1. Introduction
Camellia oleifera Abel., a member of the Theaceae, is an oil tree species that has been exclusively cultivated in China due to its economic value [
1]. Its fruit contains high-quality oil, commonly known as tea oil or camellia oil, which has 82%–84% unsaturated fatty acids, 68%–77% monounsaturated fatty acids, 67.7%–76.7% oleic acid, and 7%–14% polyunsaturated acid. The composition is similar to that of olive oil [
2], hence it is called eastern olive oil [
3]. Tea oil has also been widely used for cosmetic and medicinal purposes [
4]. Furthermore,
C. oleifera is tolerant to drought [
5], and it can be produced in a wide range of mountainous areas in subtropical regions. With the increasing demand for self-sufficiency in cooking oil [
3], the production of
C. oleifera has significantly increased over the last 20 years. The current cultivated area for
C. oleifera is more than three million ha in China [
6,
7].
The increasing production scope of tea oil trees, however, has encountered several problems including the lack of appropriate nutrient management programs, low crop yield, and variable oil quality [
1,
8]. Plant growth is highly dependent on mineral nutrient supply [
9]. Among the essential nutrients, nitrogen (N) is one of the most important macroelements that significantly affects plant growth, development, and product quality [
10]. Analysis of distribution of nutrients in different organs of
C. oleifera showed the most abundant element is N ranging from 3.2 to 12 g kg
−1 compared to the next most abundant, potassium, ranging from 3 to 6 g kg
−1 [
8]. Thus, N is critically important for the growth of
C. oleifera. Commercially, N is primarily applied in the form of nitrate (NO
3−), ammonium (NH
4+) or urea (CO(NH
2)
2). Applied urea will be converted to NH
4+ in soils then absorbed by plants, meaning that urea, although it is an organic N source, is considered the same as NH
4+ during the absorption process [
11]. A large body of evidence suggests that both rates and forms of N fertilizers can affect plant growth and development [
11,
12,
13,
14,
15]. In general, NO
3− is considered to be the main N source for most crops [
16], whereas plants adapted to acidic soils prefer NH
4+-N and those adapted to high pH soils prefer NO
3−-N [
9].
Current fertilizer programs for
C. oleifera production have focused on the formulation of N, P
2O
5, and K
2O ratios and application rates [
17,
18]. Little attention has been given to the forms of N or the ratio of N forms on
C. oleifera growth. As
C. oleifera has adapted to red-acidic soils with pH around 5 [
19], we hypothesized that
C. oleifera would prefer NH
4+ over NO
3− for its growth. The objective of this study was to test this hypothesis using uniform seedlings of
C. oleifera as a model. Seedlings were fertilized with solutions comprised of six ratios of NO
3− and NH
4+ for five months. Plant growth parameters and physiological characteristics were evaluated. Contrary to our hypothesis,
C. oleifera prefers both NO
3− and NH
4+ as N sources at a ratio of 1:1.
2. Materials and Methods
2.1. Experiment Site
The experiment was conducted at the experimental station of the National Engineering Research Center for Oil-tea Camellia, Changsha city, Hunan province, China (113°01′ E and 28°06′ N). The location has a subtropical monsoon climate with mild temperatures and distinct seasons. The annual lowest, highest, and average temperatures are −12 °C, 40.6 °C, and 17.3 °C, respectively. Annual average rainfall is approximately 1422 mm, the frost-free period is 275 days, and the annual average relative humidity is 80%.
2.2. Plant Materials and Experimental Treatments
Uniform seedlings of Camellia oleifera ‘Xianglin 27′ were grown from seeds in early March 2015 and transplanted to containers filled with a soil-based substrate (medium). The substrate was composed of yellow earth soil with sand, silt, and clay in a ratio of 2:3:1, perlite, and peat in a 3:1:1 ratio based on volume and had a pH of 5.8. The containers had a volume of 0.6 L with a height of 12 cm and diameter of 8 cm. Seedlings were grown in a greenhouse with a maximum photosynthetic radiation of 1000 μmol m−2 s−1, temperature ranging from 20 to 25 °C, and relative humidity of 50%–80%.
To determine effects of different ratios of NO
3− and NH
4+ on
C. oleifera growth, six solutions were prepared weekly and used for fertigation (i.e., both fertilization and irrigation occurred at the same time) of seedlings. All solutions consisted of 261.39 mg·L
−1 K
2SO
4, 136.09 mg·L
−1 KH
2PO
4, 221.98 mg·L
−1 CaCl
2, 120.37 mg·L
−1 MgSO
4, 1.38 mg·L
−1 MnSO
4, 2.86 mg·L
−1 H
3BO
3, 0.12 mg·L
−1 ZnSO
4, 0.05 mg·L
−1 CuSO
4, 0.017 mg·L
−1 Na
2MoO
4, 10.94 mg·L
−1 FeSO
4, and 7 μmol C
2H
4N
4 (cyanoguanidine, a nitrification inhibitor) but varied in NO
3− and NH
4+ ratios as indicated in
Table 1. N concentration in each solution was 8 mM except the control treatment (T0) devoid of N.
After three months of growth, disease and pest free seedlings were selected and arranged in a randomized complete block design with three replications, and each replication had 10 seedlings per treatment. Seedlings were fertigated with six nutrient solutions, respectively. The fertigation started in mid-June 2015, at a rate of 150 mL per container weekly. If additional irrigation was needed, seedlings were irrigated with tap water. Besides fertigation, plants were well managed by pulling any weeds by hand.
2.3. Seedling Growth Measurements
Canopy heights of seedlings and stem diameters at the ground level were recorded using rule and Vernier caliper before fertigation and at the end of fertigation (November 2015). Canopy and stem increases were calculated based on their differences between before and after fertigation. Before the conclusion of the experiment, leaf samples were collected for analysis of physiological parameters indicated below. Seedlings were then harvested by cutting shoots from the substrate surface, and roots were recovered by inverting the containers in water, gently agitating the root system and washing the roots free from the substrate. Roots and shoots were placed in paper bags, initially dried at 105 °C for 30 min, and then dried to a constant mass at 80 °C. Root and shoot dry weights were recorded.
2.4. Analysis of Physiological Parameters
Total N contents in leaves of seedlings were determined using the Kjeldahl method [
20]. For analysis of chlorophyll content, enzymatic activity, and content of soluble saccharides and proteins, leaf samples collected from each replication were immediately weighed and frozen in liquid N. Chlorophyll a and b (Chl a and Chl b) were analyzed using the acetone-ethanol-mixture method [
21], soluble saccharides were assayed by anthrone colorimetry [
22,
23], soluble proteins were tested using the Coomassie brilliant-blue G-250 staining method [
24], peroxidase (POD) activity was examined using the guaiacol method [
25], superoxide dismutase (SOD) activity was analyzed by the nitroblue-tetrazolium photoreduction method [
26], nitrate reductase activity was determined by spectrophotometry [
27], and activities of N assimilation enzymes including nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthase (GOGAT) were assayed using spectrophotometry methods [
22,
28]. All the analyses had three replicates.
2.5. Data Analysis
Plant growth data, dry weights, and physiological parameters mentioned above were subjected to analysis of variance using SPSS 13.0 for Windows (SPSS, Chicago, IL, USA). When significant differences occurred among treatments per parameter, means were separated using Fisher’s Protected Least Significant Differences (LSD) at p < 0.05 level.
4. Discussion
Camellia oleifera is one of four major woody oil plants in the world. Its production has significantly increased over the last 20 years in China and is expected to expand from 3.67 million ha to 4.67 million ha by 2020 [
29]. The increased production requires science-based information on nutrient management. N is an essential nutrient for plant growth, and large quantities of N fertilizers are applied to ensure high crop productivity [
30]. Commercial N fertilizers are comprised of NO
3−, NH
4+ or CO(NH
2)
2. N form or the ratios of NO
3− and NH
4+ can significantly affect plant growth and development [
31]. How different ratios of NO
3− and NH
4+ could affect
C. oleifera growth has not been investigated thus far. The present work studied the effects of different ratios of NO
3− and NH
4+ on seedling growth of a
C. oleifera cultivar Xianglin 27. Results showed that canopy height and stem diameter increased, and shoot and root dry weights were the highest for the seedlings fertigated with the solution containing an equal ratio of NO
3− and NH
4+. The increased plant biomass was accompanied with enhanced uptake of N, higher chlorophyll content, increased activity of SOD, POD, NR, GS, and GOGAT as well as the highest concentration of saccharides and soluble proteins in leaf tissue studied.
Our results differ from the conventional thought that plants adapted to acidic soils prefer NH
4+-N. This is based on the fact that NH
4+ has a positive charge, and when roots take up NH
4+, an identically charged molecule, in this case H
+, is released from roots to maintain a balanced pH inside the plant cells. The release of H
+ keeps the rhizosphere pH at acidic levels, thus maintaining a suitable pH environment for root growth. Another reason is that NH
4+ is generally a major form of N in acidic soils. In a study of N forms and root-zone pH on growth and N uptake of tea (
Camellia sinensis (L.) O. Kuntze) plants, a close relative of
C. oleiferea that is also tolerant to acidic soils, Ruan et al. [
32] reported that plants fertilized with NO
3− exhibited yellowish leaves and reduced growth compared to those receiving NH
4+ or NO
3− and NH
4+ regardless of root-zone pH. Absorption of NO
3− was 2 to 3.4 fold slower than NH
4+ when supplied separately, and 6 to 16 fold slower when supplied simultaneously. Additionally, supply of NO
3− reduced chlorophyll content and GS activity [
32]. Coffee plants (
Coffea arabica L.) fed with NH
4+ also absorbed and assimilated more N than plants fed with NH
4+ and NO
3− or NO
3− only [
33]. Our study did not examine the specific absorption rate of NO
3− and NH
4+ but showed that seedling growth of
C. oleiferea was the greatest when NO
3− and NH
4+ were applied at the 1:1 ratio, and NO
3− and NH
4+ applied separately had much lower dry weight accumulation and lower contents of total N, chlorophyll, soluble saccharides and proteins as well as lower enzymatic activities of POD, SOD, NR, GS, and GOGAT. Our results agreed with those for
C. sinensis that application of both NH
4+ and NO
3− increased N uptake and increased biomass accumulation [
32] but differed from those that held that application of NH
4+ alone was equal or better than the application of NH
4+ or NO
3−.
Recently, increasing evidence has suggested that mixtures of NO
3− and NH
4+ are beneficial for plant growth compared to NO
3− or NH
4+ alone [
31], a finding which may be attributed to several factors: (1) Nitrate is a nutrient, and plants have developed a whole set of systems for uptake, transport, and assimilation of NO
3−. (2) There is a complementary effect between NO
3− and NH
4+ as reported by Lima et al. [
34]. Application of NH
4+ stimulates lateral root branching, whereas NO
3− stimulates lateral root elongation. When NO
3− and NH
4+ are applied together, concomitant enhancement of branching and elongation of lateral roots occurs, suggesting that the application of NO
3− and NH
4+ has local, complementary effects on root growth [
31]. (3) The presence of NO
3− enhances NH
4+ uptake and decreases NH
4+ efflux. A study with rice (
Oryza sativa L.) showed that an increased total NH
4+ uptake occurred concomitantly with a decrease in total NH
4+ efflux in the presence of NO
3−, thus enhancing net NH
4+ uptake [
35]. (4) Nitrate is a local and systemic signal molecule. A wide range of genes can be regulated by NO
3−, which significantly affect plant growth and development [
36]. Although the present study did not investigate N effects at molecular levels, our results suggest that the increased uptake of N in seedlings fertigated with an equal ratio of NO
3− and NH
4+ could enhance NR and GS/GOGAT cycle, increase chlorophyll content and Rubisco activity and thus raise soluble saccharides and proteins, and subsequently plant growth, i.e., dry matter accumulation. Additionally, SOD and POD can scavenge reactive oxygen species (ROS), protect cell membrane structure, and maintain the ROS metabolism balance in plants. The increased activities of SOD and POD may safeguard plants from ROS damage. (5) Root-associated microbes could be another factor influencing N uptake, such as mycorrhizal fungi [
37]. Roots of
Rhododendron fortunei Lindl., also an acid-loving plant, can effectively use NO
3− due to the presence of ericoid mycorrhizal fungus (
Oidiodendron maius) that can fully utilize NO
3− [
38,
39]. It is unknown if
C. sinensis and
C. oleiferea have different rhizosphere mycorrhizal fungi or other microbes. Nevertheless, further research to test the aforementioned possibilities is needed, and information gathered from such tests will improve current nutrient management programs for
C. oleifera production.