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Growth Responses of the Perennial Grass, Phalaris Aquatica L., to Cutting Frequency and Influence on Secondary Metabolites and Antioxidant Activity
S. Sai Kachout1,* S. Ben Youssef1, S. Khnissi1, K. Guenni1, A. Zoghlami1, A. Ennajah2, N. Ghorbel3, J. Anchang4, N. P. Hanan4
Published in Journal of the ASABE 66(3): 577-587 (doi: 10.13031/ja.15370). Copyright 2023 American Society of Agricultural and Biological Engineers.
1National Institute of Agronomic Research of Tunis, INRAT, Tunis, Tunisia.
2Institut Supérieur des études Préparatoires en Biologie et Géologie, Tunis, Tunisia.
3Faculty of Mathematical, Physical, and Natural Sciences of Tunis, Tunis, Tunisia.
4Department of Plant and Environmental Sciences PES, New Mexico State University, Las Cruces, New Mexico, USA.
*Correspondence: salmasey@yahoo.fr
The authors have paid for open access for this article. This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License https://creative commons.org/licenses/by-nc-nd/4.0/
Submitted for review on 27 September 2022 as manuscript number ES 15370; approved for publication as a Research Article by Associate Editor Dr. Lin Wei and Community Editor Dr. Kasiviswanathan Muthukumarappan of the Energy Systems Community of ASABE on 17 February 2023.
Highlights
Abstract. Perennial grasses are the key to the economic and environmental sustainability of pastures for livestock, and in arid and semi-arid environments, they can provide multiple ecosystem services more effectively than production systems based on annual crops. The objective of this study was to evaluate the effect of different defoliation frequencies on forage production and nutritive value of the Phalaris aquatica L. variety Soukra under field conditions in Tunis, Tunisia, over a period of 12 weeks. We tested four defoliation frequencies: (1) severe, (2) moderate, (3) infrequent, and (4) control. The growth responses measured were plant tiller number (NT), dry matter production (DM), and relative leaf regrowth rate (RLR). DM under the severe and moderate defoliation frequencies was 7% and 41% less than under control defoliation, respectively. However, DM production under infrequent defoliation was 91% and 43% higher than under severe and moderate defoliation. The relative leaf regrowth rate was affected by defoliation frequency; the highest regrowth rate was under severe treatment. However, tillering of P. aquatica was reduced under the severe and moderate frequencies of defoliation. Under increased defoliation frequencies, concentrations of secondary metabolites significantly decreased; total polyphenol content, flavonoid content, and tannin contents were higher in control and infrequent than in moderate and severe treatments. Antioxidant activity also decreased significantly with defoliation compared to the control treatment. There were no significant differences (P > 0.05) in ABTS (3-ethylbenzothiazoline-6-sulfonic acid) among the defoliation frequencies. Pearson's r correlation and PCA (Principal component analysis) data revealed that growth parameters, secondary metabolites, and antioxidant activity have positive and negative correlations in distinguishing the control and defoliation treatments. Results indicate that P. aquatica management should target moderate harvest rates in the adoption of perennial grass forage production systems in Tunisia. Use of perennial grasses for forage production can contribute to sustained production, food security, and rural livelihoods, and move farming systems towards providing multiple economic, environmental, and social benefits.
Mediterranean pastures consist mainly of annual species, which undergo strong variations in their nutritional value during the year (Molle and Floch, 2008). The global effort to perennialize crop production offers an alternative to existing, highly disturbed annual grain production systems and has the potential to address soil degradation, reduce inputs, enhance ecosystem function, and increase resource-use efficiency while maintaining food security (Crews et al., 2018; Duchene et al., 2019).
Perennial species represent a paradigm shift in agriculture and hold great potential to move towards sustainable production systems, particularly in arid and semi-arid regions. Annual crop production systems often require significant inputs in terms of irrigation water, synthetic mineral fertilizers, labor, and soil cultivation that can reduce soil cover and disrupt soil health and carbon sequestration (Toensmeier, 2017; Crews et al., 2018). Once established, perennial crops can contribute to improved soil structure and water retention capacity while also providing climate mitigation and adaptation services that promote biodiversity and ecosystem functions (Batello and al., 2013). Among the various types of lignocellulosic biomass, perennial grasses are considered an ideal source of bioenergy and bioproducts (Efthymia 2021). In general, perennial grasses are drought-resistant crops and have recently been attracting growing interest due to their extensive environmental benefits. Grass reseeding using native perennial species has been identified as a practical ecological strategy for restoring degraded African rangelands, enhancing vegetation cover, and forage production (Kevin et al., 2021). High-yielding, deep-rooted, perennial, herbaceous plants prevent erosion, build soil organic matter, support diverse belowground microbial communities, and produce seeds and biomass that can be harvested mechanically (e.g., Crews and Cattani, 2018; Crews et al., 2016; Pimentel et al., 2012). There are various ways in which perennial crops can be incorporated into agricultural systems, including rotation with annuals, in perennial monocrops, or in perennial polycultures (Cattani, 2014; Ryan et al., 2018). However, Llewellyn et al. (2014) concluded that there is potential for greater use of forage shrubs on soils marginal for cropping, but improved plant options or species mixtures are likely to be needed, in particular, options offering improved feed quality, together with a clearer demonstration of the economic value, if substantial increases in perennial forage shrub plantings are to occur.
Perennial pasture grasses, such as Phalaris (Phalaris aquatica) is broadly utilized in Australian farming systems, avoiding the cost and labor disadvantages of annual grasses and enhancing farming system sustainability (Reed et al., 2008). Alternative cropping systems, including perennial polycultures, may also be valuable for the conservation of Plant Genetic Resources for Agriculture (PGRFA) (FAO, 2009; Heywood, 2011; Jackson and Ford-Lloyd, 1990). Perennial grasses help maintain the long-term fertility of agricultural soils. They have many benefits as an energy crop; they are easy to grow, harvest, and process. A successfully established, vigorous, and well managed perennial pasture can be expected to survive and provide increased production and environmental benefits for many years. Perennial hay, forage, and pasture crops are attracting greater attention of researchers, and farmers are looking for alternative crops to improve soil and water quality and farm profitability. Perennial grasses and legumes are the most important sources of fibrous energy for ruminants in Tunisia and may be consumed directly via grazing or eaten after conservation as hay or silage (Chakroun et al., 1995).
Phalaris aquatica L. is a perennial cool season herbaceous plant, known by the common names bulbous canary grass or harding grass, native to the Mediterranean area, which has been used for forage breeding programs due to its high biomass yield potential and drought tolerance (Pappas et al., 2013; Oram et al., 2009). It has been naturalized in South Africa, Australia, New Zealand, Northern Europe, and Northern America (USDA, NRCS, 2013) and has been extensively used as nutritional fodder (Oram et al., 2009). Phalaris is one of the most important sown perennial grasses; it is valued for its high productivity (Anderson et al., 1999; Reed et al., 2008) and ability to survive drought (Robinson and Simpson, 1966; Hutchinson, 1970). Nevertheless, stresses related to climate and soil interact frequently with grazing pressure to reduce persistence of phalaris. It displays high productivity throughout the cooler seasons, and nutritive quality comparable to other temperate grasses during this period (Hayes et al., 2010a). only 1 hayes in ref Generally, early harvesting increases the “greenness” of perennial grasses, thus increasing the potential suitability for anaerobic digestion, owing to lower C/N ratio, lignification, and higher protein content (Nizami et al., 2009). An excellent ability to survive periods of summer drought is a major favorable characteristic of phalaris, attributable to partial dormancy of buds located at the bases of reproductive tillers, combined with a deep root system capable of accessing sub-soil moisture (Oram et al., 2009; Hill, 1989; Reed et al., 2008).
Grazing systems need to be constructed to favor Phalaris persistence, avoiding grazing during sensitive periods. The critical period for grazing of Phalaris is during reproductive development, predominantly between stem elongation and ear emergence (Kemp and Culvenor, 1994). The study by Virgona and Bowcher (2000) suggests that the ability for Phalaris to maintain basal cover, increased with an increasing grazing interval. In treatments where grazing frequency was short and the plants had minimal recovery time, the basal cover was reduced to approximately 76% of the population present the previous spring. Similarly, Hill et al. (2004) found that lenient grazing regimes resulted in increases of Phalaris basal area. Studies have found that rotational grazing systems will favor perennial plant survival when compared to the same rate of set-stocking (Virgona, and Bowcher, 2000; Cullen et al., 2005a). This is predominately due to the plants ability to regrow between grazing periods, allowing for carbohydrate reserves to be replenished in preparation for summer dormancy. Species such as Phalaris are also highly tolerant of set stocking grazing regimes, which could be an advantage in cropping situations. They are broadly adapted and highly productive relative to most other alternatives (Hill, 1989; Reed et al., 2008). The nature of defoliation events (severity, frequency, and type of tissue removed) determines how the plant will recover (Richards, 1993).
Defoliation may initiate physiological recovery and chemical defense mechanisms that allow a plant to improve fitness after damage. Such responses may result in changes in plant resource allocation that influence growth and foliar chemistry. Secondary metabolites (terpenoids, cardenolides, alkaloids, flavonoid and phenolics) are organic compounds which are not necessary for plant growth, but essential for reducing plant palatability and as a deterrent against insect herbivory (Wuyts et al., 2004). According to Wu and Baldwin (2010), plants produce approximately 500,000 secondary metabolites, most of which are powerful pest-resistant chemicals, and the level of secondary metabolites varies depending on plant species and plant tissues. In many cases, plant chemical defenses reduce palatability for large herbivores, including domestic livestock, impacting growth, development, and digestion. Phalaris aquatica is known to cause toxicity in livestock in the form of acute or chronic staggers or sudden death neurological (SDN) syndrome (Read et al., 2020). Feeds containing natural phytocompounds such as condensed and hydrolysable tannins represent a sustainable means of reducing the environmental impacts of ruminants; tannin-containing feeds reduce enteric CH4 emissions and urinary N excretion (Maamouri et al., 2011; Tan et al., 2011; Aguerre et al., 2016). Tannins in particular can negatively affect the feeding and digestion of starch and other nutrients (Huang et al., 2018). By contrast, secondary metabolite profiles in some forage plants enhance large herbivore (including domestic livestock) consumption, and livestock growth and development can be enhanced when animals are offered a variety of forage species with different kinds and amounts of secondary metabolites (Provenza et al., 2009; Provenza and Villalba, 2010; Meuret and Provenza, 2015). Physiological indicators of secondary metabolites measured in Phalaris suggest the presence of a variety of phenolics, flavonoids, and tannins.
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| Figure 1. Monthly minimum and maximum temperature (°C) and precipitation (mm) over the experimental period. |
The aim of this experiment was to evaluate Phalaris growth and productivity and compare Phalaris response to continuous defoliation over a 12-week period.
Materials and Methods
Study Site
The experiment was conducted on the experimental fields the Experimental Station of INRAT (35°87 N, 9°96 E) in Tunisia. The study site (fig. 1) has a long-term mean annual precipitation of 250 mm/year, most of which falls in the cooler winter months.
Treatments and Experimental Design
The experiment was conducted using a randomized block design and three defoliation treatments across four sampling dates, over 12 weeks in order to achieve this in a sustainable way, a cutting regime must be selected which has the least impact on the crop canopy during the growing season. Four replicates of each defoliation treatment were established in February to May of 2021, with a plot size of 1.5 by 2 meters. The cultivar that was used is soukra; year of release is 1965; releasing organization is INRAT, Tunisia; Characteristics: Large seedlings, vigorous winter growth, early flowering, sheds ripe seeds.
Defoliation treatments were imposed using hand shears starting 40 d after the tillers were transplanted and with varying frequency and timing depending on the defoliation intensity treatment (table 1). To measure responses to defoliation treatments, at each harvest time biomass fresh weight was determined in a 1m2 destructive sampling sub-plot within each plot. Individual plant subsamples (10 stems) were also partitioned to assess leaf number and biomass and stem biomass. Harvested biomass was dried in an air oven at 40°C for 96 h and weighed. Dry matter for control was measured by harvesting the total plot herbage mass on four replicates after 12-week interval on 10 May. Hand shears were used for cutting the herbage to a height of 180cm.
The relative growth rate (RGR, g g-1 day-1) growth analysis is conducted on plants that have been subjected to significant losses in biomass and leaf area between harvests. The method is particularly useful to analyze the effects of defoliation on growth and biomass allocation.
Plant relative growth rate (RGR) depends on biomass allocation to leaves (leaf mass fraction, LMF), efficient construction of leaf surface area (specific leaf area, SLA), and biomass growth per unit leaf area (net assimilation rate, NAR). (Evans, 1972):
(1)
Determination of Total Phenols
Total phenol contents were assessed in 5 g of P. aquatica plant samples. Samples were extracted with a cold mortar
and pestle with 70% methanol containing 1% HCl, and then sonicated for 25 min. After centrifugation at 12.100× g for 20 min at 5 °C, the pellets were sonicated, and supernatants were pooled together and filtered using 0.45 µm Minisart filters. Folin-Ciocalteu reagent (125 µL) was added, and after 1 min, 1,250µl of aqueous Na2CO3 solution (7% w/v) was added. The flask was kept for 1 h at ambient temperature after the contents had been mixed thoroughly. Absorbance was read at 760 nm. Results were expressed as milligrams of gallic acid equivalent per gram dry weight of air-dried material (mg GAE/g). Each crude extract from each treatment was analyzed in triplicate.
Determination of Total Flavonoids
The quantification of total flavonoids in extracted samples of P. aquatica was determined according to Dewento et al. (2002). An aliquot (250 µL) of the diluted extract was added to a 5 mL volumetric flask and mixed with 75 µL of NaNO2 (5% w/v). After 5 min, 150 µL of AlCl3 (10% v/v) was added. After 1 min of mixing, NaOH (1 M; 1 mL) was added, and the total volume was made up to 5 mL with distilled water. The solution was mixed well, and the absorbance was measured against blank solution at 510 nm. Total flavonoids content was expressed as milligrams of quercetin equivalent per gram dry weight of air-dried material (mg QE/g DW).
DPPH Radical-Scavenging Assay
DPPH (1,1-Diphenyl-2-picrylhydrazyl) scavenging activities of samples were evaluated following Hatano et al. (1988). Diluted extracts (1:5; 50 µL) were added to an aqueous methanolic solution (82%) of DPPH. The macrocuvettes were covered, shaken well, and maintained in darkness for 30 min; the absorbance was measured at 517 nm. The percentage scavenging of DPPH was calculated according to the formula:
| Table 1. Definition of defoliation treatments during the experimental period. | ||
| Defoliation Treatment | Cutting Date | Number of Cuttings |
| S (Frequent, every 3 weeks) | 15 Feb;08 Mar;29 Mar;19Apr;10 May 2021 | 5 cut |
| M (Moderate, every 6 weeks) | 15 Feb; 29 Mar;10 May 2021 | 3 cut |
| L (Infrequent, every 10 weeks) | 01 Mar; 10 May 2021 | 2 cut |
| C (Control) | 10 May 2021 | 1 cut |
(2)
ABTS Free Radical Scavenging Activity
The ABTS (3-ethylbenzothiazoline-6-sulfonic acid) was determined using the protocol described by Arnao et al. (2001). A stock solution of 7 mM, 10 ml ABTS, and 2.4 mM, 10 ml potassium persulphate were prepared with distilled water and mixed in order to generate the ABTS free radical (ABTS•+). The resulting solution was incubated in the dark at room temperature for 12 h until the reaction was complete, as observed by a constant absorbance. The ABTS•+ solution (1 ml) was further diluted in 50 ml of methanol. To 200 µl of the infusion, 800 µl of the ABTS solution was added, mixed, and incubated for 30 min. The absorbance was read at 734 nm. The free radical scavenging capacity was compared with that of BHA, and the percentage inhibition was calculated as:
(3)
where
Ac = ABTS•+ solution
As = ABTS•+ extract mixture/standard.
A negative control was prepared by replacing each fraction or standard with an equal volume of methanol.
Data Analysis
The effects of defoliation frequencies were evaluated by one-way ANOVA, and significant differences between means were assessed by the least significant difference test at 95% confidence level (p = 0.05). Bars shown in figures represent the standard error of the means of three independent experiments (n = 4), each analyzed twice. Pearson correlation analysis and principal component analysis (PCA) were carried out to evaluate the relationship between plant traits and experimental treatments.
Results
Dry Matter (DM) Production
Dry matter production was decreased by defoliation frequencies (P<0.05) (fig. 2). The harvest production resulting from the control treatment resulted in greater biomass than other treatments, and the severe treatment had smaller DM production than the moderate and infrequent harvests (fig. 2). The DM concentration of 12-week-old herbage was 3500 g/plot greater than that of 3, 6, and 10 weeks of regrowth. These responses to maturity likely explain greater DM concentration of single (control) vs. multiple harvest treatments in the current experiment.
Relative Leaf Regrowth Rate
Relative leaf regrowth rate was defined as the time taken for one leaf per perennial Phalaris tiller to fully emerge following defoliation. The relative leaf regrowth rate (mg g-1 d-1) was affected by defoliation frequency (fig. 3). Phalaris aquatica maintained the highest regrowth rate throughout the experiment under severe treatment; it had a regrowth rate intermediate between moderate and infrequent frequencies of defoliation, while the control was not significantly different from moderate and infrequent treatment (fig. 3). All frequencies of defoliation showed similar responses to the period of defoliation, with regrowth rates being highest after a three-week period (severe), but no significant difference between moderate, infrequent, and control treatments (fig. 3).
Dry Biomass Partitioning Between Leaf and Stem Production
The main effect of different frequencies of defoliation on the distribution of dry matter accumulated in leaves and stems in the interval between harvests was not significant (fig. 4). The Phalaris plants showed opposite effects: severe defoliation resulted in proportionally increased partitioning of the total dry mass increment to leaves, but partitioning to stems was proportionally greater with infrequent defoliation (fig. 4). In control, shading resulted in stems receiving an increased fraction of the total increment in dry mass (fig. 4). With moderate treatment, there was a small decrease in the proportional distribution of dry matter to stems.
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| Figure 2. Mean dry matter (DM) production (g/plot) of P. aquatica cv. ’Soukra’ under field conditions when exposed to different defoliation frequencies during growth. Data are means of four replicates; error bars indicate ±1 SD. Fisher LSD test was used to detect differences among means to a significance level of 5%; different letters indicate statistical differences between sampling. |
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| Figure 3. Effect of frequency of defoliation on relative leaf regrowth rate (mg g-1 d-1) of defoliated plants of P. aquatica cv. ’Soukra’. Data are means of four observations; error bars indicate ±1 SD. Fisher LSD test was used to detect differences among means to a significance level of 5%. |
Tiller Number
Tillers per 3 m2 were measured to assess the possible variations in individual plant tillering between February and May. The effects of cutting frequency on tiller number per m2 are illustrated in figure 5. Infrequent defoliation did not significantly influence individual plant tillering, indicating that there was minimal variation between individual plants throughout the treatments. Tillering of P. aquatica was reduced under the severe and moderate frequency of defoliation, mostly due to the increased delay between the appearance of a daughter tiller and the appearance of its subtending leaf. Although the tillering rate is decreased by defoliation, it is not stopped even under severe defoliation (fig. 5). Thus, under a sequence of repeated intense defoliation of plants, the tiller number per plant continues to decrease, and the leaf elongation rate progressively declines, leading to a major decrease in leaf length and therefore tiller size.
| Table 2. Secondary metabolites and antioxidant activity of P. aquatica under different defoliation frequencies.[a],[b] | ||||||||
| Defoliation Frequencies | Secondary Metabolites/ | Antioxidant Activity | DPPH | |||||
| Total Polyphenol Content | Flavonoid Content | Tannin Contents | ABTS | |||||
| S (Frequent) | 3.4 ± 0.2bc | 1.4 ± 0.2b | 1.3 ± 0.0d | 91a | 21.2 ± 8.0c | |||
| M (Moderate) | 3.9 ± 0.3b | 1.7 ± 0.5ab | 1.5 ± 0.0c | 92a | 27.1 ± 7.4a | |||
| I (Infrequent) | 4.1 ± 0.2a | 1.9 ± 0.3a | 1.6 ± 0.0b | 95a | 26.5 ± 7.3ab | |||
| C (Control) | 4.2 ± 0.1a | 2.1 ± 0.4a | 1.9 ± 0.2a | 93a | 29.5 ± 8.3a | |||
| [a] TPC = total polyphenol content, FC = flavonoid content, TC = tannin contents, ABTS = 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, DPPH = 2,2'-diphenyl-1-picrylhydrazyl, S = frequent, M = moderate, l = infrequent, and C = control. [b] Means not followed by the same lower case letter are different (P<0.05). | ||||||||
Effects of Defoliation Treatments on the Secondary Metabolites and Antioxidant Activity
All the considered secondary metabolites and antioxidant activity parameters varied according to the harvest frequency. Both total polyphenol, flavonoid, and tannin contents were higher in control and infrequent treatments than in moderate and severe treatments at each harvest, showing downward trends with the harvest frequency, with the only exception of flavonoid content in leaves at moderate defoliation that was not statistically different from that of infrequent and control treatments (table 2).
Regarding antioxidant activity (ABTS, DPPH), all parameters showed decreasing concentrations in leaves with increasing harvest frequency. DPPH varied from 29.5 in the control (no defoliation) to 21.2 in severe defoliation treatments, while in moderate and infrequent treatments, the concentration was statistically similar (about 27). In leaves, the ABTS concentration was rather stable, ranging from 91 in severe to 93 in control treatments without significant differences (table 2).
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| Figure 4. Dry biomass partitioning of P. aquatica cv. ’Soukra’ harvested at different frequency of defoliation. Values with the same letter are not significantly different (p = 0.05). Data are means of four observations; error bars indicate ±1 SD. |
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| Figure 5. Effects of defoliation frequency on number of tillers per plant of the Phalaris cv. ’Soukra’. Data are means of four observations; error bars indicate ±1 SD. Fisher LSD test was used to detect differences among means to a significance level of 5%. |
Correlation Between Growth Parameters, Secondary Metabolites and Antioxidant Activity
Pearson correlations were calculated to determine the relationship among the growth parameters and physiological responses. Regarding correlations among growth parameters, FM showed positive correlations with NT and DBPS (dry biomass partitioning of stem). RLR was only positively correlated with DBPS in leaves plant (fig. 6). A positive correlation between NT and DBPS was also highlighted. The secondary metabolites: TPC, FC, and TC concentration show significant correlation with the growth parameters, as shown in the correlation matrix (fig. 6). ABTS was negatively correlated with growth parameters, while DPPH was positively correlated with DM, NT, and DBPS. Conversely, DPPH in leaves of Phalaris was positively correlated with all secondary metabolites.
Principal component analysis was performed using the relative values of all growth parameters, secondary metabolites, and antioxidant activity of Phalaris aquatica under field conditions over defoliation frequencies to represent physiological responses. To evaluate the contributions of each parameter in the control and defoliation-treated Phalaris plants, we performed PCA using five growth parameters (DM, RLR, NT, DBPS, and DBPL), three secondary metabolites (TPC, FC, and TC), and two-antioxidant activities (DPPH and ABTS) (figure 7). The effect of defoliation on secondary metabolites profiles was driven primarily by decreases in condensed tannins and varied total polyphenol and flavonoid contents between defoliation treatments. Antioxidant activity profiles were also influenced by defoliation frequency as well as the interaction with growth responses.
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| Figure 6. Pearson’s r correlation between growth parameters, secondary metabolites, and antioxidant activity of Phalaris aquatica under field conditions over defoliation frequencies. Bold values show significant correlations (p < 0.05): DM = dry matter, RLR = relative leaf regrowth rate, NT = number of tillers, DBPL = dry biomass partitioning of leaf, DBPS = dry biomass partitioning of stems, TPC = total polyphenol content, FC = flavonoid content, TC = tannin contents, ABTS = 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and DPPH = 2,2'-diphenyl-1-picrylhydrazyl. |
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| Figure 7. Principal component analysis (PCA) for growth responses in Phalaris plant. PC1–PC2 variables loading plots during different defoliation frequencies. PC1–PC2: the first and second principal component: DM = dry matter, RLR = relative leaf regrowth rate, NT = number of tillers, DBPL = dry biomass partitioning of leaf, DBPS = dry biomass partitioning of stems, TPC = total polyphenol content, FC = flavonoid content, TC = tannin contents, ABTS = 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and DPPH = 2,2'-diphenyl-1-picrylhydrazyl. |
Discussion
Under current scenarios of climate change for Tunisia, with the likelihood of longer and more severe droughts, increased adoption of Phalaris may be anticipated for the creation of sustainable grasslands. Perennial grasses are excellent protectors of the soil surface and prevent erosion, with turf-forming species having the greatest effect. Perennial grasses are characterized by their ability to regrow after harvest. Such plants usually provide more ground coverage and have a longer growing season than annual crops; they also possess an extensive root system in the soil. Noelle et al. (2020) showed that a 10-day grazing period during the growing season is an effective strategy to avoid second defoliations on individual perennial grass plants while maintaining sufficient forage for use during the dormant winter grazing season. Defoliation in maturity often increases DM concentration due to higher stem mass. Recently, considerable effort has been made to select Phalaris, a productive perennial grass species with good drought survival, for tolerance of the other major stresses that adversely affect persistence (Culvenor and Simpson, 2014). However, timing and harvest frequencies significantly affected plant maturity and consequently extractable CP contents and protein concentrate yields (Nielsen et al., 2021). The higher relative herbage regrowth rate of Phalaris following severe defoliation was attributed to increased light penetration into the sward and the greater photosynthetic efficiency of the smaller and younger leaves after severe defoliation. Although the effect of defoliation severity on tiller number in the current study was significant, there was a trend toward greater tiller number in plots defoliated infrequently (6 tillers/plant) for swards defoliated severely (3 tillers /plant respectively). The relative growth rate (RGR) is an important trait distinguishing plant species’ strategy (Reich et al., 2003a). Species with high growth rates are more competitive in acquiring resources, whereas species with low growth rates are more conservative with the scarce resources they have obtained (Reich et al., 2003b; Poorter and Garnier, 2007) or are able to overcome disturbance events by allocating resources to storage rather than to growth (Poorter and Kitajima, 2007).
Harvest frequency and timing are likely to be important management practices in grass biomass production systems. Harvest frequency affects biomass yield (Woodard and Prine, 1991), and multiple or delayed harvests (Knoll et al., 2012) could be used to extend the portion of the year. Multiple harvests per year, however, may reduce the persistence (Woodard and Prine, 1991) of perennial species and increase nutrient concentration, leading to greater nutrient removal in harvested biomass (Guretzky et al., 2011; Kering et al., 2013). In contrast, harvesting switchgrass (Panicum virgatum L.) only once per year after plant senescence resulted in nutrient remobilization from aboveground biomass to storage organs, thereby reducing nutrient removal (Guretzky et al., 2011; Kering et al., 2012a) and amount of fertilizer required the following growing season (Knoll et al., 2012, Kering et al., 2012b).
Later maturity often increases FM concentration due to higher stem mass, leaf senescence, and leaf abscission (Knoll et al., 2012; Woodard et al., 1993a). These responses to maturity likely explain greater FM concentration of single vs. multiple harvest treatments in the current experiment. For example, when five perennial C4 grasses were harvested at either 8 or 12 weeks following seedling emergence, the DM concentration of 12-week-old herbage was 36 g/kg greater than that of 8-week regrowth (Costa and Gomide, 1991). Phalaris decreased tiller number per plant in the defoliated treatment, suggesting that this species was capable of some development under severe defoliation. Cullen et al. (2005b) identified that an increase in either tiller density or tiller dry weight would increase the overall yield of the sward. This study also observed a negative linear correlation between tiller density and tiller weight.
Defoliation frequency is an important management factor that affects DM production (Nevens and Rehuel, 2003) and nutritive value (Turner et al., 2006a) of forage grasses by changing the morphology and physiology of plants (Ahmed et al., 2001). Generally, increasing defoliation frequency has been shown to have a beneficial effect on the nutritive value of many grass species (Pontes et al., 2007).
As discussed earlier, defoliation may initiate physiological and chemical defense mechanisms that allow a plant to improve fitness after damage, impacting plant resource allocation, growth, and foliar chemistry. We found that plants of Phalaris respond to stress in various ways, including general decreases in concentrations of most secondary metabolites. Although all the herbage harvested during the study was considered to have a high secondary metabolites value, the herbage harvested from plots that had been previously frequently defoliated had lower concentrations of tannins, flavonoids, and other secondary metabolites. We also found a shift in allocation patterns where severely defoliated plants allocated a higher fraction of production to replacing lost leaves. However, the effect was small and did not affect secondary metabolites concentration or antioxidant activity.
Due to the paucity of information on the phenolic composition of P. aquatica, we compared our findings with published data on other species of grasses from the family Poaceae. Harvest time typically influences both biomass yield and quality of perennial grasses (Kandel et al., 2013b; Ragaglini et al., 2014; Massé et al., 2010). However, stem biomass of P. aquatica was higher than leaf biomass at all the considered harvest times, with the remarkable exception of the biomass regrown after the cut in May, due to the reduced stem elongation and the high juvenility of the crop (Nizami et al., 2009).
Differences in response to defoliation frequency among grass species may be attributed to differences in the developmental rates of leaves (Fulkerson and Donaghy, 2001). Thus, the recommended defoliation interval for Dactylis glomerata is the four-leaf stage in order to maximize rates of regrowth (Turner et al. 2006a), and two to three leaves per tiller for Lolium perenne (Donaghy and Fulkerson, 1997). Depending on the purpose for which Phalaris is cropped, different management strategies can be hypothesized, involving different harvest frequencies and harvest times that can significantly affect biomass characteristics. In addition to breeding efforts to increase forage yields and other desirable traits in perennial forages, research is needed to assess the potential for managing perennial forages and to better understand the environmental benefits that can be realized with perennial crops. Nonetheless, our results can be used to improve training programs, guide research activities, and facilitate the development of perennial forage cropping systems.
Conclusion
In conclusion, there was interaction between growth parameters and defoliation severity for herbage regrowth of P. aquatica. High DM production was obtained under infrequent defoliation treatment; the relative leaf regrowth rate was affected by defoliation frequency, with the highest relative regrowth rate under severe defoliation. Although defoliation contributed to the difference in herbage regrowth derived from variation in the frequency of defoliation. The effect of defoliation severity on herbage regrowth was associated with variation of secondary metabolite content and antioxidant activity. These results suggest that Phalaris may be suited to conservation pasture systems, but for optimum production, the interval between cuts should not exceed about 6 weeks in order to maximize rates of regrowth. Further cultivar improvements are needed to make additional productivity and sustainability gains, particularly utilizing local germplasm from Tunisia. Multidisciplinary investigations are needed to identify the best-adapted and most productive perennial grasses, cultivars, and mixtures for animal production in each region, along with the most appropriate grazing management. This work must be oriented toward greater integration between basic and applied science, which will also provide greater opportunities for innovative solutions to reach farmers.
Acknowledgments
We thank A. Zoghlami and S. BenYoussef for their inspiration and encouragement in the inventory project. We also acknowledge the research assistants at INRAT and INRGREF for their valuable help. Special thanks go to J. Anchang and N. Hanan at PES for their insightful comments and revisions. This work was supported by the PEER Project, with funding from the National Aeronautics and Space Administration (NASA, USA), through the Partnerships for Enhanced Engagement in Research (PEER) program cooperative agreement number: AID-OAA-A-11–00012. The authors declare no conflict of interest, and the funders had no role in the study design, data collection and analysis, manuscript writing, or decision to publish. SSK, SBY, and AZ designed the research, SK and KG collected samples, AE and NG analyzed samples, and SSK analyzed the results and drafted the manuscript. JA and NH provided revisions to the manuscript. All authors consented to publish in this journal, and all data analyzed in the study are available upon reasonable request from the corresponding author.
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