – – –
21.9b 24.5a 26.1a 5.5c 5.6c 5.3c
– – – – – –
– – – – – –
– – – – – –
4.9b 7.4a 6.2a 0.7c 0.8c 1.2c
– – – – – –
– – – – – –
16.2a 17.2a 17.4a 8.9d 10.6c 12.7b
I0 I100 I0 I100
– – – –
– – – –
23.3a 25.0a 8.9b 2.0c
– – – –
– – – –
– – – –
5.6b 6.7a 1.3c 0.7d
– – – –
– – – –
15.8b 18.1a 10.7c 10.7c
EH
LH
EH LH
% DM = dry matter content in percentage. Probability level (P) as resulting from three-way ANOVA. ns = not significant; *, **, *** significant at P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001, respectively. When F of interaction resulted not significant, main effects are reported.
restoration at levels higher than 60%, zeroed the differences in the light response curves between well-watered and stressed plants. Despite the modelling approach concerning physiological behavior did not show differences related to ecotypes, the three Arundo donax clones exhibited differences in terms of biometric traits, biomass production and ash content. Yield potential varied significantly between clones, as Morocco (26.4 t ha−1) emerged as the most productive. Expressing the yield of Sicily and Tuscany as a percentage of the best performing Morocco, the relative performances were equal to 83% and 70%, respectively. The observed values, related to a 2 year old A. donax, were similar to those reported by Cosentino et al. (2006; 2014), Hidalgo and Fernandez (2000) and Erickson et al. (2012), but slightly lower when compared to 30 t ha−1 reported by Dragoni et al. (2015) for a two-year crop, and significantly lower to 38-40 t ha−1 reported by Angelini et al. (2009) for a mature crop (> 4 year old). The harvest period affected significantly the biomass production, and the lower yield was obtained with the late harvest (76% of the early yield). This result is similar to the results obtained in the same environment by Borin et al. (2013), who reported that the late harvest was about 77% lower relatively to the early one. The author argued that this reduction could be ascribed to leaves and stalks falling to the ground when the harvest was delayed from autumn to late winter. Interestingly, in our study, irrigation appeared as the less affecting factor for yields, as the rain-fed treatment reached 89% of the performance reported by well-watered treatment. In contrast, an open field experiment involving nine ecotypes of different provenance (Italy, Greece, France) grown in a Xeric Mediterranean environment, showed that the ratio of rain-fed to irrigated yield ranged between 54% and
superimposable trends AN-sat and Gs-sat vs. ASWC30-90, showed that CO2 diffusion limitations contributed to the reduction of the assimilation capacity. Several studies in various species reported earlier stomatal closure than photosynthesis depletion as a consequence of water stress (Medrano et al., 2002; Flexas et al., 2004; Aganchich et al., 2009; Romero-Munar et al., 2018). This is clearly showed also in our study, as in the range of 65-43% of ASWC (Fig. 3), net assimilation is not affected by reduced stomatal conductance. Whereas, as water stress progresses and, in turn, stomatal conductance further declines, photosynthesis becomes progressively inhibited. Interestingly, the physiological responses to ASWC are closely in keeping with those obtained with the classical technique of fraction of transpirable soil water (Sinclair and Ludlow, 1986; Brilli et al., 2007; Centritto et al., 2011; Brilli et al., 2013; Catola et al., 2018), indicating that the ASWC method could be successfully used in field studies to determine the crop kinetic responses to drought. Some authors asserted that in perennial plant species, one of the most important drought survival strategies is the ability to recover from the stress when ASWC is restored, and selecting traits for high recuperative ability may be of economic importance equally to selecting for improved growth during drought (Moreira et al. 1990; Norris and Thomas 1982). The full recovery of photosynthesis after a severe drought stress was reported for sorghum (Fracasso et al., 2016), whereas other authors reported limited recovery in cotton (Ennahli and Earl, 2005), in turfgrass (Hua et al., 2010), or, with different behavior depending on variety, in tobacco (van Rensburg and Kruger, 1993). Our findings showed a recuperative ability of Arundo donax that can be inferred by what observed on October measurements, when ASWC
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quality improvement of the biomass. The differences emerged in terms of biomass quality (i.e. dry matter and ash content) could be adopted as driving lines of crop management approaches depending on final destination of harvested biomass. When the quantity is the main goal (e.g. biogas production, biochemical compound extraction, etc.), product lost, due to the fall of leaves and stalks usually observed in late harvest, is obviously to be considered as a negative aspect. On the contrary, when the objective to reach is a lower amount of ash for combustion purpose, the late harvest could be preferred solution.
76% (Curt et al., 2018). The unusually large amount of precipitation occurred starting from September, surely masked the irrigation effects in our trial. Nevertheless, these reduced differences could be also explained by the capacity of the rain-fed treatment to grow, in our environment, in autumn/winter and to achieve a full recovery of net assimilation after soil water restoring. Stem height (described for the same experiment by Haworth et al., 2019) showed a higher daily increment in the stressed treatment following the late summer precipitations. As a consequence, the rain-fed plants were able to maintain a higher photosynthesis and, in turn, a higher growth rate later in the year compared with irrigated plants that, in contrast, began to senescence earlier. We noticed clear differences in biometric traits among clones, and Morocco showed higher values of stem height and basal diameter and a low leaves yield, while Tuscany exhibited higher stems density (number m−2). These results confirmed the data already recorded during the establishment of the crop (previous year - data not reported) and are in keeping with the results reported by Zegada-Lizarazu et al. (2018) obtained with the same clones used in our experiment. They observed taller plant, a significant minor number of tillers (-38%) and 27% higher dry biomass in the Moroccan ecotype than in Tuscany one, cultivated for two consecutive years in well-watered condition in rhizotrons. The authors stated that the higher productivity of Morocco could be related to the plant higher volume (i.e. height and diameter), and suggested that these traits should be taken into account in ecotypes selection activities. Cosentino et al. (2006), Pilu et al. (2014), and Fabbrini et al. (2019) indicated that stem height and basal diameter had a moderate-to-high heritability. Consequently, these could be useful traits for clonal selection activities. Moreover, the Moroccan ecotype had more xylem vessels with a higher diameter and a lower structural density than the Tuscan ecotype (Haworth et al., 2017b), allowing a higher hydraulic conductivity that may favor rapid growth. Furthermore, clonal selection may take advantage also of inheritable phenotypic traits, such as leaf area index, stem dry mass, stem density, number of nodes per stem, stem height and diameter, described by Fabbrini et al. (2019). The use of A. donax as biofuel shows some difficulties connected to the high ash content, that reduces thermal conversion efficiency and may cause problems to combustion process (Coulson et al., 2004; Smith and Slater, 2011). Nevertheless, new transformation processes and biorefinery approach for added value compound extraction contribute to expand the potential use of the crop escaping quality limitation (Antonetti et al., 2015). The amount of ash, remarkable when compared with other energy crops, comes mainly from leaf tissues (Coulson et al., 2004; Monti et al., 2008; Nassi o Di Nasso et al., 2010). Amaducci and Perego (2015) reported significant difference among clones for the plant ash content ranging from 5.3% to 8.1%. Monti et al. (2008) reported values for ash content of 11.3% and 3.2% for leaves and stems, respectively. Furthermore, Corno et al. (2014) indicated a decrease in the total plant ash content along the growing season, ranging from 9.9% DM for the May samples to 3% DM for the March samples. In comparable field conditions, Scordia et al. (2012) reported a value of 5.9% in the whole plant ash content of the late harvest. In respect to the studied quality aspects, Morocco emerged for its positive traits. The lower ash content associated with the reduced leaves biomass indicates that this clone may potentially have the higher thermal conversion efficiency. Moreover, the lower ash content measured at late harvest in the whole biomass can also influence the technical decisions concerning crop management. In our results, the amount of ash in leaves were 17.0 and 10.7% on DM basis for early and late harvest, respectively, whereas stems showed values around 4% irrespectively of the harvest dates. The observed reduction in the leaf yield at late harvest resulted in a relevant reduction in total amount of ash per hectare (about 45% of the total ash content of the early harvest, i.e. 0.84 vs. 1.85 t ha−1, respectively). Therefore, the yield reduction observed in late harvest produced a
5. Conclusion The modelling approach used in this study, focused on the effect of soil water content on net assimilation efficiency, contributed to define some features of the crop. The capacity to sustain high net assimilation rates untill low ASCW and the capacity to full recover net photosynthesis after a severe drought stress, emerged as crop traits contributing to explain the drought resistant aptitude of A. donax. However, these traits are inherent of the species, thus limiting the clonal selection for genetic improvement of A. donax. The results related to biometric and productive traits, emphasize the multipurpose value of the Moroccan ecotype reporting best performances in terms of both biomass yield and ash content. The coinciding positive performance emerged for this clone in literature, suggests the superiority of this ecotype over other clones in our environment. Funding This work was funded by the EU FP7 project WATBIO (Development of improved perennial non-food biomass and bioproduct crops for water stressed environments – no. 311929). References Aganchich, B., Wahbi, S., Loreto, F., Centritto, M., 2009. Partial root zone drying: regulation of photosynthetic limitations and antioxidant enzymatic activities in young olive (Olea europaea) saplings. Tree Physiol. 29, 685–696. Amaducci, S., Perego, A., 2015. Field evaluation of Arundo donax clones for bioenergy production. Ind. Crop. Prod. 75, 122–128. Angelini, L.G., Ceccarini, L., Nassi o Di Nasso, N., Bonari, E., 2009. Comparison of Arundo donax L. And Miscanthus × giganteus in a long-term field experiment in Central Italy: analysis of productive characteristics and energy balance. Biomass Bioenerg 33, 635–643. Antonetti, C., Bonari, E., Licursi, D., Nassi o Di Nasso, A., Raspolli Galletti, A.M., 2015. Hydrothermal conversion of giant reed to furfural and levulinic acid: optimization of the process under microwave irradiation and investigation of distinctive agronomic parameters. Molecules 20, 21232–21253. https://doi.org/10.3390/ molecules201219760. Balogh, E., Herr, J.M., Czako, M., Marton, L., 2012. Defective development of male and female gametophytes in Arundo donax L. (Poaceae). Biomass Bioenerg. 45, 265–269. Brilli, F., Barta, C., Fortunati, A., Lerdau, M., Loreto, F., Centritto, M., 2007. Response of isoprene emission and carbon metabolism to drought in white poplar (Populus alba) saplings. New Phytol. 175, 244–254. Brilli, F., Tsonev, T., Mahmood, T., Velikova, V., Loreto, F., Centritto, M., 2013. Ultradian variation of isoprene emission, photosynthesis, mesophyll conductance and optimum temperature sensitivity for isoprene emission in water-stressed Eucalyptus citriodora saplings. J. Exp. Bot. 64, 519–528. Borin, M., Barbera, A.C., Milani, M., Molari, G., Zimbone, S.M., Toscano, A., 2013. Biomass production and N balance of giant reed (Arundo donax L.) under high water and N input in Mediterranean environments. Eur. J. Agric. 51, 117–119. Catola, S., Centritto, M., Cascone, P., Ranieri, A., Loreto, F., Calamai, L., Balestrini, R., Guerrieri, E., 2018. Effects of single or combined water deficit and aphid attack on tomato volatile organic compound (VOC) emission and plant-plant communication. Environ. Exp. Bot. 153, 54–62. Centritto, M., Brilli, F., Fodale, R., Loreto, F., 2011. Different sensitivity of isoprene emission, respiration, and photosynthesis to high growth temperature coupled with drought stress in black poplar (Populus nigra). Tree Physiol. 31, 275–286. Corno, L., Pilu, R., Adani, F., 2014. Arundo donax L.: a non-food crop for bioenergy and bio-compound production. Biotechnol. Adv. 32, 1535–1549. Cosentino, S.L., Copani, V., D’Agosta, G.M., Sanzone, E., Mantineo, M., 2006. First results on evaluation of Arundo donax L. clones collected in Southern Italy. Ind. Crop. Prod. 23, 212–222. Cosentino, S.L., Copani, V., Patanè, C., Mantineo, M., D’Agosta, G.M., 2008. Agronomic, energetic and environmental aspects of biomass energy cropssuitable for Italian
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Open field experiment for the evaluation of Arundo donax ecotypes ecophysiology and yield as affected by soil water content
T
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Ezio Riggia, , Giovanni Avolaa, Giovanni Marinoa, Matthew Hawortha, Salvatore Luciano Cosentinob, Mauro Centrittoc a
National Research Council of Italy, Institute of BioEconomy, (CNR - IBE), via Paolo Gaifami 18, 95126, Catania, Italy Dipartimento di Agricoltura, Alimentazione e Ambiente (Di3A), Università degli Studi di Catania, via Valdisavoia 5, 95123, Catania, Italy c National Research Council of Italy, Institute for Sustainable Plant Protection (CNR - IPSP), Strada delle Cacce 73, 10135, Torino, Italy b
A R T I C LE I N FO
A B S T R A C T
Keywords: Giant reed Available soil water content Physiological response Light response curve Biomass yield Ash content
The high photosynthetic efficiency and the drought tolerance of Arundo donax L. contribute to its potential use as a suitable biomass crop for drought prone environments. A modelling approach to study net assimilation and stomatal conductance as influenced by available soil water content (ASWC) can be used to obtain threshold values to support the management of irrigation practice. Physiological model parameters, morphological (stem height and max diameter, number of leaves per stem), and productive traits (yield and biomass ash content), can support ecotypes selection activity, crucial for genetic improvement in a crop, such as A. donax, widely distributed in natural habitats, but characterized by no viable seeds. To this end, an open field experiment has been conducted comparing three A. donax L. clones collected from habitats representing a gradient of increasing temperature and declining water availability (Morocco; South Italy, and North Italy). Two irrigation treatments (rain-fed, and well-watered) and two different harvest dates (December and March) were also imposed as experimental factors. Physiological model involved light response curve approach measuring net assimilation and stomatal conductance under different light intensity and different actual ASWC. The obtained physiological curve parameters in relation to ASWC (maximum net assimilation - AN-sat, photosynthetic quantum efficiency - Qapp, and maximum stomatal conductance - Gs-sat) were well fitted by a two-segmented piecewise regressions. The segment breakpoints were computed at 43%, 39% and 65% of ASWC, for AN-sat, Qapp, and Gs-sat, respectively. This means that between 65% and 43% of ASWC, only stomatal conductance appeared to be influenced by the decrease of ASWC, reducing stomata opening, whereas the efficiency of net assimilation resulted unaffected by water depletion. The capacity to sustain high net assimilation rates untill low ASCW and the capacity to full recover net photosynthesis after a severe drought stress, emerged as crop traits contributing to explain the drought resistant aptitude of A. donax. No differences have been reported for any of the studied physiological parameters within ecotypes. On the contrary, the three clones exhibited differences in terms of biometric characters, biomass production and its ash content. The results of the study emphasize the multipurpose value of Moroccan ecotype reporting best performances in terms of both quantity and quality (highest biomass yield - 26.4 t ha−1 - lowest ash content 4.8%).
1. Introduction Giant reed (Arundo donax L.) is a perennial grass attracting the interest of scientific and industrial community due to its multipurpose attitude related to high biomass yield, phytodepuration properties, strong adaptation capacity to environmental conditions, greenhouse gas (GHG) emissions mitigation, and lower tillage requirement than
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traditional crop (Cosentino et al., 2006, 2008, 2014; Angelini et al., 2009; Mantineo et al., 2009; Fabbrini et al., 2019; Haworth et al., 2019). However, giant reed diffusion at a commercial scale is counteracted by many challenges recently reviewed by Ge et al. (2016). The main constrains include potential negative environmental impacts (invasive species attitude), negative biomass quality traits (limiting
Corresponding author. E-mail address: [email protected] (E. Riggi).
https://doi.org/10.1016/j.indcrop.2019.111630 Received 23 May 2019; Received in revised form 29 July 2019; Accepted 30 July 2019 Available online 07 August 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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precipitation per annum), in Catania, Sicily, South Italy (S - 26 °C mean summer temperature, approx. 400 mm precipitation) and in Florence, Tuscany, North Italy (T - mean summer temperature of 23 °C, approx. 800 mm precipitation), during March 2015. On April 2015 the rhizomes were planted in 4 x 3 m sized elementary unit at a distance of 50 cm from each other in row and 80 cm between rows (final density: 2.5 plants m−2) at the agricultural research station of the University of Catania (37°25′N 15°03′E; 10 m a.s.l.). To minimize edge effects, a 2.4 m thick border of three rows of A. donax, was planted around the experimental field. The soil was a typical clay-rich Xerofluvents soil (USDA, 1999) with 49.3%, 22.4% and 28.3% in sand, silt and clay, respectively and a bulk density of 1.1 g cm−3. In February 2016, the stems were cut at a height of 10 cm and new stems were allowed to develop for the second year growing season, representing the studied period in this paper. Irrigation was applied as main plot separating rain-fed and irrigated plot (3.0 m apart), and ecotypes were arranged in a randomized design within each main plot. During the year of establishment (2015), soil water availability was optimally kept in all treatments to allow root expansion and to achieve the desired plant density. During the trial (2016-17), water supplied to I100 was 722 mm. Irrigation was applied adopting a micro flow dispensing system with emitters at 0.5 m and 8 l h−1 flow rate. The irrigation schedule and calculation along the period between plant re-growth up to the last harvest (12 months) was determined according to Cosentino et al. (2014).
transformation processes), but also limited experiences in crop management strategies (starting from propagation high costs). Furthermore, being sterile, this species has a limited genetic variability (Balogh et al., 2012; Mariani et al., 2010), dramatically reducing the potential support of genetic improvement to its diffusion as a multipurpose crop. Nevertheless, high correlations between parent–progeny for some biometrical traits (culm height and diameter) have been assessed and they could be proposed as drivers for clonal selection (Pilu et al., 2014). Differences in biomass quality traits (ash content, biomass methane and biogas potential) have recently emerged in large collections of Arundo donax, from different geographical areas in Europe and China (Amaducci and Perego, 2015; Cosentino et al., 2016; Curt et al., 2018). The ranges of observed differences between clones, and the genetic control of ash and hemicellulose content, reported in some energy crop (Fabio et al., 2017), sustain further studies to ascertain the heritability of these characters. Moreover, photosynthetic capacity markedly characterizes giant reed with its high assimilation rate, uncommon for a C3 species, and for its tolerance to stressing environmental condition (Rossa et al., 1998; Nackley et al., 2014). However, the studies conducted on leaf level gas exchange, punctually measuring photosynthesis and transpiration rate, were not able to reveal relevant differences between clones (Haworth et al., 2017a, 2017b, 2017c). Nevertheless, knowledge on the relationship between soil water availability and physiological and productive traits could greatly contribute to describe plant responses to environmental drivers, but also to precisely define crop needs in term of irrigation. In this view, ecophysiological studies, within agricultural context, could support plant selection process to contribute to ideotype definition and mining (Haworth et al., 2018a). In a recent work, Fabbrini et al. (2019) proposed for semi-arid environment with dry and hot season, the stomatal resistance as a selection driver discriminating between ecotypes with different attitude to regulate water transpiration. The physiological responses to soil water status tend not to be linear (Jones, 2007), and species-specific thresholds could be ascertained for assimilation rate, leaf transpiration and stomatal conductance (Veihmeyer, 1956; Ritchie, 1974; Flexas et al., 2004; Cosentino et al., 2016; Romero-Munar et al., 2018; Haworth et al., 2018b). In this view, gas exchange response-curve to environmental variables could contribute to obtain a deeper interpretation of ecophysiological dynamic, escaping the effects of momentary ambient conditions. According to Rascher et al. (2000), the so-called cardinal points obtained in light response curve, resulting from a set of different light level measurements, provide highly useful data for the ecophysiological characterization of plant ecophysiology. We conducted an open field experiment on three clones of giant reed (collected from areas with different thermopluviometric characteristics) under contrasting irrigation treatments in order to test whether available soil water content (ASWC) could be used to determine threshold values to be emplaced in the irrigation management. Thus, the influence of ASWC on leaf level gas exchange was investigated adopting a modelling approach. Moreover, physiological model parameters, biometric, and productive traits were also studied to discriminate between ecotypes.
2.2. Leaf gas exchange measurements In this paper we report the physiological measurements conducted from the shoot emergence stage (occurred on early April 2016), to the early October. During this seven-month period, eight measuring sessions were performed at the beginning and end of April and May, at the end of June and July, and finally at the beginning of September and October, respectively. In each session, the measurements were made on three consecutive sunny days (one per ecotype on both irrigation levels), from 08:00 to 14:00 h solar time. Measurements of net assimilation rate and stomatal conductance were carried out using a LiCor Li6400XT (Li-Cor, Inc., Nebraska, USA) fitted with a 2 cm2 cuvette (LCF- 6400-40, Licor, U.S.A.). The leaf cuvette was clamped on the proximal segment of the lamina, just above the ligule, of the newest fully expanded leaf of three labelled stems per plot, monitored for the entire seven-month period. Leaves were acclimated to an incident photon flux of 2000 μmol m−2 s−1 prior to measurements and the CO2 concentration, temperature and relative air humidity in leaf chamber were kept at 400 μmol mol–1, 25 ± 0.5 °C and 60 ± 1%, respectively. The measurements were performed at photosynthetic photon flux density (PPFD) levels of 2000, 1500, 800, 400, 200, 100, 75, 50, 25, 0 μmol m−2 s-1 adjusted automatically by light-emitting diode (90% red light, 630 nm; 10% blue light, 470 nm) light source. To allow stomata acclimate to light level, intercellular CO2 concentration (Ci) was used as trigging value, and data were recorded when Ci reached ± 15% of the value obtained in the previous step. Determination of a single PPFD complete set (10 levels) took approximately 45 min including a dark adaptation period of 10 min.
2. Materials and method 2.1. Studied factors, experimental design and growth conditions
2.3. Light Response curve parameters calculation
The following factors were studied in a split-split-plot experimental design three time replicated: (i) Irrigation: I0 - rain-fed, I100 - 100% ETm restoration; (ii) Ecotypes: 3 clonal stands; (iii) Harvest dates: December 19th 2016 (early –EH) and March 9th 2017 (late –LH). Rhizomes were collected from single clonal stands in Marrakesh, Morocco (M - 30 °C mean summer temperature, approx. 200 mm
To describe the response of net assimilation rate to light intensity (AN:PPFD response curve), the best fit was found in the following three parameters modified rectangular hyperbolic model equation: y = y0 + a(1 – ebx) 2
(1)
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using GraphPad Prism 5 (GraphPad Software Inc., CA, USA). The same formula, expressed in a ‘physiological’ form, becomes
2.6. Statistical analysis
AN = Rd + AN-sat (1−e−(Qapp/AN−sat)PPFD)
For each of the physiological measurement sessions (8 dates), the AN:PPFD and Gs:PPFD curves were calculated for each of the 6 combinations Ecotypes x ASWC. Then, we performed a pairwise comparison of the curves for each date by means of the extra sum-of-square F test (Motulsky and Christopoulos, 2003). Fischer coefficient values as resulted from the Anova of linear regression relating ASWC in different soil layer (0–30, 30–60, 60–90, 0–60, 0–90, and 30–90 cm) vs. AN-sat, were, preliminarily, calculated to select the soil layer resulting in the most affecting the assimilation rate variations. To describe the effects of the explanatory variable ASWC on the calculated parameters of AN and Gs:PPFD response curves, a piecewise model and an iterative technique, based on two-segment, discontinuous piecewise-linear approximations (fixed design segmented regression) was applied by means of GraphPad Prism 5. The iterative technique, minimizing the residual sum of squares, leads to choose optimal breakpoints of the two segments expressing different behaviors in response to ASWC. Breakpoints are the values of ASWC where the slope of the linear function significantly changes, so representing threshold levels of available soil water content determining relevant differences in plant physiological behaviors. The statistic was applied on the entire data range and to each ecotype separately to verify any differences between clones. When significant breakpoints emerged, multiple regression analysis was conducted for each of the two segments generated with piecewiselinear analysis adopting ASWC and plant growth stage as independent variables. Irrigation, ecotypes and harvest period effects on biometrical traits, yield and ash content were tested by a three-way ANOVA using CoStat version 6.4 software (CoHost Software, Inc.) and means were separated using the Student-Newman-Keuls test (SNK).
(2)
where Rd is the dark respiration (μmol CO2 m−2 s-1), AN-sat is the maximum photosynthetic rate (μmol CO2 m−2 s-1) and Qapp is the ‘apparent’ light efficiency representing the maximum quantum yield achieved in the linear part of the curve and calculated from the first derivative of the equation ∂ AN= ∂ PPFD at PPFD = 0 (Tosserams et al., 2001). Similar approach has been applied to describe the response of stomatal conductance rate to light intensity (Gs:PPFD) and the best fit was found in the following two parameters modified rectangular hyperbolic model equation: Gs= Gs-sat (1 – eb
PPFD
)
(3) −2
-1
s ), and b a where Gs-sat is the maximum conductance rate (mol m constant value. The abovementioned parameters have been calculated on the entire data set of the replicates for each ecotype/available soil water content combination for each date.
2.4. Available soil water content (ASWC) and plant growth stage On the same date of physiological measurements, and on the same row where plants with measured leaves were located, soil samples were collected at three depths down to 0.90 m (0-0.30, 0.30-0.60 and 0.600.90 m). No deeper sampling were performed, assuming that, during the year after the crop establishment, Arundo crop was able to capture all the available water up to 100 cm soil depth (Monti and Zatta, 2009; Cosentino et al., 2014). The soil water content (SWC) was thermogravimetrically measured weighting sample from the three layers before and after oven drying at 105 °C until constant weight. Soil hydrological parameters were 11% (wilting point) and 27% (field capacity) of dry soil weight, and the available soil water content was calculated according to the following formula:
measured soil water content − wilting point x 100 ASWC = field capacity − wilting point
3. Results 3.1. Meteorological data During the trial, the average temperature raised from 10 °C of March to about 25 °C of July, where the maximum temperature peaked with more than 36 °C for few days (Fig. 1). Rainfalls during the active vegetative growth of giant reed reached a total amount equal to 117 mm from the shooting of March, when the 60% of the precipitation of the period was recorded, to August. Hereafter, from September to the first decade of October an amount of 146 mm was measured. The following autumn and winter seasons were unusually rainy, with more than 460 mm of precipitation.
(4)
According to the main scope of the experiment, available soil water content was calculated for each of the sampled layers separately and then aggregated in 0–90, 0–60 and 30–90 cm layers to focus on the layer showing the greatest influence on the most representative physiological parameter studied in the paper: the maximum photosynthetic rate obtained in the light response curve (AN-sat). On each gas exchange measurement day, number of nodes per stem has been registered and finally related to the number of nodes measured at final harvest (March 2017), to represent plant growth stage (%).
3.2. Available soil water content The available soil water content varied greatly in relation to soil depth and strongly increased with depth in absence of water restitution (Fig. 1). During spring, ASWC ranged from 70% (early April) to 38% (late May) in the average of the two deeper layers. On July, the rain-fed plots ranged between no available water content in the top layer, and 15.3% in the other layers. Whereas, the irrigated plots reported more than 75% of ASWC in the entire explored layer (0–90 cm). The rain events from late August to early October increased ASWC to values greater than 60%, even in rain-fed plots.
2.5. Biometric and biomass measurements At each harvest time, stem height and basal diameter, green leaves number per shoot and shoot number per m2, biomass fresh and dry weight was determined on a sample of 4.80 m2 in each plot (two consecutive rows), cutting the stems 5 cm above the ground level. Plant samples were partitioned into stems and leaves (lamina), in order to determine the proportion of the different components on the aboveground biomass. Dry matter content was obtained by oven drying at 70 °C until constant weight. Ash content in stems and lamina (separated) was determined placing samples in porcelain crucibles and combusted in a chamber furnace at temperature of 550 ± 10 °C according to the standard method UNI EN ISO 18122:2016.
3.3. Light response curves and related parameters vs ASWC The AN:PPFD response curves were well fitted by the modified rectangular hyperbolic model as indicated by R2 values (> 0.9), and the related physiological parameters were calculated. Then, to model the 3
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Fig. 1. Meteorological data during the trial, available soil water content (ASWC) in relation to soil depth, in irrigated (white circle) and rain-fed (black circle) plots.
The results showed fully superimposable trend for AN-sat and Qapp, and the intersection point for the two segments was computed at 43% and 39% ASWC30-90, respectively (Table 2). The causes of the divergent trends observed in the arounds of the breakpoints were mined applying a multiple regression involving soil water content and plant growth stage as leading factors. The Pearson correlation (Table 2) emphasized significant positive response to ASWC30-90 for both the assimilation parameters (AN-sat and Qapp) only when ASWC30-90 decline from breakpoint to extreme water stress. In the divergent segments (from breakpoints to maximum ASWC30-90) both parameters resulted not affected by ASWC30-90, whereas significant negative correlation emerged between AN-sat and plant growth stage (-0.0725 μmol CO2 m−2 s-1 for each 1% of the ratio actual vs. final nodes number). A higher breakpoint value resulted for the trend of Gs-sat variation in relation to soil water content, and stomata remained fully open until 65% of ASWC30-90, then, as water availability decreases, they begin to narrow until the almost stomatal closure (0.12 mol m–2 s–1) when the ASWC reached values around the 12%. The multivariate analysis showed no plant growth stage effects for both the divergent segments, whereas a strong ASWC30-90 effect emerged only for values lower than breakpoint when high correlation values have been calculated (r = 0.940; p < 0.001).
Table 1 Anova of linear regression (Fischer coefficient values) of the AN-sat vs. Available Soil Water content in relation to soil depth.
Probability level (*, **, *** significant at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively).
physiological response to the soil water content, we related the curve parameter AN-sat to the ASWC in different soil depth. The results of the regressions (Table 1) clearly showed that the top layer (0-0.30 m) tends to be less related [F (1, 42) = 4.23; p < 0.05)] to the physiological studied parameter whereas the narrowest relationship resulted for the 0.30-0.90 m layer. Consequently, all the calculated light response curves parameters were regressed to the selected layer (ASWC30-90). In Fig. 2, we plotted the AN:PPFD response curves of four representative days (early May, July, September and October). The pairwise comparison of curves by the extra sum-of-square F test led to emphasize significant differences exclusively for July and September. Precipitation that preceded the October measuring session lead to an ASWC always higher than 59%. In these conditions, no differences emerged between curves within each ecotypes in relation to soil water content. The light response curves parameters were then regressed in relation to ASWC30-90 (Fig. 3) combining all ecotypes, and the data were well interpolated by a two-segmented piecewise regressions for all the parameters, except dark respiration. Extra sum of square F-test of regressions ascertained no significant differences related to ecotype for all the two-segmented piecewise regressions parameters (breakpoints, slope 1 segment, slope 2 segment).
3.4. Biometric characters, yield and ash content Contrary to what has just been reported for physiological parameters, clones distinguished themselves when biometric variables and biomass yield and ash content were taken into account. On late harvest (Table 3), higher plants were measured (322.6 and 300.8 cm at LH and EH, respectively) confirming the continuous growing of Arundo, in Mediterranean environment, also during winter 4
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Fig. 2. AN:PPFD response curves for all the studied treatments in four representative dates. Average of the available soil water content in the 0.30-0.90 m soil depth (ASWC30-90) in irrigated (white circle) and rain-fed (black circle) plots.
larger biomass amount was collected on early harvest (25.4 t ha−1) than on late one (19.2 t ha−1). Furthermore, slight but significant higher values were registered in watered (23.6 t ha−1) than in rain-fed plots (21 t ha−1). A relevant reduction in terms of leaves could be observed comparing early and late harvests. In this case, significant interactions “Harvest Date x Ecotype” and “Harvest Date x Irrigation”, emphasized low leaves yield at LH in the average of all the other treatments (0.9 t ha−1), but also a lower amount in Morocco (4.9 t ha−1) when compared to the other ecotypes (7.4 and 6.2 t ha−1 for S and T, respectively) at EH. Moreover, consistently with the previous reported reduction of green leaves number, the leaves yield resulted larger in rain-fed treatment only for late harvest. The dry matter content at harvest showed that only the irrigation treatments resulted in low but significant differences between rain-fed and irrigated (52% and 51%, respectively). The ash content in the whole above-ground dry biomass was significantly affected by Harvest Date and Ecotypes and, to a lesser extent, by Irrigation, and no interaction emerged. In particular, on LH was
time. Stems resulted 50 cm taller in irrigated than in rain-fed plots (336 and 288 cm, respectively). The largest differences emerged for ecotypes and the tallest one, Morocco, resulted 19% higher than the lowest (Tuscany). Paralleling what already stated for plant height, Moroccan ecotype (16.9 mm) and late harvest (15.9 mm) reported the highest values of stem basal diameter. Ecotypes was the only factor affecting the density of stems, and the Tuscany exhibited the highest value (28.7 stem m−2), whereas 22 stem m−2 were counted for Sicilian and Moroccan clones, averaging the remaining factors. For all the studied ecotypes five green leaves were measured at LH, averaging irrigation levels, but the significant effect of interaction “Harvest Date x Irrigation”, put into evidence that rain-fed plant obtained 4-fold green leaves number when compared to irrigated (8.9 and 2 respectively). Relevant differences emerged in biomass yield in response to ecotypes, to harvesting periods and, in a lower extent, to irrigation, without interaction effects. Moroccan ecotype resulted the most productive and Tuscan the less (26.4 and 18.6 t ha−1, respectively), and 5
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observed a 40% reduction when compared to EH (4.37 and 7.27%, respectively), and Sicily and Tuscany ecotype (undifferentiated) contained 32% more of ash content when compared to Morocco (6.34 and 4.8%, respectively).
4. Discussion In this open field experiment, the light response curves approach was adopted to obtain a deeper interpretation of gas exchange dynamic, escaping the effects of momentary ambient conditions. According to Rascher et al. (2000), the so-called cardinal points obtained, resulting from a set of different light level measurements (10 in our study), provide highly useful data for the ecophysiological characterization of the gas exchange behavior of the studied crop. The large amount of light response recordings (more than 1400 during the plant growing season), converged to a range of calculated parameters comparable to the values described by different authors. In particular, in well-watered conditions, the range of AN-sat and Gs-sat (18–32 μmol CO2 m–2 s–1, and 0.5-0.99 mol m−2 s-1, respectively) resulted in agreement with the maximum rates (27–30 μmol CO2 m–2 s–1 and 0.6-1.1 mol m−2 s-1 for AN and Gs, respectively) measured in open field studies (Cosentino et al., 2016; Webster et al., 2016; Nackley et al., 2014; Erickson et al., 2012). Relationship concerning gas exchange at leaf level and actual ASWC were studied by means of the picewise-2segmented regressions that provided quantitative estimate of assimilation and transpiration capacity, and led to a quantification of ASWC thresholds, supporting the management of irrigation practice. The breakpoint estimated by the piecewise regression analysis for assimilation activity (43% and 39% of ASWC for AN-sat and Qapp, respectively) resulted in accordance with those obtained by Fracasso et al. (2017) on sorghum, a C4 species, grown in a controlled environment. In the abovementioned experiment, two genotypes with different sensitivity to drought stress showed significant reduction in net assimilation when ASWC decreased under 45%. However, contrary to our findings, Cosentino et al. (2106) reported a higher threshold in an open field experiment on giant reed, as a decrease in net assimilation was registered when ASWC dropped below 60%. The calculated Gs-sat breakpoint quantified the soil water content range (from maximum value to 65%) corresponding to a condition of fully stomata opening similar to what obtained by Cosentino et al. (2016). Therefore, between 65% and 43% of ASWC30-90, AN-sat and Gs trends diverged and only conductance appeared to be influenced by the decrease of ASWC, reducing stomata opening, whereas the efficiency of photosynthesis appeared unaffected by water depletion. At AN-sat breakpoint (43%) and until lowest ASWC recorded values, the fully
Fig. 3. Two-segmented piecewise regressions for AN-sat, maximum photosynthetic rate (μmol CO2 m−2 s-1), Qapp, ‘apparent’ light efficiency, Gs-sat, maximum conductance rate (mol m−2 s-1), Rd dark respiration (μmol CO2 m−2 s-1), in response to ASWC.
Table 2 Results of piecewise 2-segmented and multiple regressions on the main physiological light response curve parameters (ecotypes combined). piecewise 2-segmented regression
Multiple regression Plant Growth Stage1
ASWC breakpoint ASWC (%)
P
ASWC (%)
r
P
b
r
P
b
AN-sat
43
< 0.001
Rd Qapp
– 39
0.827 < 0.0001
Gs-sat
65
< 0.0001
min-43 43-Max – min-39 39-Max min-65 65-Max
0.863 −0.364 – 0.535 0.040 0.940 −0.301
< 0.001 0.059 – 0.027 0.837 < 0.001 0.468
36.4*** −8.19 ns – 0.04 * 0.01 ns 1.63 *** −0.43 ns
−0.844 −0.644 – −0.551 −0.171 −0.118 −0.070
< 0.001 0.001 – 0.022 0.375 0.566 0.869
−18.8*** −7.25*** – −0.03 * −0.01 ns 0.15 ns 0.01 ns
1
Plant growth stage = actual/final number of nodes at harvest (March 2017) per stem. r Pearson correlation coefficient of the multiple regression analysis. P Probability level of the Pearson correlation coefficient. b Regression coefficient of the multiple regression (ns = not significant; *, **, *** significant at P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively). 6
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Table 3 Anova results (probability level) and the average values for studied biometric, productive parameters and ash content at harvests. Height cm
Stem ∅ mm
Stems N m−2
Green Leaves N stem−1
Biomass Dry Matter Whole plant
Main effects
Ash Stem
Leaves
Whole
Stem
Leaves
t ha−1
% DM
t ha−1
t ha−1
%
%
%
Harvest Date Irrigation Ecotypes Interaction HD x I HD x E IxE HD x I x E
* *** ***
*** ns ***
ns ns ***
*** *** *
*** * ***
ns ** ns
ns ** ***
*** ns **
*** * ***
** *** ***
*** ** ***
ns ns ns ns
ns ns ns ns
ns ns ns ns
*** * ns ns
ns ns ns ns
ns ns ns ns
ns ns ns ns
** ** ns ns
ns ns ns ns
ns ns ns ns
** * ns ns
EH LH
300.8b 322.6a
13.9b 15.9a
23.9x 24.9x
– –
25.4a 19.2b
51.6a 51.2a
19.3x 18.3x
– –
7.27a 4.37b
4.32a 3.98b
– –
I0 I100
287.5b 336.0a
15.2 14.5
23.7x 25.1x
– –
21.0b 23.6a
52.2a 50.6b
17.5b 20.1a
– –
5.60b 6.05a
3.91a 4.40b
– –
M S T
356.5a 302.3b 276.4c
16.9a 14.4b 13.1c
21.1b 23.4b 28.7a
– – –
26.4a 22.0b 18.6c
51.9a 51.5a 50.9a
23.6a 18.0b 14.8c
– – –
4.80b 6.22a 6.45a
3.78b 4.30a 4.39a
– – –
M S T M S T
– – – – – –
– – – – – –
21.9b 24.5a 26.1a 5.5c 5.6c 5.3c
– – – – – –
– – – – – –
– – – – – –
4.9b 7.4a 6.2a 0.7c 0.8c 1.2c
– – – – – –
– – – – – –
16.2a 17.2a 17.4a 8.9d 10.6c 12.7b
I0 I100 I0 I100
– – – –
– – – –
23.3a 25.0a 8.9b 2.0c
– – – –
– – – –
– – – –
5.6b 6.7a 1.3c 0.7d
– – – –
– – – –
15.8b 18.1a 10.7c 10.7c
EH
LH
EH LH
% DM = dry matter content in percentage. Probability level (P) as resulting from three-way ANOVA. ns = not significant; *, **, *** significant at P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001, respectively. When F of interaction resulted not significant, main effects are reported.
restoration at levels higher than 60%, zeroed the differences in the light response curves between well-watered and stressed plants. Despite the modelling approach concerning physiological behavior did not show differences related to ecotypes, the three Arundo donax clones exhibited differences in terms of biometric traits, biomass production and ash content. Yield potential varied significantly between clones, as Morocco (26.4 t ha−1) emerged as the most productive. Expressing the yield of Sicily and Tuscany as a percentage of the best performing Morocco, the relative performances were equal to 83% and 70%, respectively. The observed values, related to a 2 year old A. donax, were similar to those reported by Cosentino et al. (2006; 2014), Hidalgo and Fernandez (2000) and Erickson et al. (2012), but slightly lower when compared to 30 t ha−1 reported by Dragoni et al. (2015) for a two-year crop, and significantly lower to 38-40 t ha−1 reported by Angelini et al. (2009) for a mature crop (> 4 year old). The harvest period affected significantly the biomass production, and the lower yield was obtained with the late harvest (76% of the early yield). This result is similar to the results obtained in the same environment by Borin et al. (2013), who reported that the late harvest was about 77% lower relatively to the early one. The author argued that this reduction could be ascribed to leaves and stalks falling to the ground when the harvest was delayed from autumn to late winter. Interestingly, in our study, irrigation appeared as the less affecting factor for yields, as the rain-fed treatment reached 89% of the performance reported by well-watered treatment. In contrast, an open field experiment involving nine ecotypes of different provenance (Italy, Greece, France) grown in a Xeric Mediterranean environment, showed that the ratio of rain-fed to irrigated yield ranged between 54% and
superimposable trends AN-sat and Gs-sat vs. ASWC30-90, showed that CO2 diffusion limitations contributed to the reduction of the assimilation capacity. Several studies in various species reported earlier stomatal closure than photosynthesis depletion as a consequence of water stress (Medrano et al., 2002; Flexas et al., 2004; Aganchich et al., 2009; Romero-Munar et al., 2018). This is clearly showed also in our study, as in the range of 65-43% of ASWC (Fig. 3), net assimilation is not affected by reduced stomatal conductance. Whereas, as water stress progresses and, in turn, stomatal conductance further declines, photosynthesis becomes progressively inhibited. Interestingly, the physiological responses to ASWC are closely in keeping with those obtained with the classical technique of fraction of transpirable soil water (Sinclair and Ludlow, 1986; Brilli et al., 2007; Centritto et al., 2011; Brilli et al., 2013; Catola et al., 2018), indicating that the ASWC method could be successfully used in field studies to determine the crop kinetic responses to drought. Some authors asserted that in perennial plant species, one of the most important drought survival strategies is the ability to recover from the stress when ASWC is restored, and selecting traits for high recuperative ability may be of economic importance equally to selecting for improved growth during drought (Moreira et al. 1990; Norris and Thomas 1982). The full recovery of photosynthesis after a severe drought stress was reported for sorghum (Fracasso et al., 2016), whereas other authors reported limited recovery in cotton (Ennahli and Earl, 2005), in turfgrass (Hua et al., 2010), or, with different behavior depending on variety, in tobacco (van Rensburg and Kruger, 1993). Our findings showed a recuperative ability of Arundo donax that can be inferred by what observed on October measurements, when ASWC
7
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quality improvement of the biomass. The differences emerged in terms of biomass quality (i.e. dry matter and ash content) could be adopted as driving lines of crop management approaches depending on final destination of harvested biomass. When the quantity is the main goal (e.g. biogas production, biochemical compound extraction, etc.), product lost, due to the fall of leaves and stalks usually observed in late harvest, is obviously to be considered as a negative aspect. On the contrary, when the objective to reach is a lower amount of ash for combustion purpose, the late harvest could be preferred solution.
76% (Curt et al., 2018). The unusually large amount of precipitation occurred starting from September, surely masked the irrigation effects in our trial. Nevertheless, these reduced differences could be also explained by the capacity of the rain-fed treatment to grow, in our environment, in autumn/winter and to achieve a full recovery of net assimilation after soil water restoring. Stem height (described for the same experiment by Haworth et al., 2019) showed a higher daily increment in the stressed treatment following the late summer precipitations. As a consequence, the rain-fed plants were able to maintain a higher photosynthesis and, in turn, a higher growth rate later in the year compared with irrigated plants that, in contrast, began to senescence earlier. We noticed clear differences in biometric traits among clones, and Morocco showed higher values of stem height and basal diameter and a low leaves yield, while Tuscany exhibited higher stems density (number m−2). These results confirmed the data already recorded during the establishment of the crop (previous year - data not reported) and are in keeping with the results reported by Zegada-Lizarazu et al. (2018) obtained with the same clones used in our experiment. They observed taller plant, a significant minor number of tillers (-38%) and 27% higher dry biomass in the Moroccan ecotype than in Tuscany one, cultivated for two consecutive years in well-watered condition in rhizotrons. The authors stated that the higher productivity of Morocco could be related to the plant higher volume (i.e. height and diameter), and suggested that these traits should be taken into account in ecotypes selection activities. Cosentino et al. (2006), Pilu et al. (2014), and Fabbrini et al. (2019) indicated that stem height and basal diameter had a moderate-to-high heritability. Consequently, these could be useful traits for clonal selection activities. Moreover, the Moroccan ecotype had more xylem vessels with a higher diameter and a lower structural density than the Tuscan ecotype (Haworth et al., 2017b), allowing a higher hydraulic conductivity that may favor rapid growth. Furthermore, clonal selection may take advantage also of inheritable phenotypic traits, such as leaf area index, stem dry mass, stem density, number of nodes per stem, stem height and diameter, described by Fabbrini et al. (2019). The use of A. donax as biofuel shows some difficulties connected to the high ash content, that reduces thermal conversion efficiency and may cause problems to combustion process (Coulson et al., 2004; Smith and Slater, 2011). Nevertheless, new transformation processes and biorefinery approach for added value compound extraction contribute to expand the potential use of the crop escaping quality limitation (Antonetti et al., 2015). The amount of ash, remarkable when compared with other energy crops, comes mainly from leaf tissues (Coulson et al., 2004; Monti et al., 2008; Nassi o Di Nasso et al., 2010). Amaducci and Perego (2015) reported significant difference among clones for the plant ash content ranging from 5.3% to 8.1%. Monti et al. (2008) reported values for ash content of 11.3% and 3.2% for leaves and stems, respectively. Furthermore, Corno et al. (2014) indicated a decrease in the total plant ash content along the growing season, ranging from 9.9% DM for the May samples to 3% DM for the March samples. In comparable field conditions, Scordia et al. (2012) reported a value of 5.9% in the whole plant ash content of the late harvest. In respect to the studied quality aspects, Morocco emerged for its positive traits. The lower ash content associated with the reduced leaves biomass indicates that this clone may potentially have the higher thermal conversion efficiency. Moreover, the lower ash content measured at late harvest in the whole biomass can also influence the technical decisions concerning crop management. In our results, the amount of ash in leaves were 17.0 and 10.7% on DM basis for early and late harvest, respectively, whereas stems showed values around 4% irrespectively of the harvest dates. The observed reduction in the leaf yield at late harvest resulted in a relevant reduction in total amount of ash per hectare (about 45% of the total ash content of the early harvest, i.e. 0.84 vs. 1.85 t ha−1, respectively). Therefore, the yield reduction observed in late harvest produced a
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