Optimum nitrogen fertilization of Calophyllum inophyllum seedlings under greenhouse conditions

Abstract

A greenhouse pot experiment was conducted to study the effects of nitrogen fertilization on Calophy- llum inophyllum seedlings grown with 0, 50, 100, 150, 200, 300, 400 and 600 mg N per seedling according to exponential functions. Seedling height, root collar diameter, leaf area and total biomass increased with increasing fertilization from 0 to 200 mg N per seedling and decreased with further increase in fertilization from 300 to 600 mg N per seedling. The net photosynthetic rate, stomatal conductance, intercellular CO₂ concentration and transpiration rate of C. inophyllum seedlings showed a unimodal parabolic trend, with peak values of 7.29 mmol·m⁻²·s⁻¹, 0.071 mol·m⁻²·s⁻¹, 220 mmol·mol⁻¹ and 1.34 mmol·m⁻²·s⁻¹, respectively, when the rate of fertilization was 200 mg N per seedling. Photosynthetic gas exchange parameters were significantly different among nitrogen treatments. Based on the critical values of leaf N and P concentration and N/P ratio, the optimum amount of nitrogen of C. inophyllum seedlings was 200–400 mg per seedling for leaf N and P concentration, and 100–400 mg per seedling for N/P ratio. It was concluded that 200–400 mg N per seedling was the most suitable nitrogen range for C. inophyllum seedlings.

Keywords

Calophyllum inophyllum / growth / nitrogen fertilization / nutrient status / photosynthesis

Introduction

Nitrogen is a key factor affecting seedling growth and development^[ 1– 3^], and is important in regulating plant growth and metabolism. Suitable nitrogen fertilization can lead to high-quality seedling production^[ 4, 5^].Therefore, the amount and effectiveness of nitrogen fertilizer has been a strong research focus. Currently in seedling production, nitrogen use efficiency is low because of inappropriate fertilization methods. Studies have shown that 32%–85% of nitrogen fertilizer cannot be absorbed or used by forest seedlings in the nurseries^[ 6^].

During the seedling growth, fertilization methods have usually involved supplying fertilizers in equal doses. This method results in a surplus of nutrients at the beginning followed by deficiency later in seedling growth^[ 7^]. Since the 1980s, researchers have discovered that exponential fertilization can actively enhances nutrient utilization efficiency^[ 8^], including black spruce (Picea mariana)^[ 9– 11^], white spruce (Picea glauca)^[ 12^], Norway spruce (Picea abies)^[ 13^] and birch (Betula alnoides)^[ 14^].

Calophyllum inophyllum is a salt-tolerant and wind-resistant evergreen species distributed in the tropical and subtropical regions of Asia, Oceania and South America^[ 15^]. In China, it is mainly found in Hainan Island^[ 16^]. C. inophyllum has a high value for development and utilization because of its ecological, medicinal and seed oil functions. Researchers have extensively investigated the biological characteristics^[ 17^], ecological functions^[ 18, 19^], medicinal efficacy^[ 20, 21^] and economic values^[ 22, 23^] of C. inophyllum. However, little is known about its nutrient requirements, which potentially creates inefficiencies in its silviculture.

The objective of this study was to determine the optimum nitrogen fertilization for C. inophyllum seedlings grown under greenhouse conditions. Responses of seedlings to nitrogen fertilizer treatments, including growth characteristics, photosynthetic gas exchange parameters and nutrient status, were examined. Optimum fertilization was determined by the critical value method to provide a theoretical basis for fertilization practice and cultivation technology for this species.

Materials and methods

Plant materials and growth condition

Healthy one-month old seedlings of C. inophyllum about 6.5 cm in height were grown in a greenhouse at the Research Institute of Tropical Forestry, Guangzhou, China. The seedlings were cultured in 12 × 15 cm round, tapered plastic pots filled with peat, vermiculite and pearlite (3:2:2 v/v). Each pot was irrigated to 75%–80% of field capacity determined gravimetrically at transplanting^[ 24^]. Plant Prod (Plant Products Co. Ltd, Brampton, Ontario, Canada) soluble fertilizer (N:P₂O₅:K₂O, 20:10:20) was applied twice per week. Plastic bags were placed inside the pots to prevent drainage of water and nutrients. The average daily temperature in the greenhouse ranged from 23 to 38°C and night temperature from 14 to 22°C. Relative humidity ranged from 34% to 78%. To reduce the edge effects, the positions of the posts were rotated every two weeks. Fungal diseases were prevented by injecting a 0.2% solution of carbendazim into the growth medium.

Experimental design and fertilizer treatments

The fertilizer was dissolved in distilled water and the same volume carefully applied to seedlings using a syringe. To ensure similar moisture conditions of the seedlings, each seedling pot was weighed and an appropriate amount of water added.

Eight nutrient treatments (N1 to N8= 0, 50, 100, 150, 200, 300, 400 and 600 mg N per seedling) were applied to seedlings with an exponentially increasing amount applied from June to December 2010 (Table 1). Each treatment had four replicates; each replicate consisted of 12 seedlings, totalling 384 seedlings across all treatments. Fertilizer treatments commenced two weeks after transplanting and were continued for 24 weeks.

The fertilizer treatments followed exponential functions to match nutrient supply with seedling growth according to Salifu and Timmer^[ 11^].

(1) {{\displaystyle N}}_t={{\displaystyle N}}_s(e^{rt-\mathrm{1}})-{{\displaystyle N}}_{(t-\mathrm{1})}

where N_t is the amount of N to be added at time t for a given relative additional rate r,N_s is the initial quantity of N at the commencement of the treatment, and N_(t-1)is the cumulative amount of N added. r was calculated using the method described by Dumroese et al.^[ 25^].

(2) {{\displaystyle N}}_T={{\displaystyle N}}_s(e^{rt-\mathrm{1}})

where N_T is the desired amount to be added over the number of fertilizer applications (t = 12). N_s was predetermined from an additional 60 seedlings at 12.1 mg N per seedling.

Measurements

Root collar diameter and height of all seedlings were measured at the end of the experiment. Total leaf area of four seedlings randomly selected from each treatment was determined using a hand-held laser leaf area meter (CI-203, CID Bio-Science, Inc., Camas, WA, USA), and net photosynthesis,transpiration rate, stomatal conductance and intercellular CO₂ concentration were also measured using a portable photosynthesis measurement system (Licor-6400, Li-cor Biosciences, Lincoln, NE, USA). Four seedlings of each treatment were harvested and separated into leaves, stems and roots, and then oven-dried at 65°C for 48 h to estimate biomass production. The dried seedling samples were finely milled using a H₂SO₄ and K₂SO₄-CuSO₄ mixture catalyst to determine total N by the diffusion method, and were wet-digested using HNO₃-HClO₄ mixture solution to analyze P by the molybdenum blue method and K by atomic absorption.

Data analysis

After checking for normality and homoscedasticity, the effects of nutrient application on seedling height, root collar diameter, leaf area and biomass were assessed using a one-way analysis of variance by SPSS 16.0 (IBM, Armonk, NY, USA). Significant differences among treatment means were further assessed using Duncan’s multiple range tests at the 5% level. Effects of nitrogen fertilization on photosynthetic gas exchange parameters and leaf nutrient status were plotted using Excel 2003 (Microsoft Corp. Redmond, WA, USA). Regression equations between nutrient concentration or ratio and total biomass of the seedlings were fitted and then diagnostic equations were accepted that were statistically significant. The critical value and the optimum concentration range were calculated using the nutrient concentration which corresponded to 90% maximum biomass from the equation^[ 26, 27^].

Results

Root collar diameter, height and leaf area

The root collar diameter, height and leaf area at the end of experiment of C. inophyllum seedlings are shown in Table 2.The values recorded for these growth parameters increased with an increase in nitrogen fertilizer peaking at N4 to N5 (150–200 mg N per seedling) and then decreased with further increase in the nitrogen fertilizer levels.

Biomass

Total biomass of C. inophyllum seedlings increased with increasing nitrogen fertilizer from N1 to N5, and decreased with further increase in the nitrogen fertilization from N6 to N8 (Fig. 1). The greatest biomass production was observed in N5 with 10.4 g per seedling compared to the 5.5 g per seedling in N1.

Photosynthetic gas exchange parameters

The net photosynthetic rate, stomatal conductance, intercellular CO₂ concentration and transpiration rate of C. inophyllum seedlings showed a unimodal parabolic trend. The peak values recorded for 200 mg N per seedling were 7.29 mmol·m^-2·s^-1, 0.071 mol·m^-2·s^-1, 220 mmol·mol ^-1 and 1.34 mmol·m^-2·s^-1(Fig. 2).

Leaf nutrient status

As the amount of nitrogen supplied increased, leaf nitrogen concentration increased from 6.28 mg·g^-1 in N1 to 19.4 mg·g^-1 in N8 (Table 3). Leaf nitrogen content increased with increasing nitrogen fertilization from N1 to N5, followed by a slight decline in N6, then increased again from N7 to N8. In contrast, leaf biomass of the seedlings increased with increasing nitrogen fertilizer from N1 to N5 then declined from N6 to N8.

The optimal nitrogen determined by the critical value

There were parabolic relationships between biomass and leaf N and P concentration, and N/P ratio as illustrated by the scatter plots (Fig. 3).

There were significant differences (P<0.01) between leaf N and P concentrations, and N/P ratio (Table 4). The parabolic relationships between leaf K concentration, N/K and P/K ratio and seedling biomass were not significantly different.

The critical values of the leaf N and P concentrations, and N/P ratio of C. inophyllum seedlings were 9.49 mg·g^-1, 0.48 mg·g^-1 and 17.2, respectively, and their optimum concentration ranges were 9.49–17.04 mg·g^-1, 0.48–0.73 mg·g^-1 and 17.2–23.7, respectively. Therefore, given these optimum concentration ranges, the optimum amount of nitrogen for seedlings for these three measures were 200–400 mg per seedling, 200–400 mg per seedling and 100–400 mg per seedling, respectively.

Discussion

Numerous research results have shown that nitrogen influences the growth of young trees^[ 3, 5, 28^]. In our study, we observed that seedling height, root collar diameter, leaf area and biomass of C. inophyllum seedlings were enhanced by nitrogen fertilization. Dosage of 200 mg N per seedling appears to be the critical point for optimum growth of the seedlings. When the application was more than 200 mg N per seedling, excessive nitrogen reduced carbon assimilation and RuBP carboxylase activity, resulting in decreased photosynthesis and increased seedling respiration. In addition, photosynthesis was weakened while respiration of seedlings was strengthened. The growth of young leaves and seedlings was slow, thus the appropriate amount of nitrogen fertilizer can promote seedling growth and development. Our results are in accord with those reported by other researchers^[ 29– 31^].

The peak values of net photosynethetic rate, stomatal conductance, intercellular CO₂ concentration and transpiration rate of C. inophyllum seedlings were obtained with fertilization of 200 mg N per seedling (N5). Nitrogen fertilizer treatments had significant effects on the photosynthetic gas exchange parameters (Fig. 2), which suggests that an appropriate amount of nitrogen can enhance the photosynthetic capacity of C. inophyllum seedlings. Brown et al. concluded that the photosynthetic rates increased linearly with nitrogen addition to a maximum at 21 mg·g^-1 and declined at higher rates of nitrogen addition when the seedlings of the evergreen conifers Picea sitchensis, Thuja plicata and Tsuga heterophylla were fertillized with nitrogen using exponentially increasing rates^[ 32^]. Nakaji et al. found the net photosynthetic rate of Pinus densiflora was reduced by the high nitrogen treatments in the first half of the growing season, and suggested that the reduced photosynthesis under high nitrogen dosage was mainly due to the decline of both CO₂ fixation and photosystem II activity in the chloroplasts^[ 33^].

Most studies have used exponential fertilization to indicate the relationship between nitrogen concentration and supply. Almost all these studies considered that seedling biomass and nitrogen content had an increase-steady-decline trend, with nitrogen concentration rising continuously as nitrogen supply increased^[ 34^]. We came to the same conclusion from the findings presented here (Fig. 3). Salifu and Jacobs studied the exponential fertilization in P. mariana and observed the optimum nitrogen range was 30–64 mg N per seedling^[ 35^]. These authors also found the most appropriate amount of nitrogen for Quercus rubra was 25–100 mg N per seedling. Wei et al. reported that 24.3–33.7 mg N per seedling resulted in the greatest biomass and highest nitrogen efficiency in Larix olgensis^[ 30^]. Chen et al. determined that the suitable nitrogen range for B. alnoideswas 200–400 mg N per seedling using exponential fertilization^[ 14^]. In our research, leaf biomass reached its peak at N5 treatment (200 mg N per seedling), with the leaf nutrient level being satisfactory for seedling growth. From N6 to N8, leaf biomass of C. inophyllum seedlings decreased while the leaf nitrogen concentration increased, indicating that leaf nutrient status was in the luxury consumption under these conditions. This luxury nutrient consumption by seedlings and the concentration effect resulting from reduction in the leaf biomass may cause a continuing increase of leaf nitrogen concentration. Therefore, we conclude that 200–400 mg N per seedling is the most appropriate nitrogen range for C. inophyllum seedlings. All results from this and other studies indicate that there are marked differences for nutrient requirements between different tree species^[ 13, 36^].

The optimum nitrogen range in our study was determined by the early growth performance of newly-transplanted seedlings. However, it should be noted that there are likely to be differences between greenhouse and field environments^[ 37^]. Further study in the field is thus warranted to provide a sound theoretical basis for fertilization of C. inophyllum.

Conclusions

Nitrogen fertilization had significant effects on the height, root collar diameter, leaf area, total biomass, net photosynthetic rate, stomatal conductance, intercellular CO₂ concentration. Transpiration rate of C. inophyllum seedlings, and the peak values of the eight measures were obtained with fertilization of 200 mg N per seedling. It was concluded that 200–400 mg N per seedling was the most suitable nitrogen fertilization range for C. inophyllum seedlings.

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