Nitrogen Requirement and Nitrogen Form Preference by Radermachera hainanensis and R. sinica Plug Seedlings

Excessive fertilizer application not only increases production costs but also contributes to water pollution. Nitrogen significantly influences photosynthesis and the growth rate of plants. The proper nitrogen concentration requirements and the nitrate to ammonium ratio have yet to be determined for Radermachera species, which are widely used as ornamental foliage plants or in outdoor landscapes in tropical or subtropical regions. These woody plant species have a longer production cycle, and proper nitrogen fertilizer management for seedling trays can accelerate production. In this research, two species of Radermachera were treated with nine nutrient solution nitrogen concentrations and five nitrate to ammonium ratios to determine the proper nitrogen requirements for Radermachera. The information acquired from this study should contribute to a better understanding of nitrogen effects on photosynthesis and to accelerate growth, achieving uniform young Radermachera seedlings.

Radermachera sinica, commonly known as the China Doll, and Radermachera hainanensis are native to Southeast Asia. Both species are valued for their attractive, delicate, pinnately compound leaves and are often cultivated as ornamental foliage plants or outdoor landscape plants in tropical or subtropical regions. China Doll was introduced to Europe, America, and New Zealand in the 1980s and 1990s (Griffith 2006, Thomas et al. 1995). Radermachera hainanensis has recently been among the top ten ranked potted plants in Taiwan. Radermachera plants are primarily propagated from seeds. The use of plug production offers several advantages, such as uniform production and the reduction of transplanting shock. Mineral nutrient management during plug production can impact seedling growth and quality (Styer and Koranski 1997). Growers are eager to explore nutritional management methods to accelerate growth and achieve uniform young Radermachera seedlings.

The nutritional requirements of container-grown R. sinica have been previously studied. Chase and Poole (1989) demonstrated that plant height and quality were not affected within a wide range of soluble salts, with leachate electrical conductivity (EC) ranging from 0.3 to 4.4 dS·m⁻¹. Thomas et al. (1995) also found that R. sinica is an adaptable species that can thrive with low nitrogen (N) levels. However, a strong growth response is observed when additional N is provided, while phosphorus (P) and potassium (K) levels have little influence on growth. In cases of nitrogen deficiency or excess, photosynthesis may be reduced, actual photosystem II (PSII) photochemical efficiency decreases, the maximum photochemical quantum yield (Fv/Fm) decreases, and reactive oxygen species are generated, causing damage to the photosystem in cassava (Cruz et al. 2003). Currently, information regarding effects of nitrogen concentration on the growth and PSII efficiency of Radermachera plug seedlings is lacking.

Plants primarily absorb and utilize two forms of nitrogen: nitrate (NO₃^-) and ammonium (NH₄⁺). Foliage plants have shown varying growth responses to nitrogen form. For example, the best growth and quality of peacock plant (Calathea makoyana E. Morr. & Boom) were achieved with NH₄-N or urea-N and poorest with NO₃-N (Conover and Poole 1982). Growth and grade of ‘Silver Queen’ aglaonema (Aglaonema commutatum Schott) and heartleaf philodendron [Philodendron scandens subsp. oxycardium (Schott) G.S. Bunting] improved when they received N sources containing 25% to 100% NH₄-N or 100% urea-N compared to 100% NO₃-N (Conover and Poole 1986). Dumb cane (Dieffenbachia maculata Sweet) ‘Camille’ grew equally well regardless of N form (Conover and Poole 1986), whereas dumb cane [D. seguine (Jacq.) Schott] ‘Tropical Snow’ showed a preference for NH₄-N (Jiménez and Lao 2005). The nitrogen form can impact rhizosphere pH, plant growth, and photosynthesis (Guo et al. 2007). While there is ample research on nitrogen preferences for various foliage plants, there is currently no report on the nitrogen preference for Radermachera species.

Nitrogen fertilizer has the most significant influence on the growth and quality of potted Radermachera (Thomas et al. 1995) and many plug seedlings of bedding plants (van Iersel et al. 1999). Therefore, the objectives of this study were to evaluate the effects of N concentration and the NO₃-N to NH₄-N ratio on medium pH and EC, growth and photosynthetic parameters of the plug seedlings in the two Radermachera species.

Seeds of Radermachera hainanensis and R. sinica were sown into 72-celled plug trays (4.5 × 4.5 × 3 cm or 1.8 × 1.8 × 1.2 in) containing 3 peat moss (pH balanced peat moss, Klasmann-Deilmann GmbH, Geeste, Germany): 1 perlite (No.2, Nanhai Vermiculite Industrial Co., New Taipei City, Taiwan): 1 vermiculite (No.2, Nanhai Vermiculite Industrial Co., New Taipei City, Taiwan). Plug trays were placed in a chamber at 24 C (75 F) under 80 µmol·m⁻²·s⁻¹PPF (photosynthetic photon flux) (7.4 ft-c) conditions. When seedlings reached one pair of leaves, they were transferred into a phytotron at 30/25 C (86/77 F) and 260 µmol·m⁻²·s⁻¹PPF (24.2 ft-c) at plant height to receive N treatments.

Seedlings were supplied with nutrient solution formulated as per Johnson et al. (1957). The concentration of NH₄NO₃ was altered to prepare nutrient solutions with 0, 4, 8, 12, 16, 20, 24, 28, and 32 mM N. The nutrient solutions also contained 2 mM NaH₂PO₄, 3 mM K₂SO₄, 1 mM MgSO₄·7H₂O, and 4 mM CaSO₄·2H₂O, along with micronutrients specified by Johnson et al. (1957). The corresponding EC of each solution, measured with a conductivity meter (Model SC-170, Suntex Instruments Co., Taiwan) was 1.7, 1.9, 2.1, 2.3, 2.5, 2.7, 2.9, 3.2, and 3.4 dS·m⁻¹, respectively. The pH of the nutrient solution was adjusted to 6.5 using 1 N NaOH before each application. Each plant received 50 mL (1.7 oz) of nutrient solution once a week. The treatments were replicated three times, with nine plants per treatment. Measurements were taken on three randomly sampled plants per treatment, with three replicates.

Changes in medium EC and pH during the experiment were monitored using the press extraction method (Scoggins 1999). Leachate was collected from plug cells, and the EC and pH of the leachate were measured using a conductivity meter and a pH/oxidation-reduction potential meter (SP-2300, Suntex Instruments Co.).

At 55 d after treatments began, two recently fully expanded leaves from each plant were sampled to measure chlorophyll fluorescence parameters using a portable chlorophyll fluorometer Mini-Pam (Heinz Walz GmbH, Effeltrich, Germany). Minimal fluorescence (Fo) was determined after 30 min dark adaptation, while maximal fluorescence (Fm) was measured after a saturation pulse (ca. 18,000 µmol·m⁻²·s⁻¹ or 1,672 ft-c) at 25 C (77 F). The fluorescence ratio Fv/Fm, with Fv = Fm - Fo being the variable fluorescence, was then calculated. Photochemical quenching (qP) and non-photochemical quenching (qN) were also measured.

At 60 d after treatments were initiated, the relative chlorophyll content of the most recently fully expanded leaf was also measured in situ with a chlorophyll meter (SPAD-502, Minolta Camera Co., Tokyo, Japan). Leaf area of the most recently fully expanded leaf was calculated using ImageJ (National Institute of Health, Bethesda, MA). Plants were divided into above-ground (shoot) and below-ground (roots) parts, oven-dried at 70 C (158 F) until constant, and dry weights were recorded. The root-to-shoot ratio was calculated as root dry weight divided by shoot dry weight. This experiment was arranged in a completely randomized design, and regression analysis was used to describe the relationships between nutrient solution N concentration and growth and PSII parameters.

Nutrient solutions with various nitrate to ammonium ratio including 0:100, 25:75, 50:50, 75:25, and 100:0 (in percentages) ratios. Nutrient solutions supplied with, in mM, 16 N, 1 P, 8 K, 6 Ca, and 1 Mg. Ca(NO₃)₂·4H₂O, (NH₄)₂SO₄, KNO₃, NaH₂PO₄, K₂SO₄, MgSO₄·7H₂O and CaSO₄·2H₂O were nutrient sources. The actual EC values of these solutions were measured using a conductivity meter (Model SC-170, Suntex Instruments Co.), as 2.26, 2.57, 2.66, 3.02, and 3.42 dS·m⁻¹ for 0:100, 25:75, 50:50, 75:25 and 100:0 (nitrate: ammonium), respectively. Treatments were replicated three times, with nine plants per treatment. Three plants per treatment were randomly sampled for measurements, with three replicates.

Medium EC and pH were monitored as described above. Leaf area and relative chlorophyll content of the most recently fully expanded leaf was determined and chlorophyll fluorescence parameters were also measured as previously described. Photosynthetic parameters were assessed using a portable photosynthesis system (LI-6400, LI-COR, Lincoln, NE, USA). Air was pumped through the desiccant (Drierites, W.A. Hammond Drierite Co., Xenia, OH, USA) and soda lime [a mixture of sodium hydroxide (NaOH) and calcium hydroxide (Ca(OH)₂)] (LI-COR) to eliminate excess water vapor and CO₂. Light intensity within the leaf chamber was set at 800 µmol·m⁻²·s⁻¹PPF (74.3 ft-c) and a reference CO₂ concentration of 400 µmol·mol⁻¹ was provided. The air flow rate was set at 600 µmol·s⁻¹, and the measurement area was set at 3 cm² (0.465 in²). Temperature at the leaf surface was maintained at an average of 25.3 C (77.5 F). This allowed for the measurement of the net CO₂ assimilation rate (Pn), stomatal conductance (g_s), transpiration rate (E), and intercellular CO₂ concentration (C_i) of the most recently fully expanded leaves. The plants were divided into shoot and roots, dried, and dry weights were determined as previously described. The experiment was arranged in a completely randomized design. Regression analysis was performed with SigmaPlot 10.0 (Systat Software Inc., Palo Alto, CA, USA) using the percentage NH₄-N as the independent variable.

Both species under N deficiency exhibited smaller leaf areas (Fig. 1A and F) and lighter green leaves as indicated by the lowest SPAD-502 value (Fig. 1B and G). This suggests that nitrogen deficiency inhibits cell expansion and chlorophyll synthesis (Yanagida et al. 2011). Shoot and root dry weights were significantly reduced, while the root-to-shoot ratio increased, in response to the N-deficient treatment (Fig. 1C-E, H-J). Similar examples for preferential partitioning of photosynthetic carbon to the roots and an increase in root-to-shoot ratio are well-documented for other species under N deficiency (Chen et al. 2018, Hermans et al. 2006, Yeh et al. 2000).

Leaf area, SPAD-502 value, shoot and root dry weights increased as nutrient solution N concentration increased and plateaued at 12-16 mM N treatments and then declined gradually thereafter (Fig. 1A-D, F-I). These results are consistent with Thomas et al. (1995), demonstrating that N nutrition has a strong growth-promoting effect. More pronounced declines in leaf area, SPAD-502, and root dry weight were observed in R. hainanensis receiving 20 mM or higher N treatments (Fig. 1A, B, D). Excessive N accumulation in the rhizosphere may cause leaf necrosis from excess salt levels and limit root water uptake, and result in low water turgor for leaf expansion (Mengel and Kirkby 2001). Radermachera sinica is reported to have higher water requirements than other foliage plants (Poole and Conover 1992). It was observed that R. hainanensis, probably due to its faster growth and larger leaflets, is more susceptible to drought stress compared to R. sinica.

Minimal fluorescence (Fo) and qN decreased, while Fm and qP increased, with solution N concentration increasing from 0 to 12-16 mM N (Fig. 2). Nitrogen-deficient R. hainanensis exhibited the lowest Fv/Fm (Fig. 2C). Both species treated with 16 mM or higher N concentration exhibited increased Fo and qN and decreased Fm and qP (Fig. 2). Depending on the species/cultivar, plants under different stress conditions can exhibit increases in Fo and/or qN but decreases in Fm and/or qP (Adams III and Demmig-Adams 2004). The growth response to N concentration of Radermachera was well correlated with PSII parameters, indicating that N application can alter photosynthesis and growth by regulating PSII. Similar results, where N levels alter PSII, have been reported for other plants (Chen et al. 2018, Cruz et al. 2003, Shangguan et al. 2000, Tantray et al. 2020).

For each treatment, the leachate EC value of the medium increased on the day of fertilization and then decreased afterward (Fig. 3A and C). This decrease in leachate EC value was attributed to the absorption of mineral nutrients by seedlings or leaching out of the pot from irrigation, and the leachate EC value increased again only until the next fertilization. The leachate EC increased with increasing nutrient solution N concentration. Throughout the experimental period for both species, the leachate EC values for treatments with 8-16 mM N fell within the recommended range of EC values (0.9-2.5 dS·m⁻¹) for plug seedlings (Scoggins 1999). For treatments with N concentrations exceeding 20 mM, the leachate EC values after 28 d exceeded the upper limit of the recommended range by Scoggins (1999).

The leachate pH remained constant during the experiment with nitrogen-deficient or 4 mM N treatments (Fig. 3B and D), indicating insufficient nutrition for plant uptake. The leachate pH for well-grown plants under 12-16 mM N treatments ranged from 5.0 to 5.5 (Fig. 3B and D), closely aligning with the medium pH of 4.8 reported for R. sinica (Thomas et al. 1995). The leachate pH decreased with increasing nutrient solution N concentration from 8 to 32 mM. This might be attributed to the roots absorbing more cations, leading to the release of H⁺ from the roots to maintain ionic balance in the plant (Feng et al. 2020).

Both species with sole NO₃-N or NH₄-N exhibited reduced leaf area, SPAD-502 value, and plant dry weight compared to 50% NH₄-N: 50% NO₃-N treatment (Table 1). The restriction in leaf expansion may be due to a reduced ability of N absorption in the roots under NO₃-N only treatment (Ruan et al. 2007). High NH₄-N ratios may inhibit shoot growth and leaf expansion, mainly due to reduced osmotic regulation (Raab and Terry 1994, Walch-Liu et al. 2000). Also, high NH₄-N ratios may inhibit K absorption and cause toxicity symptoms for many plant species (Mengel and Kirkby 2001). No foliar symptoms of K deficiency, such as marginal necrosis and irregular spots, occurred, suggesting that the absorption of K was likely not severely reduced by applying sole NH₄-N to Radermachera species. However, it was observed that some plants of R. hainanensis with 100% NH₄-N expressed downward leaf curling, possibly related to NH₄ toxicity. Maximum growth was obtained with 50% NH₄-N: 50% NO₃-N for both species (Table 1). Foliage plant species show a great diversity of growth response according to N sources. For example, best growth and grade of heartleaf philodendron also occurred with 50% NH₄-N: 50% NO₃-N (Conover and Poole 1986). Growth and grade of Boston fern [Nephrolepis exaltata (L.) Schott] was not influenced by N source, whereas peacock plant had best growth and quality with NH₄-N or urea than with NO₃-N (Conover and Poole 1982, 1986).

The general trends of changes in photosynthetic parameters and growth in response to N form and ratio were similar for both species (Table 2). Notably, R. sinica supplied with 100% NH₄-N had a higher Pn, g_s, and E than those with 100% NO₃-N (Table 2), similar to a report for French bean (Phaseolus vulgaris L.) (Guo et al. 2002). This may explain why growth of R. sinica was improved with NH₄-N over NO₃-N (Table 1). N form or ratio altered g_s, but not C_i (Table 2), indicating N source affects photosynthesis through regulating stomatal opening and closure for Radermachera. However, best growth and photosynthetic performance of Radermachera were obtained with both NH₄⁺ and NO₃^– sources (Tables 1 and 2). These results agree with Marschner et al. (1986), who reported that a mixture of different N forms increases the efficiency of N use.

Throughout the experimental period, the leachate EC values for treatments with 25% NH₄-N: 75% NO₃-N or 50% NH₄-N: 50% NO₃-N fell within the recommended range of EC values (0.9-2.5 dS·m⁻¹) for plug seedlings, as suggested by Scoggins (1999). For treatments with higher NH₄-N ratios, the leachate EC values exceeded the upper limit of the recommended range (Fig. 4A and C; Scoggins 1999). This may explain why reduced growth occurred in plants with 75% NH₄-N: 25% NO₃-N or 100% NH₄-N (Table 1).

It is proposed that NO₃^– uptake is mediated by a 2 H⁺/1 NO₃^– symport to a plant cell, followed by the release of one H⁺ ion from the cell, resulting in increased medium pH (Crawford and Glass 1998). However, the leachate pH from the medium with 100% NO₃-N treatment did not increase substantially and it remained between 5.9 and 6.6 (Fig. 4B and D). In addition, the leachate electrical conductivity (EC) values with sole NO₃-N did not change pronouncedly compared to treatments containing NH₄-N (Fig. 4A and C), implying that Radermachera prefers NH₄-N over NO₃-N under suitable medium EC ranges for growth. The leachate pH values decreased with NH₄-N treatments over the experimental period (Fig. 4). This medium acidification might be attributed to the roots absorbing more NH₄⁺ and other cations, leading to the release of H⁺ from the roots to maintain ionic balance in the plant (Feng et al. 2020, Mengel and Kirkby 2001, Stegani et al. 2019). Similar examples, a decrease in leachate pH with increasing N concentration linked to the absorption of a significant amount of NH₄⁺, have been reported in woody plants such as tea plant [Camellia sinensis (L.) Kuntze] (Ruan et al. 2007) and mountain azalea [Rhododendron canescens (Michx.) Sweet] (Clark et al. 2003).

In conclusion, nutrient solution N concentration significantly affects the growth performance of plug seedlings in Radermachera hainanensis and R. sinica. Weekly application of a nutrient solution with 12-16 mM N resulted in maximum leaf growth and plant dry weight, along with improved PSII electron transfer efficiency. The EC of medium leachate should be kept below 2.5 dS·m⁻¹. Both best growth and photosynthetic performance of Radermachera seedlings were obtained with 50% NH₄-N: 50% NO₃-N.