Mean Leaf Angles Affect Irrigation Efficiency and Physiological Responses of Tropical Species Seedling

Abstract

In forest nurseries, irrigation management becomes more complex as different seedlings of tropical species, with different architectures, are grown close to each other. In this context, there are gaps in knowledge about the physiological responses of species with different mean leaf angles when subjected to different irrigation depths. Thus, this work aimed to analyze whether mean leaf angles affect irrigation efficiency and, consequently, physiological responses of tree seedlings. Six species with different mean leaf angles were submitted to three irrigation depths (6, 9, and 12 mm) applied daily by micro-sprinklers in a completely randomized design in a split plot scheme. The following variables were evaluated: leaf water potential, stomatal conductance, relative water content in the leaf, daily transpiration, leaching fraction, and total dry mass. In tree species seedlings with positive mean leaf angles, smaller irrigation depths are already able to increase leaf water potential, stomatal conductance, leaf relative water content, and transpiration efficiency. In contrast, when the mean leaf angles are negative, it is necessary to apply larger irrigation depths so that seedling physiological responses do not reduce the production of total dry mass.

1. Introduction

Most reforestation projects use seedlings produced in forest nurseries [1] and the rapid establishment of seedlings in forest regeneration or afforestation sites after planting is a prerequisite for successful reforestation [2]. Seedling production intensively demands water and nutrients [3] and the handling of these inputs needs to be appropriate to species, time of year, type of container, and substrate [4,5]. In view of this great demand, a reduction in the availability of surface and groundwater is expected, as well as an increase in regulations regarding the flow of water and nutrients [6]. In this context, irrigation efficiency in forest nurseries needs to be substantially improved in order to allow them to maintain profitability and minimize environmental impact [7,8].

Managing micro-sprinkler irrigation systems in forest nurseries is especially challenging because different species, at different growth stages, are grown nearby and usually submitted to the same water management [9]. In micro-sprinkler irrigation, differences in water use and abstraction between species may mean that the appropriate deficit for one species is harmful to the other [10]. The lack of knowledge about the amount of water required and how seedling architecture affects water capture limits efficiency improvements, that is, it does not help nurseries to reduce the amount of water applied and leachate fertilizers [11]. Plant architecture is defined as a three-dimensional organization of its body involving the branching pattern, size, shape, and leaf position [12]. Regarding leaf position, the angles formed between leaf surface and horizontal plane are defined as mean leaf angles [13].

Most studies have investigated leaf angle as a functional characteristic that influences light interception [14,15,16]. The effect of leaf angle on light interception efficiency, photosynthetic rate, and yield has been investigated on cereal crops [17], yet significant knowledge gaps remain in understanding the effects of this complex trait in tropical tree seedlings.

A recent study with tree seedlings revealed that the mean leaf angle and the irrigation depths are factors that can provide differentiated growth [18]. The growth of plants is a consequence of several physiological processes controlled by environmental conditions and the genetic characteristics of each plant species [19].

To provide the information necessary for making sound restoration site decisions, a seedling quality program needs to combine morphological and physiological attributes [20]. The physiological attributes can also be used to relate seedling quality to field performance after planting [21]. Improved survival is the result of greater drought resistance and improved seedling nutrition at planting, which increases the speed with which seedlings can overcome planting stress, become established, and grow on the forest restoration site [22].

In this sense, physiological studies with tropical tree species with different mean leaf angles subjected to different irrigation depths have received little attention. Here, we hypothesized that tree seedlings with different mean leaf angles might show differences in their water capture and, consequently, in their physiological responses.

2. Materials and Methods

2.1. Study Site and Mean Leaf Angle Characterization

The experiment was conducted in the forest nursery of the School of Agriculture of São Paulo State University (UNESP), municipality of Botucatu, Brazil, located at the geographical coordinates 22°51′ S and 48°26′ W and an altitude of 786 m (Figure 1). According to the Köppen classification, the region’s climate is Cfa type—hot temperate (mesothermic) humid climate with rains in the summer and drought in the winter. The average temperature in the hottest month is above 22 °C, with potential evapotranspiration of 945.15 mm, actual evapotranspiration of 940.0 mm, water deficit of 5.10 mm, and water surplus of 488.30 mm [23].

To characterize the mean leaf angle of the six tree species, 30 seedlings of each species with standard size (approximately 25 cm) for field planting were measured with a plastic protractor according to [13]. The angles formed between leaf surface and horizontal plane (θ = 0° → horizontal plane; θ = 90° → vertically upwards; θ = −90° → vertically downwards) were measured on the first and second fully expanded leaves, in the top direction to the bottom of each seedling. Tree seedling species with different mean leaf angles used in this study are shown in Figure 2. In Figure 3a,b we show how the seedlings with different mean leaf angles were measured, and in Figure 3c,d we graphically represent our hypothesis.

2.2. Experimental Design and Treatments

The experimental design adopted in the experiment was completely randomized in a split plot scheme, in which irrigation depths (6, 9, and 12 mm) were allocated in the main plots and mean leaf angles in the subplots (of the 6 species), resulting in 18 treatments.

The daily irrigation depths were applied by a micro-sprinkler irrigation system at 10:00 am and, at 3:00 pm, each bed received an irrigation depth (main plot) and in each bed, the six species (subplots) were randomly distributed. Each species had four trays, the nine central seedlings being considered useful for evaluation. In total, there were 36 seedlings per irrigation depth and 648 in the experiment.

2.3. Nursery Culture

To start seedling production, seeds were collected in fragments of Atlantic Forest and Cerrado in the State of São Paulo, Brazil. After beneficiation, sowing was carried out in polyethylene trays with 288 cells (15 cm³ per cell) filled with a substrate composed of sphagnum peat, carbonized rice husk, and vermiculite in the proportion (2:1:1 v:v:v), staying in a shade house with 50% shade and application of 6 mm irrigation depth day⁻¹ in 12 installments. At 30 days after sowing, seedlings of each species were transplanted into polyethylene tubes, with 120 cm³, packed in polyethylene trays with a capacity of 108 cells, previously filled with the same substrate. The occupancy density of the trays was 115 tubes m⁻².

Before starting the application of the irrigation depths, we guaranteed that the average height value (± standard deviation; cm) of each species did not differ between the plots because after initial measuring the seedling’s height, we applied the Scott-Knott statistical test, at 5% of significance, and we verified that there was no difference between the plots: Ficus luschnathiana (76°) 1.2 ± 0.6, Sapindus saponaria (57°) 11.5 ± 2.8, Iochroma arborescens (54°) 0.5 ± 0.3, Luehea grandiflora (−1°) 2.4 ± 0.8, Alchornea glandulosa (−44°) 13.3 ± 2.2, and Cecropia pachystachya (−64°) 1.9 ± 0.7.

To start the application of irrigation depths, seedlings were transferred to suspended beds, mini tunnel type, with a 150-micron diffuser agricultural film cover to prevent rain interference and micro-sprinklers with a flow rate of 129 L h⁻¹. The irrigation system was driven by an electrical panel, programmed to apply the specified irrigation depths.

Growth fertilization started with the irrigation depths of the treatments. All seedlings received a nutrient solution applied twice a week for 158 days using the Venturi dilution system. The growth solution was composed of purified monoamoniophosphate, magnesium sulfate, potassium chloride, calcium nitrate, urea, boric acid, sodium molybdate, manganese sulfate, zinc sulfate, copper sulfate, and iron sulfate, in concentrations of 295, 84, 200, 160, 38, 52, 4.6, 3.9, 1.2, 0.6, 0.3, and 25 mg L⁻¹ of N, P, K, Ca, Mg, S, B, Mn, Zn, Cu, Mo, and Fe, respectively. In the hardening phase, fertilization with potassium chloride at a concentration of 700 mg L⁻¹ of K was applied for 45 days by the same fertigation system.

At the end of the production cycle, 203 days after the start application of the irrigation depths were evaluated: leaf water potential, stomatal conductance, relative water content in the leaf, daily transpiration, leaching fraction, and the total dry mass.

2.4. Physiological Analysis

Leaf water potential (Ψ; MPa) was evaluated in five seedlings of each irrigation depth by mean leaf angle with the aid of a psychrometer model Dewpoint Potential Meter (WP4-T) (Decagon Devices, Pullman, WA, USA). At 12:00 h (midday), the first and second fully expanded leaves were collected in the top direction for the base of each seedling.

Stomatal conductance (g_s; mmoL m⁻² s⁻¹) was evaluated in five seedlings of each irrigation depth by mean leaf angle with the aid of a Leaf Porometer (Decagon Devices/Pullman, WA, USA) in a state of dynamic equilibrium. At 10:00 am, the evaluation was carried out in the first and second leaves fully expanded from top to bottom.

Regarding leaf relative water content (RWC; %), the evaluation was carried out in five seedlings of each irrigation depth by mean leaf angle, with the first and second leaves fully expanded from top to bottom. The leaf limbs were cut into a rectangle (3 × 4 cm) and weighed immediately to obtain fresh mass (FM). Then, samples were placed in Petri dishes, with filter paper, immersed in deionized water, and stored for 24 h at 5 °C for rehydration, according to [24]. After this period, samples were weighed to obtain the turgid mass (TM), and then, to obtain the dry mass (DM), they were taken to an oven (70 °C) until reaching a constant mass. The determination of RWC was made according to [25], using the formula:

$$\mathrm{RWC}=\frac{\left(\mathrm{FM}-\mathrm{DM}\right)}{\left(\mathrm{TM}-\mathrm{DM}\right)}\times 100$$

(1)

Daily transpiration (T; mg of H₂O m⁻² leaf s⁻¹) was evaluated by the gravimetric method, according to [26] with some modifications, in 10 seedlings of each irrigation depth by mean leaf angle. At 6:00 pm, seedlings were irrigated by capillarity until complete substrate saturation. After draining, tubes were wrapped in plastic bags and sealed with masking tape on the seedling’s neck, in order to prevent water from evaporating into the atmosphere. At 7:00 am the next day, the initial mass of each set (Im; mg) consisting of seedling + tube + plastic bag + masking tape was weighed and then kept in full sun. The final weighing of sets (Fm; mg) was performed 24 h (t; seconds) after the first, and then, all leaves of each seedling were detached from the stem to measure leaf area (LA; cm²) with the aid of an Area Meter LI-COR^®, model LI-3100C. Daily transpiration was calculated using the formula:

$$\mathrm{T}=\frac{\frac{\mathrm{Im}-\mathrm{Fm}}{\mathrm{LA}}}{\mathrm{t}}$$

(2)

2.5. Leaching Fraction

The leaching fraction (%) was measured in 15 seedlings of each irrigation depth by mean leaf angle. The leaching fraction is defined as the amount of water and nutrients (solution) that runs down the bottom of the container divided by the total amount of solution applied to the container [27]. To quantify the solution that flowed through the bottom of the tube after irrigation, plastic bags were attached with rubber bands to the tube. The total amount of solution applied to the tube was measured using the amount retained in the substrate after irrigation plus the amount of solution drained from the bottom of the tube. In order to quantify the amount of solution retained in the substrate, the mass of the tube + seedling + plastic bag was weighed on a precision electronic scale before and after each irrigation event.

2.6. Total Dry Mass

To measure the total dry mass (g), sectioning was done in the seedling stem region, separating aerial and root parts. Once cleaned of substrate particles, the root system and aerial part were packed in paper bags and taken to a forced circulation oven at 70 °C, until they reached constant mass, and weighed on a precision electronic scale. Through the sum of aerial and root dry mass, the seedlings’ total dry mass was determined.

2.7. Data Analysis

The normality of data was tested with the Shapiro–Wilk test [28]. The data were submitted for analysis of variance, and when the value of the F test [29] was significant (p < 0.05), the Scott-Knott test [30] was applied to compare treatments (p < 0.05). Analyses were performed using the STATISTICA software package [31].

3. Results

In the analysis of variance, the mean leaf angles and irrigation depths interacted significantly (p < 0.05) in all studied variables, indicating dependence between the effects of these factors.

3.1. Leaching Fraction

The increase in irrigation depths increased, at different levels, the leaching fractions, except for Cecropia pachystachya (−64°), where the leaching fractions did not differentiate. The species with negative mean leaf angles (−64, −44, and −1°), in all irrigation depths, provided, at different levels, lower leaching fractions than species with positive mean leaf angles (76, 57, and 54°), indicating that the negative angles made it difficult for water to reach the substrate and consequently affecting the irrigation efficiency (

Table 1. Effect of interaction between mean leaf angle and irrigation depth on leaching fraction (%) of seedlings (mean ± standard deviation).

Mean Leaf Angles (°)	Leaching Fraction (%)
	Irrigation Depths (mm)
	6	9	12
76	22.1 ± 3.8 Bc 1	34.0 ± 2.2 Bb	66.8 ± 1.7 Aa
57	45.9 ± 0.8 Ab	47.5 ± 0.7 Ab	53.0 ± 0.2 Ba
54	24.7 ± 1.6 Bb	42.3 ± 0.5 Aa	42.9 ± 1.4 Ba
−1	3.9 ± 0.4 Db	13.5 ± 4.2 Ca	16.8 ± 3.8 Ca
−44	11.3 ± 2.6 Cb	17.6 ± 1.9 Ca	19.2 ± 3.8 Ca
−64	6.5 ± 1.2 Da	7.2 ± 3.7 Da	7.3 ± 2.5 Da

¹ Means followed by the same capital letter in the column and the same lowercase letter in the row are not different from each other according to Scott-Knott’s test (p < 0.05).

).

3.2. Physiological Analysis

In species with positive mean leaf angles (76, 57, and 54°), irrigation depths did not provide differentiated water potential, indicating that these angles facilitated water arrival in the substrate and guaranteed the same water status to seedlings, even in the smallest depth irrigation applied (6 mm). In species with mean leaf angles of −1° and −64°, the 6 mm irrigation depth reduced leaf water potential, while in species with a mean leaf angle of −44°, the 6 and 9 mm depths did not differ from each other and produced lower water potentials of the 12 mm irrigation depth (Figure 4).

For stomatal conductance (g_s), irrigation depths applied to species with positive mean leaf angles (76, 57, and 54°) did not differ. On the other hand, in species with negative leaf angles (−64, −44, and −1°), the 6 mm irrigation depth reduced the seedlings’ stomatal conductance (Figure 5).

Regarding relative water content, the irrigation depths applied to species with positive mean leaf angles (76, 57, and 54°) did not differ, showing the same behavior as for leaf water potential. In species with mean leaf angles of −1° and −44°, the 6 mm irrigation depth reduced relative water content, while in species with mean leaf angle −64°, the 12 mm irrigation depth was necessary to increase this variable (Figure 6).

The increase in irrigation depths increased, at different levels, daily transpiration in all mean leaf angles. In species with leaf angles of 76, 57, 54, and −44°, the highest transpiration was observed in the 12 mm irrigation depth, while in species with −1 and −64° angles, irrigation depths of 9 and 12 mm did not differ to produce the highest transpiration (Figure 7).

3.3. Total Dry Mass

Regarding total dry mass, in species with positive mean leaf angles (76, 57, and 54°), irrigation depths of 6 and 9 mm did not differ from each other and produced a greater amount of biomass. In these species, the larger irrigation depth (12 mm) had a negative effect, since it generated a high fraction of leachate (

Table 2. Effect of interaction between mean leaf angle and irrigation depth on total dry mass (g) of seedlings (mean ± standard deviation).

Mean Leaf Angles (°)	Total Dry Mass (g)
	Irrigation Depths (mm)
	6	9	12
76	9.85 ± 0.75 Aa 1	9.26 ± 0.69 Aa	7.66 ± 0.55 Bb
57	8.52 ± 0.51 Ba	8.22 ± 0.75 Ba	7.09 ± 0.34 Cb
54	4.49 ± 0.34 Da	4.13 ± 0.19 Ea	3.67 ± 0.19 Eb
−1	2.36 ± 0.50 Fb	2.54 ± 0.22 Fb	3.37 ± 0.53 Fa
−44	5.56 ± 0.54 Cc	7.07 ± 0.43 Cb	8.52 ± 0.65 Aa
−64	3.88 ± 0.34 Eb	5.87 ± 0.60 Da	6.43 ± 0.59 Da

¹ Means followed by the same capital letter in the column and the same lowercase letter in the row are not different from each other according to Scott-Knott’s test (p < 0.05).

), causing greater loss of nutrients and consequently less seedling growth. In species with mean leaf angles of −1° and −44°, a 12 mm irrigation depth was needed to increase seedlings biomass, while in species with a mean leaf angle of −64°, the 9 and 12 mm irrigation depths did not differ among themselves to raise the value of this variable (Table 2).

4. Discussion

4.1. Leaching Fraction

The effect of interaction between leaf angle and irrigation depth on leaching fraction is in agreement with a study by the authors of [18], demonstrating that tree seedlings with positive mean leaf angles facilitate the capture of irrigation water and are directed to the substrate in micro-sprinkler systems. For water to reach the plant’s root system by micro-sprinkling, it has to overcome a barrier, which is the plant canopy, with micro-sprinkler irrigation adopted in seedling nurseries being quite inefficient in this sense [32].

In [33], only 20 to 40% of the water applied by micro-sprinklings systems, in tube-type containers, is retained for plant use, in addition to, according to the authors of [34] and [35], contributing to nutrients loss by leaching, as pointed out here in this study, mainly by plants with positive mean leaf angles (76, 57, and 54°). The control of the amount and frequency of irrigation is crucial for plants’ adequate development [36]. However, the fear of damaging seedlings’ development due to water deficit leads to overestimated irrigation, based only on the nurseryman’s experience [37]. The excess of water in seedling irrigation, aside from causing the waste of water resources, also causes nutrient leaching, negatively influencing seedlings’ development [38], which can lead to loss due to diseases, mainly fungal [39]. In contrast, water deficit drastically affects plant metabolism, inducing stomatal closure and drop water loss through transpiration, which results in a reduction of photosynthetic activity and other physiological processes, which can lead the plant to a point of permanent wilt and leaf abscission, thus reducing the photosynthetically active area and its death [40].

4.2. Leaf Water Potential

The decrease in water available in the substrate reduces water supply in the plant aerial part, promoting stomatal closure, blocking the flow of CO₂, and affecting the production of photoassimilates [40]. Leaf water potential, in turn, is related to the maintenance of plant water status and to mechanisms of resistance to water deficit or excess, which can vary according to the intensity and duration of the stress, characteristics of the species, and stage of development [41]. In our study, the maintenance of high values of this parameter in species with positive mean leaf angles (76, 57, and 54°) suggests that even the smallest applied irrigation depth allowed an adequate availability of water throughout the day for these species and consequent hydration of tissues.

4.3. Stomatal Conductance

Plant stomatal control is associated with its high relationship with carbon fixation, irradiation, plant water potential, the vapor-pressure deficit in the atmosphere, and CO₂ concentration [42]. Stomatal conductance can be understood as an important mechanism of vascular plants to regulate water loss; when plants are under stress due to water deficit there is a decrease in the stomatal opening, a reduction in stomatal conductance, and a consequent reduction in photosynthetic activity [43], as observed in this experiment for species with negative leaf angles (−64, −44, and −1°). According to [44], a significant reduction in the performance of Swietenia macrophylla seedlings was too observed with the increasing levels of drought stress. Physiological attributes such as stomatal conductance, transpiration rate, and water potential were the poorest in the least irrigated treatment. Under the threats of climate change, a better understanding of the effects of abiotic stress on plant responses and environmentally friendly remedies will help nursery managers and foresters to avoid large-scale failures in different planting programs.

The reduction of stomatal conductance, in addition to affecting transpiration, can impair sap flow to the aerial part, reducing water content, the supply of nutrients, and, consequently, the production of plant biomass [45,46]. The authors of [47] suggest that in plants under constant irrigation and increasing temperatures, the reduction of stomatal conductance causes a reduction in photosynthetic rates and optimum temperature for photosynthesis. The authors of [48], in Cedrela fissilis seedlings, obtained lower values of stomatal conductance and transpiration when subjected to a daily irrigation depth of 6 mm, as confirmed by our results in species with negative mean leaf angles.

4.4. Relative Water Content

The decrease in relative water content in situations of water deficit is closely related to the efficiency of stomatal closure and solute accumulation from plant metabolism such as abscisic acid [49]. In this study, we observed that plants with negative leaf angles showed a greater influence of irrigation depths on this physiological parameter, the conformation of these species’ leaves impaired the entry of water into the substrate and, consequently, the availability of this resource for the plant. There is a direct relationship between water content in the substrate and relative water content in leaves; therefore, it is expected that the reduction of moisture in the substrate has resulted in lower values of this parameter [50]. According to the authors of [51], relative water content and leaf water potential were accurate predictors of drought mortality risk in seedlings. Relative water content is of special interest because it allows comparisons across different morphologies.

4.5. Daily Transpiration

With water available on the substrate linked to the higher incidence of radiation on the leaf, leaf temperature is increased, increasing the difference in vapor pressure between the air and the leaf, facilitating transpiration [52]. In contrast, in the case of reduced water available in the substrate, plants prevent transpiration by using several mechanisms linked to an increase in stomatal resistance, reduction in stomatal conductance, or even total closure of stomata [53]. In Eucalyptus and Corymbia seedlings, [54] verify that transpiration decreased 30–57% and photosynthesis 14–48% under water-limited conditions. Likewise, this occurred in our study with species with negative mean leaf angles.

Otherwise, in our study, we observed that plants submitted to larger irrigation depths showed greater transpiration, favoring CO₂ assimilation. In the work developed by the authors of [55] evaluating physiological responses of different Eucalyptus clones under different irrigation regimes, it was observed that, under non-limiting water conditions, stomata remained open, favoring transpiration, assimilating more CO₂, thus resulting in greater growth and biomass accumulation.

4.6. Total Dry Mass

Although seedlings of species with positive mean leaf angles (76, 57, 54°) transpired more with the 12 mm irrigation depth, it was the 6 mm irrigation depth, which did not differ from the 9 mm, that produced a greater amount of total dry mass, indicating greater efficiency of plants in moderating water loss while allowing sufficient CO₂ absorption for photosynthesis. In contrast, in species with negative mean leaf angles (−64, −44, and −1°), transpiration accompanied the production of total dry mass, making it necessary to apply the 12 mm irrigation depth to increase the value of these variables. The reduction of dry mass accumulation when plants are under stress due to water deficit is explained by the smaller number of leaves, smaller leaf area, and reduction in chlorophyll content, caused by the reduced photosynthetic activity [56,57,58].

5. Conclusions

In conclusion, our study showed that positive mean leaf angles facilitate the arrival of irrigation water in the substrate, making the application of the smallest irrigation depth (6 mm) sufficient to increase leaf water potential, stomatal conductance, leaf relative water content, transpiratory efficiency and, consequently, seedling biomass. In contrast, when mean leaf angles are negative, it is necessary to apply a larger irrigation depth (12 mm) so that seedlings’ physiological responses do not reduce the production of total dry mass.