Morphophysiology and quality of Alibertia edulis seedlings grown under light contrast and organic residue

HIGHLIGHTS: The use of chicken manure contributes for seedling Alibertia edulis production. A. edulis seedlings presented physiological adjustments in the two light contrasts. For quality A. edulis seedlings should use 6.24 g CM kg-1 under shade or 4.16 g CM kg-1 under full sun. ABSTRACT Organic residue in the substrate and the availability of light influence the production of high-quality seedlings for ex situ cultivation of native forest species. This study aimed to evaluate the effect of doses of chicken manure incorporated in the soil and shading levels on the morphophysiological responses and quality of Alibertia edulis (Rich.) A. Rich seedlings. Five doses of chicken manure (CM) were tested, 0.00, 2.08, 4.16, 6.24, and 8.32 g CM kg-1 soil, under two shade conditions, 0% (full sun) and 50% shading. At 215 days after transplanting, the maximum height obtained for A. edulis was 47.35 cm with 5.64 g CM kg-1 soil under 50% shade. Shaded seedlings had a higher number of leaves. The maximum leaf and root areas were 796.64 and 118.83 cm2 with 5.32 and 4.21 g CM kg-1 soil, respectively, both under 50% shade. Seedlings in 0% shade had lower physiological indices. Shading levels did not affect stomatal limitation, but CM contributed to physiological efficiency. Shoot biomass was higher under 50% shade with higher CM doses. The highest quality indexes occurred with 3.98 and 6.28 g kg-1 CM under 0 and 50% shade, respectively. A. edulis seedlings responded positively to the addition of CM to the soil and presented plasticity under contrasting light conditions. A. edulis seedlings produced under 50% shading with the addition of 6.24 g CM kg-1 soil presented high morphophysiological and seedling quality indicators. The addition of 4.32 g CM kg-1 soil contributed to high seedling quality under full sun conditions.


Introduction
Marmelo-do-cerrado (Alibertia edulis (Rich.) A. Rich, Rubiaceae) is a native species widely distributed in the phytophysiognomies of the savanna in Brazil.The species exhibits ecological characteristics that are useful to recover degraded areas.Their fruits are food sources consumed in natura and as candies, jellies, and unprocessed juices.
However, the composition and distribution of biodiversity in the phytophysiognomies of this region is declining owing to inadequate collection or deforestation.Therefore, planning the ex situ cultivation of A. edulis is necessary to introduce it into production systems on a sustainable basis, such as the enrichment of forests and recovery of degraded areas, that is, environments characterized by different light gradients and stressful.
Plants tend to exhibit different mechanisms when exposed to different luminous conditions; for example: under full sun, plants have high CO 2 assimilation rates and adjustments of leaf metabolism (Major & Mosseler, 2020;Santos et al., 2020a), whereas in a shaded environment, there is an increase in vegetative characteristics (Mditshwa et al., 2019), metabolic processes, and biomass production as the plants are under limiting light conditions.
Native tree species have a long growing cycle, and the gradual release of nutrients contained in organic residues, such as chicken manure (CM) (Santos et al., 2020b;Goelzer et al., 2021), present in substrate formulations can improve the microbiological, physical, and chemical attributes of the substrate (Wolschick et al., 2018;Silva et al., 2019), thereby providing adequate conditions for regulating physiological indices and increasing photoassimilates for plants.
To test the hypothesis that A. edulis presents adjustments to different luminous conditions and that the addition of CM to the substrate can positively contribute to the robustness of seedlings of this species, this study aimed to evaluate the effect of CM doses on the morphophysiological responses and quality of A. edulis seedlings produced under different shading levels.

Material and Methods
The experiment was carried out at the Faculdade de Ciências Agrárias, Universidade Federal da Grande Dourados (UFGD), Dourados, MS, Brazil.According to the Köppen classification, the climate of the region is CWa with hot summers with a rainy season, winters with moderate temperatures, and a dry season (Fietz et al., 2017).
The experiment was performed in plastic pots with a capacity of 4.2 dm 3 , filled with substrate comprising an Oxisols soil (United States, 2014), which corresponds to a Distroferric Red Latosol in the Brazilian Soil Classification System (EMBRAPA, 2018).Five CM treatments (0.00,2.08,4.16,6.24,and 8.32 g CM kg -1 soil) semi-composted with a rice husk base were incorporated into the soil as described by Santos et al. (2020b).The pots were placed under two shade levels: full sun (0%) and shaded (50%).The treatments were arranged in a 5 × 2 factorial scheme in a randomized block design with four replicates, and four plants comprised a single experimental unit.
A black-colored screen with 50% shade -Sombrite ® , was used for shading.Full sun and shaded environments exhibited the following conditions, on average, at the end of the cultivation cycle: temperature, 31.62 and 28.42 °C; relative air humidity, 65 and 78%; water vapor pressure deficit according to Sadler & Evans (1989), 4.36 and 2.33 kPa; atmospheric CO 2 concentration -C a , 419.00 and 423.30ppm; and photosynthetically active radiation -PAR, 1,245.25 and 560.12 μmol m -2 s -1 ; respectively.
Mature fruits of A. edulis were harvested randomly from mother plants (Figures 1A and D) in the Cerrado area (Farm Santa Madalena, 18° 07' 03" S, 54° 25' 07" W, altitude of 452 m) in Dourados, Mato Grosso do Sul state, Brazil.A specimen was deposited in the DDMS Herbarium of the Universidade Federal da Grande Dourados (UFGD), with voucher No. 4649.Access to genetic heritage was registered under no.A9CDAAE.
The culture treatments during the experimental period included daily irrigation to maintain 70% of the water retention capacity in the substrate, as determined by the water content retained after draining according to Souza et al. (2000), where the pots were weighed using an analytical balance.Base fertilization with fertilizers was not performed to determine the direct effect of CM doses on the soil and plants.In addition, two sprays of neem oil (Azadirachta indica A. Juss.) at 3% were applied owing to the incidence of whitefly and cochineal, with effective control.
Samples of each combination of substrate under the different shading levels were collected to characterize their chemical attributes (Table 1) according to the methodology of Silva (2009).
At 215 days after transplanting, the following characteristics were evaluated: Initial growth: Seedling height (H) was measured using a ruler graduated in mm, with the standard of measurement as the distance between the collar and the inflection point of the highest leaf.The stem diameter (D) was determined using a digital caliper (0.01 mm), and the height/diameter ratio was calculated.
Leaf area, production, and biomass: The seedlings were harvested, removed from the pots, washed to remove excess substrate from the root part, and the leaves, stems, and roots were separated.The leaf and root surface areas were evaluated using an area integrator (LI-COR, 3100 C -Area Meter).Subsequently, the organs were dried in an oven with forced airflow at 60 ± 5 °C until a constant weight was obtained for the dry mass and were then weighed on a millesimal precision scale (0.0001 g).
Data on leaf area and dry mass were used to determine the physiological indices including specific leaf mass, leaf area ratio, and specific leaf area were calculated according to Hunt (2017).
The data C i and C a were quantified using the infrared gas analyzer (IRGA), ADC, model LCi PRO (Analytical Development Co. Ltd., Hoddesdon, U.K.) between 8 and 11 a.m., considering the environmental conditions of each luminous environment.
Dickon quality index (DQI): using HDR, aerial part/root ratio (APRR), and total dry mass (TDM) were calculated (Dickson et al., 1960) using the following Eq.2: ( ) Data were subjected to analysis of variance, and when significant (F test, p ≤ 0.05), the means for shading treatments were compared using the Student's t-test.Regression analysis (linear or quadratic) was applied to clarify the interactions of shading with varying CM doses in the substrate (p ≤ 0.05).The software SISVAR 5.6 was used for the analyses.Based on the groupings of the data on the morphophysiological responses of seedlings, cluster analysis was performed using the most distant neighbor method to describe similarities between treatments.The grouping was performed employing Euclidean distances.Analyses were performed using the software PAST 3.21.

Results and Discussion
Alibertia edulis seedlings exhibited morphophysiological adjustments to light gradients and CM influenced the initial growth of seedlings.The height of A. edulis seedlings was influenced by the interaction of the factors under study, showing quadratic relationships at both shade levels.The maximum heights were 47.35 and 35.14 cm with the addition of 5.64 and 6.01 g CM kg -1 soil, under 50 and 0% shade, respectively (Figure 2A).
The highest seedling height in the shaded environment can be explained by the fact that the species belongs to the ecological succession group of initial secondary species (Leles et al., 2011) that optimize environmental resources.The lower height under full sun may be associated with hormonal imbalance because, under high irradiance, auxins tend to undergo degradation (Chandler, 2016), reducing the elongation of internodes and morphometric aspects of the plant.
The leaf and root areas were influenced by interactions between factors, with maximum values of 796.64 and 118.83 cm 2 with the addition of 5.32 and 4.21 g CM kg -1 soil, respectively, both under the shaded environment (Figures 2B and C,respectively).Shaded plants tend to increase the number of leaves per unit area, aiming to maximize their ability to intercept light and potentiate photosynthetic activity (Santos et al., 2019).
Furthermore, under conditions of low light availability, some species tend to invest a high proportion of photoassimilates in increasing leaf area to maximize the capture of available light (Bartieres et al., 2020), similar to the number of The largest root areas were found in the shaded seedlings (Figure 2C), which was related to water use efficiency.In this study environment, plants lose less water because of transpiration owing to a low water vapor pressure deficit (2.33 kPa).Furthermore, the addition of CM likely enabled the production of seedlings under 0 and 50% shading; however, at different doses, improvement in the physical attributes of the substrate would differ, as organic residues added to the soil act to reduce soil density owing to the conditioning effect (Santos et al., 2020b) and thus favor the development of the root system.Contin et al. (2021) reported that under condition of low light availability in the environment, plants invest in aerial part characteristics to ensure the partitioning of photoassimilates and carbon balance.This is consistent with the observations in A. edulis in the current study.
Stem diameter was influenced only by CM doses, with linear growth exhibiting the largest diameter of 8.12 mm with 8.32 g CM kg -1 soil (Table 2).The increased stem diameter with organic residue application favors improved translocation of photoassimilates for seedlings.Although leaf nutrient content was not quantified in A. edulis seedlings, this result may be associated with the increase in nutrients contained in organic residues, especially K (Table 1), as this nutrient contributes to plant tissue structuring and increase in mass (Cavalcante et al., 2019).
The height/diameter ratio (HDR) was influenced by the factors evaluated in the study, with a high value (5.46) found under 4.95 g CM kg -1 soil and in 50% shade (5.32) (Table 2).Chicken manure in soil was observed to favor an increase in growth in a balanced manner because growth in height was accompanied by an increase in dry mass and diameter under the same growing conditions, ensuring the robustness and stability of seedlings.
Increased HDR indicate plant etiolation as a function of cultivation conditions in the initial growth phase in a nursery, and may present the probability of damping under conditions after transplanting (Sousa et al., 2022).Although high under shaded conditions, the values for A. edulis seedlings were within the established standards, which is an important indicator for obtaining high-quality seedlings.
The benefits of organic residue to the soil on growth indicators are associated with increased microbiological activity and physical improvements in the soil (Volpiano et al., 2022), in addition to favoring the increase in nutrient content, such as that of N and K.These nutrients were present in the CM applied to A. edulis; N is associated with vegetative growth (Osorio et al., 2014), whereas K affects tissue structure (Cavalcante et al., 2019).
The aerial part/root ratio (APRR), was only influenced by the luminous environment (Table 2).The higher APRR (4.19) in the shaded environment was owing to the ability of the species to invest in the mass production of tissues in different organs to maintain physiological processes and increase aerial parts in response to water absorption and maintaining wateruse efficiency (Castro & Newton, 2015).
The leaf, stem, and root dry masses were influenced by interactions among the factors under study (Figure 3).Leaf dry mass showed a linear trend, with a high value of 7.88 g per plant, in 8.32 g CM kg -1 soil under 50% shade (Figure 3A).Under 0% shade, the response of seedlings followed a quadratic model, with a maximum value (5.95 g per plant) with 4.98 g CM kg -1 soil (Figure 3A).The maximum stem dry mass was 2.42 g per plant in 7.40 g CM kg -1 soil under 50% shade (Figure 3B).Increasing the amount of added CM contributed to an increase in nutrients, especially N, thereby ensuring vegetative growth.These high values were also correlated with increased height, number of leaves, and leaf area under the same light conditions.
In addition, with CM incorporation in the soil, there is an increase in beneficial microorganisms that accelerate nutrient cycling (Mierzwa-Hersztek et al., 2018), which promotes an increase in biomass.The improved growth of seedlings observed under the highest doses of CM was because of the better growth and root area conditions, indicating rhizosphere exploration and mass input.This is related to the fact that organic matter improves the physical attributes of soil, mainly by favoring stability, aggregation, and reduction in particle density (Wolschick et al., 2018), especially because the soil used in this experiment was an Oxisols with a clay texture.
The maximum root dry mass was 2.51 g per plant with 3.95 g CM kg -1 soil under 0% shade (Figure 3C).Increase in root mass is a mechanism used for better exploration of the rhizospheric area and optimization of water resources under full sun conditions.Regarding the organic residues, seedlings of Alibertia sessilis Schum., a native species, also showed an increase in the mass of different organs when CM was incorporated into the substrate (Mota et al., 2017), demonstrating that the addition of this organic residue contributes to the production of native tree species seedlings.However, high doses of CM reduced the potential for stem and root mass production in A. edulis seedlings.Similarly, Silva et al. ( 2019) evaluated Enterolobium contortisiliquum Vell.seedlings and verified that application of increasing doses of CM to the substrate negatively affected root mass production.
The leaf area ratio was influenced only by CM doses, with a maximum leaf area ratio of 119.05 cm 2 g -1 with the addition of 3.44 g CM kg -1 soil (Table 3).A higher leaf area ratio indicates a greater capacity for plants to grow and presents a higher proportion of photosynthetically active tissue in the leaf area (Santos et al., 2019); therefore, there is no limitation on leaf metabolism.These results can be attributed to the high chlorophyll index and leaf area observed with the addition of CM to the substrate.Stomatal limitation (SL) was affected only by CM doses and showed a quadratic relationship, in which the maximum calculated SL value (0.44) was obtained with 6.24 g CM kg -1 soil and the lowest values (0.33 and 0.40) occurred with 0.0 and 8.32 g CM kg -1 soil, respectively (Table 3).
Although shading levels did not affect the stomatal limitation (SL) of A. edulis seedlings, further confirming their physiological plasticity, the use of CM contributed to the stability of photosynthetic metabolism.When there is an increase in SL, there is a decline in intercellular CO 2 concentration, indicating higher carboxylation and photosynthesis efficiency (Berry & Downton, 1982;Song et al., 2020), that is, conversion into photoassimilates owing to improvements in the chemical attributes of the substrate and possibly nutritional status of A. edulis seedlings.
The largest specific leaf area and specific leaf mass were observed in shaded seedlings (Table 3), demonstrating that A. edulis seedlings converted a greater amount of photoassimilates per unit of photosynthetic area, verifying the hypothesis that cultivation under full sun conditions causes changes in morphophysiological responses.An increase in these indices indicates the adaptive ability of leaf tissues to optimize light capture (Moura et al., 2022) as a function of reduced incident radiation.
The DQI was influenced by the interaction between the factors under study, with the highest index values of 1.31 and 1.11 with 3.98 and 6.28 g CM kg -1 soil under 0 and 50% shade, respectively (Figure 4).The obtained high DQI values indicate that A. edulis presents morphophysiological adjustments to contrasting light conditions; that is, under both conditions of light availability, this species ensures stability in development and use of available resources regulating leaf metabolism and increasing photoassimilates.This species requires the supply of organic matter to the soil, represented here by the use of CM, to express its physiology and growth potential.The DQI values vary according to several factors such as plant age, species, and genetics.
Cluster analysis verified that there was high similarity between the same CM doses within each light environment (Figure 5).Thus, four groups were formed, in which treatments without the addition of CM (0.00 g kg -1 ) were isolated from the others (G1).When considering hierarchical groups, the shortest Euclidean distances occurred between 2.08 g CM kg -1 soil in both environments (3.01), followed by 4.16 g CM kg -1 under 50% shade and 6.24 g CM kg -1 under 0% shade with a value of 4.21.The addition of organic residue improved the chemical, physical, and microbiological attributes of the substrates and resulted in corresponding improvements in seedlings exhibiting better results for the production indicators than those of seedlings grown without CM.The results demonstrated that the use of CM in soil favored the production of A. edulis seedlings, as these showed greater similarity.
Based on these results, the addition of CM to the soil was demonstrated to be a promising strategy, mainly by contributing to the chemical attributes of the substrate and consequently to the physiology and growth of A. edulis seedlings under the evaluated light gradient.Furthermore, the potential of the species for planting in areas with different features, such as open (0% shade) and understory (50% shade), was verified.Considering its morphophysiological plasticity under different shading levels, the utility of the seedlings in both environments was evidenced by the good DQI values.Studies involving the ex situ cultivation of native fruit species from the Brazilian Cerrado are of paramount importance to conserve biodiversity in silvicultural activity and genetic resources for food, medicinal, and ecological purposes.

Conclusions
1. Alibertia edulis seedlings produced under 50% shading and with the addition of 6.24 g CM kg -1 soil presented improved morphophysiological and seedling quality indicators.
2. The addition of 4.32 g CM kg -1 soil contributed to the high quality of seedlings under full sun.
3. Light contrasts had no influence, but varying doses of CM contributed to stomatal physiological efficiency of A. edulis seedlings.

Figure 1 .
Figure 1. Adult plant (A), flowers (B), green fruits (C), mature (D-E) and seedlings (F) of A. edulis in Dourados, MS, Brazil Table 1.Chemical attributes of substrate for production A. edulis seedlings produced with doses of chicken manure (CM) and/ or under 0 and 50% shade, at 215 days after transplanting Figure 2. Plant height (A), leaf area (B) and root area (C) of A. edulis seedlings produced with doses of chicken manure (CM) under 0% (FS) or 50% (S) shade leaves.Regarding CM, an increase in nutrients in the soil contributed substantially to the production of photoassimilates, distribution, and increase in vegetative organs owing to improvements in the chemical, physical, and microbiological attributes of the substrates.The largest root areas were found in the shaded seedlings (Figure2C), which was related to water use efficiency.In this study environment, plants lose less water because of transpiration owing to a low water vapor pressure deficit (2.33 kPa).Furthermore, the addition of CM likely enabled the production of seedlings under 0 and 50% shading; however, at different doses, improvement in the physical attributes of the substrate would differ, as organic residues added to the soil act to reduce soil density owing to the conditioning effect(Santos Figure 3. Leaf (A), stem (B) and root (C) dry mass of A. edulis seedlings produced with doses of chicken manure (CM) under 0% (FS) or 50% (S) shade * -Significant at p ≤ 0.05 by F test; Means with equal letters do not differ statistically from each other by the t test at p > 0.05; C.V. -Coefficient of variation; FS -full sun (0% shade); S -shading (50% shade)

Figure 4 .
Figure 4. Dickson quality index (DQI) of A. edulis seedlings produced with doses of chicken manure (CM) under 0% (FS) or 50% (S) shade * -Significant at p ≤ 0.05 by F test; Means with equal letters do not differ statistically from each other by the t test at p > 0.05; C.V. -Coefficient of variation; FS -full sun (0% shade); S -shading (50% shade)