Introduction

Aeschynanthus longicaulis Wall. ex R.Br., belonging to the Gesneriaceae family, is native to southern Yunnan in China and to Malaysia, Myanmar, Thailand, and Vietnam (Wang et al., 1999). It produces clusters of orange flowers against dark green leaves on trailing stems. The back of the leaf is mottled, and the leaf color changes with the intensity of the light (Li et al., 2014). Like many other Aeschynanthus species, it can be grown as a hanging basket or indoor pot plant in a wide range of temperate climates and outside in tropical areas, with its trailing or pendulous stems and attractive leaf color (Mason, 2017). The best-known ornamental species of the genus Aeschynanthus is A. pulcher; the common name “lipstick plant” is given for the beautiful shape of the calyx and developing bud. There are also some hybrid cultivars, such as Aeschynanthus “Bali,” “Red Cascade,” “Hot Flash,” “Rigel,” and “Big Apple,” with different flower colors (Shalit, 2007). The plants are usually epiphytic and grow in well-drained but moist soil rich in organic matter or in soilless mixes consisting of bark, perlite, vermiculite, and/or charcoal (Shalit, 2007; Middleton, 2010).

With the popularity of Aeschynanthus in horticulture, many studies have concentrated on growth regulation methods (Schüßler, 1992); flowering regulation according to temperature and photoperiod (Welander, 1984; Gertsson, 1987; Whitton et al., 1990, 1991); and propagation through cuttings (Henselová, 2004), micropropagation (Kozak and Stelmaszczuk, 2010), and somatic embryogenesis (Cui et al., 2008). However, the fertilization of Aeschynanthus species is rarely investigated with reference to the fertilizer ratio or rate. Poole and Conover (1989) suggested a fertilizer requirement with 14-4-9 (N-P₂O₅-K₂O) g·m^-2 per month for A. pulcher in a general paper on the production of ornamental foliage plants. Aeschynanthus ‘Koral’ plants were fertilized weekly with 16N-6.9P-13.3K at 200 mg·L^-1 N in flowering research (Whitton et al., 1991). However, there is no systematic experimental research on the fertilization of this genus. Interaction between the irrigation volume and NPK rates on plant growth was only reported for Primulina yungfuensis, another genus of Gesneriaceae used as a house plant (Luo et al., 2019). The quality of ornamental plants is directly influenced by the balance of nutrients and other environmental factors. Unlike field crops, fruits, and vegetable crops, the precise nutritional requirements of ornamental plants are not well established, particularly species-specific requirements (Furtini Neto et al., 2015). Optimizing species-specific fertilization can enhance container plant production and reduce fertilizer waste (Clark and Zheng, 2015).

The quadratic regression model with a three-factor NPK fertilizer design has been widely used in field crops and in some horticultural crops, such as turfgrass (Ihtisham et al., 2018), strawberry (Wu et al., 2020), and tree seedlings (Yang et al., 2020), to determine optimal NPK ratios and rates. The “3414” fertilizer experiment design is a three-factor model with the advantages of an optimal regression design with fewer treatments and higher efficiency (Yang et al., 2020). After our previous study on leaf morphology and photosynthesis under different growth light levels (Li et al., 2014), the main objective of this study is to investigate the NPK fertilizer ratio and rate of the vegetative growth of A. longicaulis from cuttings in a “3414” fertilizer experiment.

Materials and Methods

Plant, Container Media, and Growth Environment

The plant materials were propagated by single-node cuttings in 32-cell plug trays (Fig. 1A). Similarly sized rooted cuttings with a single new shoot about 5 cm long (Fig. 1B) were selected and planted in 10 cm diameter plastic pots (10 cm top diameter× 8.5 cm bottom diameter× 9.5 cm height, two plants per pot) in May of 2017. A potting mix of 50% peat, 30% coir fiber, and 20% perlite (by volume) was used as the container media. The plants were grown in a 1000 m² greenhouse with outer and inner shade screens, pad and fans, and controllers for temperature and light. The maximum photosynthetic photon flux density (PPFD) in the greenhouse was 650 µmol·m^-2·s^-1. The highest temperature was 35°C and the lowest was 15°C during the experimental period from May to November. The experiment lasted six months and ended when the plants were ready to transplant to a larger container.

Fig. 1.

Single-node cuttings in 32-cell plug trays (A) and uniform rooted cuttings with a single 5-cm-long new shoot (B) were selected for the experiment.

NPK Fertilization Experimental Design

The “3414” fertilizer experiment design was conducted according to the recommended agriculture standard NY/T 2911-2016 (Regulations for soil testing and formulated fertilization) by the Ministry of Agriculture and Rural Affairs of the People’s Republic of China (Zhang, 2016). Four fertilization levels (0, 1, 2, 3) were established for three factors of N, P, and K. Here, 0 was the control (no fertilizer), the middle fertilizer rate was level 2 (0.3 g·L^-1 N, 0.15 g·L^-1 P, 0.3 g·L^-1 K), 0.5 times that of level 2 served as level 1 (0.15 g·L^-1 N, 0.075 g·L^-1 P, 0.15 g·L^-1 K), and 1.5 times that of level 2 was level 3 (0.45 g·L^-1 N, 0.225 g·L^-1 P, 0.45 g·L^-1 K). The “3414” fertilizer experiment design was derived from three factors and four levels of optimal design, but the total treatment was simplified to 14 (Table 1). Each treatment had nine replications (9 pots), and all treatments were laid out as a randomized complete block design.

The NPK fertilizer solutions for each treatment were made up of calcium nitrate [Ca(NO₃)₂·4H₂O] as the N source, monosodium phosphate (NaH₂PO₄) as the P source, and potassium sulfate (K₂SO₄) as the K source (Table 1). A mixed solution of MgSO₄·7H₂O (493 mg·L^-1) and micronutrients (each liter contains 20 mg NaFe-EDTA, 2.86 mg H₃BO₃, 2.13 mg MnSO₄·4H₂O, 0.22 mg ZnSO₄·7H₂O, 0.08 mg CuSO₄·5H₂O, and 0.02 mg (NH₄)₆Mo₇O₂₄·4H₂O) was applied separately to all treatments. Each solution's electrical conductivity (EC) value was measured using a portable EC meter (SX650). Fertilizer solutions were applied weekly at 50 ml·pot^-1. The moderate fertilizer amount was estimated according to the recommended rate of 14-4-9 (N-P₂O₅-K₂O) g·m^-2 per month for A. pulcher (Poole and Conover, 1989), and the total surface area of 120 pots in our experiment was approximately 1m². Additional water was supplemented by hand watering based on the moisture condition of the potting mix.

Measurement

In December of 2017, the branch number of each pot was counted, plant shoots and roots were harvested and dried at 85 °C for 48 hours, and dry weights were recorded. To determine the NPK contents, dry shoots (mix of all stems and leaves) of three pots from the same treatment were mixed and milled and subsequently passed through a 1 mm screen. Thus, three composite samples from nine pots of each treatment were used for the NPK concentration measurements. The grounded dry material (around 0.2000g) was digested using a H₂SO₄-H₂O₂ solution. Nitrogen was determined by the Kjeldahl method (Horneck and Miller, 1998). Phosphorus was determined by the phosphomolybdate-blue method (John, 1970). Potassium was determined by flame emission spectrophotometry (Horneck and Hanson, 1998).

Data Analysis

All data were subjected to an analysis of variance using SPSS 19.0. Normal distributions were assessed according to the Kolmogorov-Smirnov criterion. Where significant differences were noted, means were separated using Duncan’s multiple range test at P = 0.05. A three-factor quadratic regression model was developed among NPK levels and shoot dry weight. One-factor regression equations were calculated to evaluate the relationships among the total dry weight, root/shoot ratio, and NPK fertilizer concentration with fixed levels of the other two nutrients. Linear equations were fitted among shoot and root N, P, and K concentrations and the fertilizer’s N, P, and K concentrations.

Results

Effects of NPK Fertilizer Application on Plant Growth

The EC values of the NPK fertilizer solution varied between 0 (N0P0K0) and 3.5 mS·cm^-1 (N3P2K2) (Table 1). The shoot dry weight, root dry weight, and total dry weight were significantly increased with an increase in the N, P, and K concentrations in all cases (Table 2). However, only N and P greatly influenced the root/shoot ratio and number of branches. Only the interaction of N × K had a considerable effect on the root dry weight, and P×K significantly influenced the number of branches. The control (N0P0K0) plants mostly did not grow during the entire experimental period (0.40 g shoot dry weight). In treatment N0P2K2 without N, though P and K were at level 2, the plants only showed slight growth (0.73 g shoot dry weight). The N3P2K2 treatment led to the highest dry biomass (9.41 g shoot dry weight, 23.5 times the control) and showed better shoot growth with more branches (Fig. 2). These outcomes indicate that N had the most significant influence on the growth of shoots.

Fig. 2.

Plant growth with 14 fertilizer solution treatments. Plastic pots have a 10 cm top diameter × 8.5 cm bottom diameter × 9.5 cm height; the scale bar represents 10 cm.

The above-ground part determines the visible quality of plant growth. The shoot dry weight was closely related to the root weight, total weight, and number of branches. A three-factor quadratic regression model was developed to describe the relationship of the shoot dry weight (Y) among NPK fertilizer application concentrations.

Y = 0.52 + 1.85N + 4.36P + 6.06K + 12.2N²－37.5P²－25.5K² + 52.9NP + 13.4NK + 19.3PK (R² = 0.742)

In the equation above, both coefficients of N and N² were positive, while P² and K² had negative coefficients; it was also found that N had a more significant influence on the shoot growth than P or K.

The relationships among the fertilizer concentration and the total dry weight and root/shoot ratio were fitted as a one-factor regression (Fig. 3) when the other two fertilizer nutrients concentrations were fixed at level 2. The plant’s dry weight increased linearly with increases in the fertilizer N and P concentrations, and the root/shoot ratio decreased linearly with the fertilizer N and P concentrations. However, the K concentration did not demonstrate a good relationship with the plant’s dry weight or the root/shoot ratio with a low R², even according to a quadratic regression assessment.

Fig. 3.

Effects of N, P, and K levels on the total plant dry weight (DW) and root/shoot ratio.

Effects of Fertilizer NPK Concentrations on the Plant Tissue NPK Concentrations

All of the shoot and root NPK concentrations increased linearly with the applied fertilizer NPK concentrations when the other two nutrients were fixed at level 2 (Fig. 4). However, the application of N and P improved the N and P concentrations in plant tissue more significantly, especially in the shoot. The shoot and root N concentrations of the N level 4 treatment were four and two times greater than that of the control (level 0), respectively. The shoot P concentration increased eightfold from 0.43 mg·g^-1 in the control to 3.93 mg·g^-1 in the 0.225 g·L^-1 fertilizer P treatment. Meanwhile, the root P concentration increased twofold. Shoot and root K concentrations were only increased by 20% and 50% respectively, when the fertilizer K was increased to level 3 from level 0.

Fig. 4.

Effects of N, P, K levels on the shoot and root N, P, K contents.

Discussion

N, P, and K are the three primary macronutrients that plants need in large amounts. The NPK ratio and rate have significant impacts on plant growth outcomes. Vascular epiphytes in natural habitats are generally assumed to be nutrient deficient; an increased supply of both N and P independently stimulates vegetative growth in three epiphyte bromeliad species (Zotz and Asshoff, 2010). Considering the plant biomass and visual appearance, the N3P2K2 treatment with 0.45 g·L^-1 N (nitrate), 0.15 g·L^-1 P (P₂O₅), and 0.3 g·L^-1 K (K₂O) obtained the best result. The ratio (N:P:K = 3:1:2 = 15:5:10) was very close to the NPK ratio of 14-4-9 recommended for A. pulcher by Poole and Conover (1989). The potassium ratio was lower than that of Primulina yungfuensis (10:2.84:22.6), another Gesneriaceae genus (Luo et al., 2019).

Unlike the traditional quadratic relation of the crop yield to the nitrogen input with maximum output, the vegetative growth of A. longicaulis increased continuously with an increase in the N concentration in this experiment. However, the EC value of the N3P2K2 nutrient solution was 3.5 mS·cm^-1; increasing more N may cause salt stress. It is better to increase fertilization by the application frequency, particularly through the drip irrigation system, but as a vascular epiphyte, irrigation with too much water may easily cause root rot. Thus, it is also important to research the balance of irrigation water and fertilizer in the future.

Nitrates are often a preferred source for horticultural crop growth, and high NH₄-N levels are usually harmful to many horticultural crops. However, the appropriate NO₃-N/NH₄-N ratio benefits ornamental plant growth (Stegani et al., 2019). An application of nitrogen with NH₄-N ratios of 40% and 50% benefited the growth of epiphytic Phalaenopsis and Dendrobium, but higher proportions of ammonium resulted in decreased N, K, Ca, and Mg absorption outcomes (Mantovani et al., 2018). Nitrate-N increased the IAA/cytokinin balance, while urea and NH₄-N favored cytokinins, thus inhibiting root development in epiphytic bromeliads (Mercier et al., 1997). The nutrient solution in this study only constituted NO₃-N. Therefore, it is necessary to investigate the effect of the NO₃-N/NH₄-N ratio on the growth of epiphytic Aeschynanthus in the future.

Phosphorus can be beneficial for leaf formation in gesneriads (Primulina yungfuensis) (Luo et al., 2019). In epiphytic bromeliads, phosphorus is a limiting nutrient under natural conditions; an increased supply of P significantly improved growth and increased tissue P concentration levels (Zotz and Asshoff, 2010). The phosphorus uptake efficiency of epiphytic bromeliads is very high (Winkler and Zotz, 2008). In this study, the epiphytic A. longicaulis also showed highly efficient P uptake; the shoot P content increased 8-fold when the plants were supplied with 0.225 mg·L^-1 P. However, another gesneriad P. tabacum growing in the karst region showed a low N/P ratio (7.6) in the above-ground biomass, indicating that N was the most limiting nutrient (Liang et al., 2010).

Plants grown at a low concentration of K did not show significantly stunted vegetative growth, especially with sufficient amounts of N. However, there is very little data published on the demand for K in relation to the genus Aeschynanthus. The average K concentration of 16 field-grown species of epiphytic bromeliads is 1.1 ± 0.6% (dry weight), close to eutrophic vegetation level (Winkler and Zotz, 2010). Moreover, increasing the K concentration in the nutrient solution could not increase the Guzmania dry biomass (Lin and Yeh, 2008). This study also found that K did not influence the vegetative growth of A. longicaulis as significantly as N and P.

The interactions among P and K, N and P had significant effects on the growth of P. yungfuensis (Luo et al., 2019). In this experiment on A. longicaulis, only the interaction between N and K had a significant effect on the root dry mass, and P×K had a considerable influence on the number of branches. The NPK ratio also affected reproductive growth, particularly the flowering quality in ornamental plants (Ngapui et al., 2018). The effects of the NPK ratio and rate on the flowering should be investigated further in future studies.

Conclusions

The vegetative growth of A. longicaulis was improved by increasing the NPK application concentration. N had a more significant influence on the shoot growth and root/shoot ratio than P or K. The optimal NPK fertilizer ratio is 3-1-2 for A. longicaulis vegetative growth on soilless media. However, as water plays an important role in the plant growth, the water demand and the interaction between water and fertilizer levels should be studied further. The NO₃-N/NH₄-N ratio may also significantly influence A. longicaulis growth and also should be researched. Finally, further research is warranted to elucidate the different NPK demand levels at different plant growth stages, particularly at the flowering phase for A. longicaulis as an ornamental plant.