Effect of Nitrogen Nutritional Statuses and Waterlogging Conditions on Growth Parameters , Nitrogen Use Efficiency and Chlorophyll Fluorescence in Tamarillo Seedlings

Climate change has altered rainfall patterns causing waterlogging periods that often negatively affect the performance of horticultural crops in the Andean region in Colombia. An experiment was carried out under greenhouse conditions using three-month-old tamarillo (Solanum betaceum Cav.) seedlings, which were grown under two levels of nitrogen (N) (10 and 150 mg N∙L H2O). At 28 days after transplanting (DAT), waterlogging treatments were established when well-nourished plants (150 mg N L H2O) significantly showed a higher shoot length than poor-nourished plants (10 mg N L H2O) (~20 cm vs. ~10 cm, respectively). Three different periods of waterlogging were performed between 35 and 37, 51 and 55, and 64 and 70 DAT by covering the holes in the plastic pots to ensure a constant water depth. Results showed that well-nourished plants without waterlogging treatments through the experiment’s stress showed a greater shoot length (30 cm), total plant dry weight (7.95 g), Fv/Fm ratio (0.62) and leaf chlorophyll content (37.51 SPAD units) than poor-nourished plants without stress condition (15 cm, 5.57 g, 0.5 and 12.69 SPAD units, respectively) at the end of the experiment. Overall, waterlogging reduced leaf area and nitrogen use efficiency (about 75% and 50%, respectively) in both N levels. However, periods of waterlogging enhanced dry matter partitioning to stems (around 30-35%) in both N levels. This study showed that tamarillo plants are susceptible to landscaping situations where periods of waterlogging can be expected regardless of their N nutritional status.


Introduction
Tamarillo (Solanum betaceum Cav.) is a plant that belongs to Solanaceae family and native from Andean region.This fruit is grown commercially in countries like Brazil, Colombia, Ecuador, USA (California), Australia and New Zealand (Mejía et al., 2009;Correia and Canhoto, 2012).In Colombia, this crop occupied an area of 9000 hectares with a production of 160,000 tons in 2013 (Agronet, 2015).
Horticultural crops have been severely affected due to the increased frequency and intensity of rainfall in Colombia in recent years, as a result of climate change conditions or environmental phenomena such as La Niña, causing prolonged waterlogging periods (Floréz-Velasco et al., 2015).Waterlogging is a situation of excess water that fills soil pores and results in a reduction of available oxygen, causing different physiological responses in plants (Parad et al., 2013).Flooding has some severe consequences on plant growth and its survival strategies (Phukan et al., 2016).In this sense, the following physiological disorders have been reported under waterlogging: a lower plant growth (Aldana et al., 2014), leaf chlorophyll content (Rao and Li, 2003), and total plant dry weight (Florez-Velasco et al., 2015), as well as changes in the plant water status (Striker et al., 2005).Also, waterlogging can alter biomass allocation in plants (Wu et al., 2015) Plant nutritional status can significantly influence the plant acclimation to stressful environmental conditions.In this respect, the interaction between plant nutritional status and environmental stresses (drought, salinity, temperature, etc.) is important to understand yield losses (Cakmak, 2005).On the other hand, nitrogen (N) is one of the most essential mineral nutrients due to its many effects on plant mix of sand and white peat without nutrients (Base 1 substrate, Klasman-Deilmann GmbH, Geeste, Germany) in a relation 2:1 as substrate.Weather conditions in the greenhouse during the experiment were the following: average temperature 20 ± 4 °C, relative humidity between 60 and 80% and a natural photoperiod of 12 hours (photosynthetically active radiation at noon of 1600 µmol m -2 s -1 ).

Nitrogen treatments
At 15 days after transplanting (DAT), a group of 20 plants was chosen to establish each of the nutritional nitrogen statuses.Then, plants were watered during the experiment with ½ Hoagland nutrient solutions with the following composition: 0.25 mM calcium phosphate [Ca(H2PO4)2], 2.5 mM potassium chloride (KCl), 1.0 mM magnesium sulphate (MgSO4), 12.5 μM boric acid (H3BO3), 1.0 μM manganese sulphate (MnSO4), 1.0 μM zinc sulfate (ZnSO4), 0.25 μM copper sulphate (CuSO4), 0.2 μM ammonium molybdate [(NH4)6Mo7O24], 10 μM Fe-ethylenediamine-di-o-hydroxy phenylacetic acid and either 10 (low N) or 150 mg N L -1 H2O (high N).The two concentrations of N in the nutrient solutions used in this study were selected because similar ranges were employed in previous studies with the purpose of establishing low and high levels of nitrogen in plants (Belesky et al., 1984;Florez-Velasco et al., 2015).Also, the fertilizer used as a source of N was 46% urea (Monomeros, Barranquilla, Colombia).Furthermore, the concentration of the other macro and micronutrients of this study has also been widely used in studies of mineral nutrition (Garcia-Castro and Restrepo-Díaz, 2013).
Plants were watered twice a week, and the irrigation volume was adjusted throughout the experiment.Tamarillo plants were watered with 150 mL plant -1 week -1 between 14 and 35 DAT and 200 mL plant -1 week -1 between 35 and 70 DAT.The water needs of the plants were estimated by the weight of the plants every 24 hours.

Waterlogging treatment
Nitrogen highly influences the growth and development of plants (Maschnner, 2013).For that reason, tamarillo plants were divided into two groups of 10 plants in each nitrogen treatment with the purpose of establishing the waterlogging conditions (with or without waterlogging) when differences on shoot length were observed between the two treatments of nitrogen fertigation (28 DAT).Three different periods of waterlogging with duration of two, four and six days were established.The periods of low availability of oxygen in the soil were developed between 35 and 37 (S1), 51 and 55 (S2), and 64 and 70 (S3) DAT, respectively.Also, plants had a recovery time of 14 days between the first two periods of stress.The conditions of oxygen deficiency in the soil were carried out by covering the holes in the plastic pots to ensure a constant water depth of 1 cm over the substrate during periods of waterlogging.At the end, four groups for different treatments were available: i) well-nourished plants with N, but without waterlogging, ii) well-nourished plants with N and with waterlogging stress, iii) poorly nourished plants with N, but without waterlogging conditions and iv) poorly nourished plants with N and with waterlogging.In general, each treatment consisted of ten plants and the experiment lasted for 70 days.
growth and yield (Fageria, 2001).N is involved in fundamental processes such as nutrients uptake, protein metabolism, photosynthesis and carbon partitioning (Maschnner, 2012).Furthermore, it has been documented that the plant N content decreases in plants under waterlogging conditions because O2 deficiency in the root zone can change the redox state of nutrients, making them unavailable (e.g.nitrogen) or potentially toxic for plants (Rao andLi, 2003: Najeeb et al., 2015).
Plant nutrient deficiency is the leading cause of poor plant growth under waterlogging conditions (Steffens et al. 2005).In this sense, it has been reported that an optimum N supply can help to ameliorate the adverse effects of waterlogging in a range of economically important crops (Percival and Keary, 2008).In wheat, a high nitrogen supply improved the plant growth compared to nonfertilized controls under waterlogging stress conditions (Huang et al., 1994).Flórez-Velasco et al., (2015) also observed that lulo plants grown with high N level (110 mg N•L −1 ) and subjected to waterlogging conditions showed a major plant height than plants cultivated with low nitrogen (10 mg N•L −1 ) under flooded periods.Finally, plant N status is essential for plant recovering after O2 deficiency in the root zone (Milroy et al., 2009).
The severity of the effects of waterlogging is also associated with the duration and/or frequency of the waterlogging event (Malik et al., 2002;Visser et al., 2003).In tomato, short periods of waterlogging (2-4 days) reduced stem growth and leaf chlorophyll content (Rao and Li, 2003).Malik et al. (2002) also reported that even short periods (as short as 3 d) of waterlogging have important long-term effects on the growth of young wheat plants.On the other hand, Ezin et al. (2010) and Aldana et al. (2014) also observed that leaf growth and plant height varied according to the flooding duration in tomato and cape gooseberry plants, respectively.
In general, studies about physiological responses of some Andean fruit trees species also belonging to Solanaceae family to waterlogging stress conditions have gained importance in recent years in Colombia due to their export potential as fresh fruit (Aldana et al., 2014;Florez-Velasco et al., 2015;Cardona et al., 2016).For that reason, it is important to enlarge the available knowledge on the interaction between nutritional status and waterlogging stress conditions (duration and/or frequency) for the economic and agricultural improvement of tamarillo plants in waterlogging affected areas.Therefore, the objective of this work was to study the interaction between N nutritional status and three short periods of waterlogging stress on the physiological traits of tamarillo seedlings.

General growing conditions
The present work was performed under controlled conditions in the greenhouses of the Faculty of Agricultural Sciences at Universidad Nacional de Colombia in Bogotá (2556 m.a.s.l.; 4° 35' 56'' N, 74° 04' 51'' W) for 12 weeks from March to May 2012.Two-month-old 'Amarillo' tamarillo seedlings were transplanted into 1-L plastic pots containing a

Determined variables
The shoot length was determined weekly from 14 to 70 DAT.At the end of the trial (70 DAT), the efficiency of photosystem II (Fv/Fm ratio) was determined by using a Fluorescence spectroscopy (Handy PEA, Hansatech Instruments, Kings Lynn, UK).Also, leaf chlorophyll content was obtained by SPAD readings as a non-destructive tool using a chlorophyll meter (SPAD-502; Minolta, Ramsey, NJ).The leaf chlorophyll content and maximum efficiency of PSII were obtained by taking a fully expanded leaf of the outer half portion of the plant.Subsequently, plants were harvested to determine the dry weights of each organ.Leaf area (LA) was also determined at the end of the experiment using a leaf area meter (LI-3100, LI -COR Inc., Lincoln, NE, USA).Likewise, the leaf area was used to calculate the leaf area ratio (LAR) by the relationship between leaf area and total plant dry weight (TPDW).Finally, nitrogen use efficiency (NUE) was also calculated using the methodology described by Florez-Velasco et al., 2015, which consisted in estimating the ratio of the total plant dry weight (TPDW) and the amount of nitrogen applied in each treatment (NUE = TPDW / N applied, g g -1 ).

Experimental design and statistical data analysis
A factorial design with two factors (nitrogen nutritional status vs. waterlogging conditions) with ten repetitions (plants) per treatment was used.All values in percentages were transformed with the arcsine formula before the analysis.Data were analyzed using the program Statistix v. 8.0 (Analytical Software, Tallahassee, FL, U.S.).Finally, the summary of the analysis of variance of the effect of waterlogging treatments and N nutritional status and their interaction on the physiological response of tamarillo seedlings to waterlogging period is shown in Table 1.

Growth parameters
Differences were observed on shoot length due to two nitrogen (N) nutritional statuses (10 vs. 150 mg N L -1 H2O) at 28 DAT.Cultivated plants with a higher N content in the nutrient solution began to show a greater height until the end of the experiment (Fig. 1).Regarding the interaction between nutritional status vs. waterlogging stress, it was observed that shoot growth was lower in tamarillo plants subjected to three waterlogging periods in both N treatments at 70 DAT.On the other hand, stem diameter showed only differences in the N status factor and waterlogging treatments.A higher stem diameter was found when tamarillo seedlings were cultivated with 150 mg N L -1 H2O.Likewise, when plants were subjected to three different periods of waterlogging, the stem diameter was also lower in comparison to plants without stressful conditions (Table 2).Aldana et al. (2014) also reported similar observations when a significant reduction in plant height and stem diameter was seen after subjecting cape gooseberry (another Solanaceae species) seedlings to eight-day waterlogging periods.Also, Florez-Velazco et al. (2015) also evaluated the effect of the interaction between waterlogging vs. nutritional N status and found that a series of short periods of oxygen deficiency in the soil caused a decrease in the shoot length of lulo plants (another Solanaceae species) cultivated with two levels of N (10 vs. 150 mg N L -1 H2O), highlighting that the most significant reduction was observed in plants that were always nitrogen-deficient.A lower plant height under waterlogging conditions can be attributed to low O2 supply which causes ionic imbalance and/or nutrient stress.Also, waterlogging stress causes a decreased N uptake, affecting the development of the cortical cells (Rao and Li, 2003).For that reason, stem diameter could show a lower diameter in waterlogging plants.In general, several studies on the effects of flooding in crop plants have showed that morphological adjustments (such as plant height, stem diameter, adventitious roots, and hypertrophic lenticels) contribute to flood acclimatization (Liu et al., 2014;Najeeb et al., 2015;Phukan et al., 2016).In the present study, plant height and stem diameter did not increase, indicating that these lacks of changes suggest that S. betaceum could not achieve flood tolerance in spite of an optimum N nutritional status.
On the other hand, differences were also obtained in the N nutritional status vs. waterlogging interaction on leaf area and leaf area ratio (LAR) at the end of the experiment (Fig. 2).Plants cultivated with high levels of nitrogen without waterlogging had greater leaf area.Also, this variable was negatively affected by waterlogging conditions since wellnourished plants showed similar leaf area values to those obtained in plants with low nitrogen and without periods of excess water stress.Finally, tamarillo plants grown with 10 mg N L -1 H2O and waterlogging had the lowest leaf area at the end of the experiment.Similar trends were also obtained in LAR.Waterlogging stress periods also caused a reduction of LAR values in tamarillo seedlings cultivated with either high or low N.The leaf area reduction in tamarillo plants at three periods of waterlogging stress may be due to a reduction in the leaf gas exchange properties (transpiration and stomatal conductance), as observed in other Solanaceae species such as tomato (Rao and Li, 2003) and lulo (Florez-Velasco et al., 2015), causing an adverse effect on the photosynthetic capacity of the plant (Alcantara et al., 2012).Also, a lower LAR under flooding periods is due to limit nutrients uptake, producing a higher biomass allocation towards stems and/or roots (Rubio et al., 1995).

Dry weights and distribution of assimilates
Similar trends to the ones observed in leaf area and LAR were also found in the nutritional status vs. waterlogging interaction on leaves, stems, and total plant dry weight (Table 2).In general, it can be seen that plants under N optimal conditions without waterlogging in soil showed higher dry weights at 70 DAT.Also, waterlogging stress reduced the accumulation of dry matter in both nutritional states (10 and 150 mg N L -1 H2O).Similar results were seen in cotton (Hocking et al., 1985) and lulo (Florez-Velasco et al., 2015).Regarding roots dry weights, differences were not found between N nutritional status (N) and waterlogging treatments (T).However, when dry weight data were analyzed as dry mass partitioning, it was observed that there were differences in the interaction NxT on root allocation.In general, leaves biomass allocation was negatively conditioned by waterlogging conditions or low nitrogen levels during growing (Fig. 3).In contrast, a greater accumulation of assimilates (about 40%), was found under soil oxygen-deficiency conditions in tamarillo stems in both nutritional states.In this aspect, Trought and Drew (1980) also observed an increase in the dry matter partitioning towards wheat plant stems exposed to a short waterlogging period (8 days) due to a higher accumulation of carbohydrates.Bailey-Serres and Voesenek (2008) has also been reported that a re-allocating biomass (especially starch) to stems and petioles under waterlogging stress because flooding promotes the conversion of starch to glucose as a metabolic acclimation under O2 deprivation.Finally, a higher dry matter partition towards roots (̴ 70%) was observed when plants were grown in an N deficient medium, despite the waterlogging condition.Plants often respond to nutritional stresses by increasing biomass partitioning towards their roots because more nutrients are absorbed when there is a greater root volume (Mengel and Kirkby, 2001).Poorter et al. (2012) also stated that a significant increase in biomass allocation towards roots at the expense of stem and, especially, of the leaf when nutrients are limiting.Data from this study showed that 70% of the biomass was found in roots of tamarillo plants grown in Table 2. Effect of the interaction between N nutritional status (10 and 150 mg N L -1 H2O) and waterlogging conditions (with or without) on some growth parameters (Stem Diameter (SD), Leaves Dry Weight (LDW), Stem Dry Weight (SDW), Roots Dry Weight (RDW) and Total Plant Dry Weight (TPDW)) in tamarillo seedlings at 70 days after transplanting y Means with different letters represent statistically significant differences according to Tukey's test (p ≤ 0.05).
x * , ** , and *** significantly different at the 0.05, 0.01 and 0.001 probability levels, respectively.NS, not significant at α = 0.05.an N deficient medium (10 mg N L -1 H2O) in both conditions of waterlogging.Similar observations were found in lulo by Parra-Coronado et al. (2015) where they reported that 70% of the dry matter was accumulated in roots under N deficiency.
Chlorophyll content, chlorophyll fluorescence and NUE Tamarillo plants grown with a high nitrogen concentration and without waterlogging periods showed the highest SPAD readings at the end of the experiment (Table 3).In this context, a low chlorophyll content under waterlogging stress may be because this abiotic stress condition causes a significant decrease in the N uptake due to a reduced root activity (Rao and Li, 2003).On the other hand, plants grown with a low N solution and without low oxygen availability conditions in the soil had the highest nitrogen use efficiency (NUE).NUE was reduced to 50% by waterlogging periods in tamarillo plants in both N levels.In this sense, the effect of the waterlogging vs. N nutritional status interaction has also been reported by Florez-Velasco et al. (2015) in lulo seedlings.They found that NUE was also higher when plants were grown under low N.A higher NUE in tamarillo plants cultivated in an N deficient medium can be due to the fact that NUE tends to increase when the N input decreases, since the plant tries to optimize its N absorption through a high-affinity nitrate transport system (Mi et al., 2007).On the other hand, our results also showed that waterlogging caused a lower NUE.Under flooded conditions, the bioavailability of N decreases due to dilution and leaching (Liu et al., 2013).Also, it is important to consider that under O2 deficiency in the root zone can change the redox state of nutrients, making them unavailable (e.g.nitrogen) (Najeeb et al., 2015).
Differences were separately observed on Fv/Fm ratio due to N nutritional status, or three short periods of waterlogging.Fv/Fm ratio diminished when plants were subjected to low N or waterlogging conditions (Fig 3).Studies have shown that chlorophyll fluorescence is a good indicator of nutrient stresses (Gorbe and Calatayud, 2012).Parra-Coronado et al. (2015) also found that Fv/Fm ratio was lower under nitrogen deficiency in lulo.Furthermore, Lima et al. (1999) stated that a drop in the Fv/Fm ratio might be caused by chlorophyll loss and a low nitrogen content created by an imbalance in the allocation of assimilated nutrients.This theory could also be applied to the poor-nourished plants (10 mg N L -1 H2O) in the present trial, which demonstrated a lower Fv/Fm ratio, as these plants had a lower chlorophyll content (Fig. 3) and accumulated less LDW (Table 2).As it was mentioned above, Fv/Fm ratio was conditioned by waterlogging treatments.Malik et al. (2015) observed that the PSII efficiency was also negatively affected by waterlogging.Also, Irfa et al. (2010) mentioned that a lower Fv/Fm ratio under waterlogging conditions may be due to an increase in the ethylene concentration in leaves because 1aminocyclopropane-1-carboxilic acid (ACC, a precursor of ethylene) is synthesized and then transported in the xylem to the aerial part where it is rapidly oxidized to ethylene in anaerobic conditions (Ahmed et al., 2006).

Conclusions
In general, this study also showed that a lower shoot length, stem diameter, leaf area, total plant dry weight, NUE, Fv/Fm ratio and chlorophyll content either with a low or high N status.This lack of response in the physiological traits under waterlogging conditions indicates that S. betaceum plants could not achieve a flood tolerance mechanism, allowing inferring that this species is susceptible to landscaping situations where periods of waterlogging can be expected in spite of an adequate nitrogen nutrition.3. Effect of the interaction between N nutritional status (10 and 110 mg N L -1 H2O) and waterlogging conditions (with or without) on leaf chlorophyll content (SPAD readings) and Nitrogen Use Efficiency (NUE) in tamarillo seedlings at 70 days after transplanting

Fig. 1 .
Fig. 1.Effect of the interaction between N nutritional status (10 (Low) and 150 (High) mg N L -1 H 2 O) and waterlogging conditions (with or without) on shoot length.For each treatment, the values are the mean of 10 plants ± standard error.Arrows indicate when waterlogging stress periods started.

Fig. 2 .Fig. 3 .
Fig. 2. Influence of the interaction N nutritional status between (10 (Low) and 150 (High) mg N L -1 H 2 O) and waterlogging conditions (with or without) on leaf area (A) and leaf area ratio (B).Each bar chart represents the mean of 10 plants.Vertical bars represent ± standard error

FFig. 4 .
Fig. 4. Influence of the effect of the interaction between waterlogging conditions (with or without) (A) and Nitrogen Levels (10 (Low) and 150 (High) mg N L -1 H 2 O) (B) on F v /F m ratio.Each bar chart represents the mean of 20 leaves replicates.Vertical bars represent ± standard error

z
Means with different letters represent statistically significant differences according to Tukey's test (p ≤ 0.05).

Table 1 .
Summary of analysis of variance of the effect of N fertigation status and waterlogging treatments on the physiological behaviour of tamarillo seedlings z *, **, and *** significantly different at the 0.05, 0.01 and 0.001 probability levels, respectively.NS, not significant at α = 0.05. *