Introduction

Irisgermanica is one of the most popular ornamental perennials of the iris genus due to its showy display with multiple colors. A subclass of I.germanica cultivars, called re-blooming iris, blooms more than once per growing season, providing a prolonged flowering period and greater ornamental value. However, there is limited information about the management of nitrogen (N) and phosphorus (P) for optimizing the growth of re-blooming iris.

Plant growth is often limited by N and P availability (Vitousek et al., 2002; Iversen et al., 2010). N is the most commonly used mineral nutrient in plants; about 1－5% of total plant dry matter consists of N. N plays a pivotal role as a constituent of proteins, nucleic acids, chlorophyll, and other compounds (Marschner, 2012). Greater N fertilization rates can significantly promote shoot growth (Wang, 1996; Dong et al., 2004; Bi et al., 2007; Scagel et al., 2011).

P is another essential macronutrient that works as a structural element in nucleic acids and phospholipids and plays an important role in energy transfer. Usually, P is associated with root growth during plant development (Zhang et al., 2002; Graciano et al., 2006; Ristvey et al., 2007).

Plant growth and productivity can be affected by the balance between nutrients (Ingestad, 1991; Dighton et al., 1993; Graciano et al., 2006; Hasanuzzaman et al., 2012). Usually, N fertilization can increase extracellular phosphatase activity and hence stimulate P uptake (Fujita et al., 2010). On the other hand, N uptake efficiency was increased by increasing P availability (Iversen et al., 2010). This indicates that the interaction of P and N availability plays an important role in growth-related processes (Cornelissen et al., 1997).

When N availability limits plant growth, uptake of other nutrients is expected to decline accordingly (Marschner, 1995). For example, insufficient N supply caused growth limitation and led to decreased uptake of P, potassium (K), sulfur (S), calcium (Ca), and magnesium (Mg) in azalea (Rhododendron L.) (Scagel et al., 2008a). By contrast, increasing N application rate improved uptake of K, Ca, S, and Mg in rhododendron (Rhododendron L.) (Scagel et al., 2011). Thus, increased N rate should accompany modified concentrations of other nutrients in a fertilizer formulation to optimize growth (Scagel et al., 2008a).

In I. germanica, high N rates had positive effects on the number of inflorescences and inflorescence stem length (Hanley et al., 2008; Zhao et al., 2016), which indicates that high N level may improve the ornamental value of I. germanica by producing more flowers. However, excessive N fertilizer will run-off to the soil and pollute the water. Thus, optimal N rates should be determined to protect the environment while optimizing ornamental performance.

N utilization was affected by application level of P in work by Ali (2002), in which a higher P rate increased the N utilization. Thus, P level could influence plant growth or uptake of other nutrients by affecting N utilization. As P availability influences N uptake efficiency, the effects of N on plant growth and development can be accurately examined with sufficient P supply. In this work, factorial combinations of three N rates and three P rates were applied to study the effects of different N and P rates and their interaction on growth, flowering, and uptake of other mineral nutrients in I. germanica.

Materials and Methods

Plant Material and Growth Conditions

This study was conducted outdoors in full sun at Starkville, MS (lat. 33°46' N, long. 88°82' W). In August 2012, rhizomes (average caliper = 4.7 cm and length = 5.8 cm) of ‘Immortality’ Tall bearded iris (Schreiner’s Iris Gardens, Salem, OR) were potted into 3.78-L (23-cm diameter; 16-cm height) round plastic pots with one rhizome per pot. The pots were filled with commercial substrate with no starter fertilizer (Fafard 2; Sun Gro Horticulture, Agawam, MA). Two weeks after transplanting, each plant was fertigated with 400 mL of nutrient solution with N-free fertilizer (1.06 mg·mL^-1; Cornell No N Formula 0-6-27; Greencare Fertilizers, Kankakee, IL) plus 10 mM N from NH₄NO₃ (Sigma Aldrich, St. Louis, MO) twice per week from August through September in 2012 to provide basic nutrients for fall growth.

Experiment Treatments

In April 2013, five plants were harvested for baseline biomass and nutrient composition before spring fertigation treatment started. From April to September 2013, nine N and P rate combinations using a 3 by 3 factorial treatment design were applied twice per week to plants with 20 replicates under each treatment in a completely randomized experimental design. The three rates of N and P were 5, 10, or 15 mM, and each treatment was designed to allow for only N or P rate to change while all other nutrients were held constant, except for chlorine (Cl^-) (different concentrations of KCl were used to maintain a constant concentration of K) Table 1. Other micronutrients [Fe (0.1 mM); Mn (0.01 mM); zinc (Zn, 10^-3 mM); Cu (10^-3 mM); B (0.05 mM)] were also added to nutrient solutions. Analytical grade chemicals were used to make fertilizer solutions.

Experiment Methods

During the growing season, flowering data (number of inflorescences and inflorescence stem length), plant height (average height of top three cluster of leaves), and relative leaf chlorophyll content measured as SPAD readings were collected. The leaf SPAD readings were taken using a chlorophyll meter (SPAD-502; Minolta Camera Co., Japan), with one of the first two fully expended leaves selected to measure SPAD.

In December 2013, four plants from each treatment were randomly selected and destructively harvested (senesced leaves were removed). During harvesting, numbers of clustering leaves (cluster of leaves) and floral meristems (FMs) visible to the naked eye were counted. Each plant was divided into leaves, roots, and rhizomes. All samples were dried in a 60°C oven until they reach a constant weight and then samples were ground to pass a 40-mesh sieve in a Wiley Mill (Thomas Scientific, Swedesboro, NJ) for nutrient analysis. Tissue N was determined by the Kjedahl method (Schuman et al., 1973), and concentrations of other macronutrients and micronutrients in plant tissues were obtained using inductively coupled plasma optical emission spectroscopy (ICP-OES) at the Soil Testing Lab of Mississippi State University. Nutrient content of each tissue was calculated by multiplying dry weight by concentration. Total plant content of each nutrient was calculated by the sum of the content in leaves, roots, and rhizomes. Nutrient uptake was estimated by subtracting the average total nutrients in April 2013 from the average total nutrients in December 2013.

In the following spring (2014), the remaining four plants from each treatment combination were grown outdoors without any fertilizer. In spring 2014, flowering (number of inflorescences and inflorescence stem length), plant height (average height of top three clustering leaves), and leaf SPAD reading data were collected.

Statistical Analysis

The experiment was conducted in a completely randomized design with factorial arrangement of treatments. N rate (three levels) and P rate (three levels) served as the two main factors. Continuous response data were analyzed using linear models with the GLM procedure of SAS (version 9.3; SAS Institute, Cary, NC), and count data were analyzed using generalized linear mixed models with the GLIMMIX procedure of SAS. If the interaction term was not significant, main effects are reported and discussed; if the interaction was significant, simple effects (the effect of a variable at each level of the other variable) are reported and discussed. Mean comparisons were made using Tukey’s honestly significant difference (HSD) at p < 0.05.

Results and Discussions

Effects of N and P on Plant Growth

In this study, an increasing N rate had positive effects on plant height in June 2013 (approximately 3 months after treatment); SPAD readings were higher in plants with 10 and 15 mM N treatment compared to those with 5 mM N in June 2013 (Table 2). Plant height was measured with a ruler, and SPAD value is an indicator of the greenness or relative chlorophyll content of leaves. In this study, the results showed that N rates had positive effects on plant height in June 2013 (with treatments of about 3 months); SPAD readings were higher in plants with 10 and 15 mM N treatment compared to 5 mM N treatments in June 2013 (Table 2).

In March 2014, the plant height and SPAD value were measured to reveal the effects of storage nutrients from last year on the plant growth in early spring. In March 2014, plant height was increased by N rates of 10 and 15 mM compared to 5 mM treatment, whereas SPAD readings were affected by the interactions between N and P rate (Table 2).

Effects of N and P on Plant Flowering Performance

In December 2013, the numbers of clustering leaves and FMs were affected by N rate but not by P rate (Table 3). The numbers of clustering leaves and FMs under N rates of 10 and 15 mM were greater than that with 5 mM.

Compared to the 5 mM N treatment rate, 10 and 15 mM treatments also increased the diameter and length of the largest axillary rhizome (Table 3). This is supported by research with Siam tulip (Curcuma alismatifolia), in which increasing N rates increased rhizome size (Ruamrungsri and Apavatjrut, 2003). Craver and Harkess (2012) indicated that floral initiation was related to rhizome size, and larger rhizomes were more likely to initiate flowering. Considering that the rhizome size was increased by the 10 and 15 mM rates, the greater number of FMs and rhizome sizes might be positively related in this study.

In spring 2013, neither N nor P rate affected the number of inflorescences (presented as stems/plant) and inflorescence stem length (Table 3). Since the flowers bloomed in late April, which is less than 1 month from when the fertilization treatments started, none of the effects of N or P treatments on florescence were due to a short treatment period.

In spring 2014, inflorescence stems were longer with 10 mM N than with 5 or 15 mM N. Neither N nor P rate affected the number of inflorescences (Table 3). The numbers of FMs were 2.8, 4.3, and 4.5 per plant with 5, 10, and 15 mM N, respectively, in December 2013, which had great potential to flower in the spring. The numbers of flower stems of each plant in spring 2014 were much lower than the number of FMs in December 2013. It is possible that storage nutrients from 2013 were not sufficient to support all the FMs to blooming in spring 2014, even though those FMs initiated in fall 2013.

Effect of N and P on concentration of macro-nutrients in December 2013

Concentrations of N, P, and Mg in leaves (Table 4); concentrations of P, K, and Mg in roots (Table 6); and concentrations of N, P, Mg, and Ca in rhizomes were affected by treatments in December 2013 (Table 8).

N concentrations in rhizomes and leaves of plants treated with 10 and 15 mM N were higher than those with 5 mM N. N concentration was not affected by either N or P rate in roots. The average N concentration in leaves and rhizomes was greater than 2% compared with less than 1% N concentration in roots.

Phosphorus concentration in roots and rhizomes was affected by the main effect of N rate: 10 and 15 mM N increased P concentration compared with 5 mM N. In leaves, P concentration was influenced by the main effects of N and P rate separately without interaction.

In roots, K concentration was highest in treatment with 5 mM N and 5 mM P, which means higher N and P rates decreased root K concentration (Table 6). In leaves and rhizomes, concentrations of Mg were influenced by the main effects of N rate. Interestingly, general trends of Mg concentration decreased with higher N rates in leaves and increased in rhizomes.

In this study, tissue N:P ratio varied from 4.7 to 7.5, 2.4 to 4, and 6 to 8.7 in leaves, roots, and rhizomes, respectively, under different N and P treatments (Tables 4, 6, and 8). In leaves and roots, higher P rate decreased tissue N:P ratio, but N rate had no influence on N:P ratio. In rhizomes, neither N nor P rate affected tissue N:P ratio.

Effect of N and P on Concentration of Micro-nutrients in December 2013

The N and P treatments had effects on concentrations of only a few micro-nutrients. Concentrations of zinc (Zn), copper (Cu), and boron (B) in leaves (Table 5), concentrations of Zn and Cu in roots (Table 7), and concentrations of iron (Fe) and Cu in rhizomes (Table 9) were affected by treatments.

Iron concentrations in leaves and roots were affected by neither N nor P treatment. In rhizomes, Fe concentration was the highest in plants receiving 10 mM N (Table 9). In leaves and roots, concentrations of Zn were influenced by the interaction of N and P treatments (Tables 4 and 8). Concentration of Cu was only affected by the main effects of N rate in leaves and interaction of N and P treatments in roots and rhizomes (Tables 5, 7, and 9).

Effect of N and P on Net Nutrient Uptake of iris ‘Immortality’ in December 2013

In this study, net uptakes of N, P, Mg, Mn, Zn, and B were only affected by the main effects of N rates. Uptake of K, Ca, and Fe was affected by neither N nor P rate. Phosphorus rate had no influence on the uptake of most nutrients, except for Cu, which was affected by the interaction between N and P rate (Table 10).

Discussion

In addition to flowers, the sword-shaped linear leaves of irises give them a high ornamental value. SPAD reading is an indicator of relative leaf chlorophyll content. A higher SPAD reading means healthier and greener leaves. In this study, the effects of N treatment on plant height and SPAD reading indicated that greater N rates promote vegetative growth in iris, which is consistent with previous studies of rhododendron (Bi et al., 2007) and Curcuma alismatifolia (Ruamrungsri and Apavatjrut, 2003).

In this study, the P rate did not affect plant height in June 2013 and March 2014. Plant height was affected by the rates of both N and P in maize (Khan et al., 2014). In the bulbous plant gloriosa lily (Gloriosa rothschildiana), N did not affect plant height, whereas low P rate reduced plant height (Ruamrungsri et al., 2011). The different responses of plants to changes in P rate indicated that the requirement for P fertilization may vary among plant species.

The P rates used in our study of 155 mg/L (5 mM), 310 mg/L (10 mM), and 465 mg/L (15 mM) were relatively high compared to the 50 and 100 mg/L P rates in Ruamrungsri et al. (2011), suggesting that a 5 mM P rate is likely high enough to satisfy the P requirement for the growth of TB iris ‘Immortality’. In many crops, the need for high P rates may have been overemphasized, which causes overapplication of P fertilizer (Wang and Konow, 2002; Ristvey et al., 2007).

N fertilizer usually promotes vegetative growth rather than reproductive growth (Bi et al., 2007; Scagel et al., 2011). In this study, N rates of 10 and 15 mM increased both number of clustering leaves and FMs compared with 5 mM, which means greater N rates also have positive effects on reproductive growth. This result is supported by the research in hybrid Phalaenopsis orchids, in which high N rate is necessary for increasing number of inflorescences (Wang, 2000).

N:P ratio has often been used in agriculture and forestry to analyze nutrient limitations (Fenn et al., 1998; Tessier and Raynal, 2003). For instance, in 1-year-old rhododendron (Rhododendron L.), N:P ratio was greater than 14:1 without N limitation, and N:P ratio was less than 9:1 when plants were N-deficient (Scagel et al., 2008b). In this study, N:P ratio was lower than 9:1, suggesting N may be the limiting factor to growth, which may explain why most growth-related data were only affected by N rate.

In this study, the uptake of most nutrients was only affected by N rates. This indicates that N altered the uptake ability and demands of the plant for other mineral nutrients. To optimize growth, increasing N rate should accompany modified doses of other nutrients in a fertilizer formulation (Scagel et al., 2008a).

P rate had no influence on nutrient uptake, including plant P uptake, although 15 mM P rate increased P concentration in leaves and roots. One explanation could be the amount of P uptake was more related to DW, which was affected by N rate. In addition, the N:P ratio in our study was low, suggesting plants in this study had a relatively high tissue concentration of P and 5 mM P rate satisfied plant demand.

Conclusions

In summary, plant height, leaf SPAD reading, and flowering performance, including number of FMs and flower stems, were mainly affected by N rate. Relatively high N fertilizer rates are recommended for growing I. germanica to obtain high ornamental value. In general, increasing N rates from 5 to 15 mM increased uptake of N, P, Mg, Mn, Zn, and B in TB iris. Thus, fertilization doses of other nutrients should be modified with changes of N rate to optimize plant growth. P rate affected concentrations of P and B in leaf, concentration of P in root, and concentration of Cu in rhizome.