E ﬀ ect of Cropping System and Humidity Level on Nitrate Content and Tipburn Incidence in Endive

: Tipburn is an important disorder caused by a calcium deﬁciency that a ﬀ ects the marketability of endives. Genotype, air relative humidity, and temperature are directly involved in tipburn occurrence. Our study aimed to investigate the e ﬀ ect of cropping systems and relative humidity on the marketable yield, nitrate accumulation, and incidence of tipburn in endives. Two cultivars were evaluated in pots (‘Cuartana’ and ‘Natacha’), two cropping systems (greenhouse and open-ﬁeld), and in two di ﬀ erent air humidity levels (high level: plants under a plastic tunnel with an extra supply of humidity with micro-sprinklers, and low level: plants without an extra supply of humidity and outside of a plastic tunnel) during two years (2013 and 2014) in di ﬀ erent growing seasons. Nitrate content was determined by reﬂectometry, and tipburn was evaluated using a qualitative scale. Results showed that tipburn was favored under greenhouse with low humidity levels, with 40–60% plants a ﬀ ected. ‘Natacha’ was more susceptible to tipburn ( > 20% plants a ﬀ ected) than ‘Cuartana’ ( < 20% plants a ﬀ ected). Leaf nitrate accumulation was favored by the highest temperatures (greenhouse). It is concluded that in our conditions, tipburn incidence in endives depends on the interaction of genotype and the environmental conditions. Nitrate content was more inﬂuenced by the temperature than by the cultivars used.


Introduction
In Spain, the annual consumption of fresh vegetables for prepared salads is about 6.67 kg person [1]. The per capita consumption in the United Kingdom, France and Italy is 12, 6, and 4 kg, respectively; however, consumption in Belgium or Croatia is 4−10 times higher. Sales of fresh vegetables for prepared salads in Spain increased 57.8% in 2018 compared to the previous year, indicating its importance as a specialty crop [2].
Vegetables are the most important source of nitrate (NO 3 − ) intake for humans, but the consumption of these compounds in high concentrations can be toxic. Leafy vegetables are especially prone to nitrate accumulation [3]. Although nitrate is relatively non-toxic, it is converted to nitrites in the digestive system, which are associated with methaemoglobinemia, and the development of stomach cancer [4,5].
The maximum allowable concentration of nitrate specified in the Official Journal of the European Union [6] is 5000 (in greenhouse) and 4000 mg NO 3 kg −1 (in open-field), when the vegetables

Materials and Methods
Field trials were conducted during two growing seasons (Table 1)  The venlo-type greenhouse had a glass cover with automatic zenith windows and a cooling-system inside that evaporate water to plants when temperatures were higher than 28 • C or lower than 14 • C.
The experiment was arranged in a factorial split-split-plot. The main-plot was the cropping system and the subplots were the humidity treatment and cultivar. Cropping system treatments included greenhouse and open-field. The greenhouse cropping system was a glass covered venlo-type greenhouse with automatic zenith window and an evaporative cooling-system (setpoints 28 • C and 14 • C). The humidity level included low level (plants grown in greenhouse and open-field without an extra supply of humidity) and high level (plants grow in the greenhouse and open-field under a plastic tunnel with supplemental humidity supplied by micro-sprinklers located at the top of the tunnel). The supplemental humidity supplied by the micro-sprinklers did not result in additional irrigation water for plants, as this water was used for evaporative cooling. Cultivars included 'Cuartana' and 'Natacha'. All main-plots and subplots treatments were assigned by complete block randomization. Each treatment combination (2 cropping system × 2 humidity levels × 2 cultivars) was replicated three times with 10 plants in each replicate for a total of 240 plants. . The volume of each irrigation and the number of irrigations were scheduled to maintain drainage between 20% and 30%. The evaluation of tipburn incidence and the determination of nitrate content were performed immediately after harvest. Tipburn was evaluated using a qualitative scale from 0 to 3 (0 = no damage, 3 = high level of damage). Only plants in the 0-1 value range were considered marketable. After harvesting, four fully expanded leaves were collected from two plants in each replication. A solution of midrib sap and distilled water (1:25) was used to determine nitrate content by reflectometry using a commercial kit with nitrate-sensitive test strips containing two reactive zone pads that turn red-violet when exposed to nitrate solution. The color intensity is proportional to the concentration of nitrate in the solution. Nitrate concentration was determined quantitatively by comparing the color of test strip reactive zones using a hand held reflectometer (RQflex ® KGaA; Merck, Darmstadt, Germany).
To determine the chlorophyll content by spectrophotometry, a 0.5-g sample of the leaf blade (was taken. Samples were ground in a mortar with acetone (90%) and centrifuged for 10 min at 4000 rpm. The supernatant was poured into volumetric flask and diluted with acetone (90%) to 50 mL. The absorbance of this solution was measured at the wavelengths A470, A645, and A663 nm. The chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids were determined according to the following equations: (open-field) and a datalogger connected to climatic sensors (Climatec Agro, Climatec, Almería, Spain) (greenhouse) with a logging interval of ten minutes.
The variation of temperature and humidity obtained in the conditions of low humidity and high humidity in two years were assessed by the mean, standard error, and confidence intervals using a statistical program (Statgraphics Centurion XV; Statgraphics Tecnologies Inc, Lawrence, NJ, USA) ( Table 2). Table 2. Ranges of temperature and humidity relative to the different conditions of humidity tested (low humidity and high humidity) and in the two cropping systems (greenhouse and open-field).

Temperature ( • C)
Humidity Relative (%) For each year and season, the significance of the effects was determined by means of ANOVA for the biomass of marketable plants, tipburn incidence, nitrate accumulation, and chlorophyll content, considering the cultivar (Cv), humidity level (HL), and crop system (CS) and their interactions using a statistical software (Statgraphics Centurion XV; Statgraphics Tecnologies, Inc., Lawrence, NJ, USA). In order to determine tipburn incidence, an angular transformation was performed: arcosin √ x , x = percentage of plants with tipburn. Mean separation was performed using least significant differences (LSD) test (p ≤ 0.05).

Results
The biomass of marketable plants was influenced by the crop system in winter of 2013 and both seasons of 2014 (p ≤ 0.01). In general, plants grown in the open field obtain higher values of marketable biomass (p ≤ 0.05); but, when the interaction HL x CS is taking into consideration (p ≤ 0.01), these differences are only obtained under low humidity conditions. The marketable biomass is 68.2% higher in the open-field system compared to the greenhouse system in the winter of 2013 and both seasons of 2014.
No statistical significance was observed for cultivar nor in the interactions (double or triple) with the cultivar. The only difference in biomass between cultivars are observed in spring of 2014, with 'Natacha' showing the highest values (p ≤ 0.01) ( Table 3). We found significant interaction of Cv × CS in open-field (p ≤ 0.05). The effect of humidity on yield is variable between years and seasons.
Results show a statistical influence of the cultivar in the tipburn incidence (p ≤ 0.01) ( Table 4). Generally, 'Natacha' presents significantly higher values of tipburn (p ≤ 0.01) ( Table 4). An interaction is detected with Cv x HL (2013 p ≤ 0.01; 2014 p ≤ 0.05) to influence tipburn where differences between cultivars are found in low humidity conditions, while no differences are detected in high humidity treatment ( Figure 1).    The performance of both cultivars was similar across the experiment, with 'Natacha' presenting higher tipburn incidence than 'Cuartana' under greenhouse conditions and no differences between cultivars in open-field (Cv x Cs interaction p ≤ 0.01) (Figure 2). The influence of the crop system is statistically significant in tipburn incidence (p ≤ 0.01; p ≤ 0.05) ( Table 4). Tipburn incidence is higher in the greenhouse (p ≤ 0.05), but this significant difference is only found in cultivar 'Natacha' (interaction Cv × CS) (Figure 2). Humidity level influenced the tipburn incidence (p ≤ 0.05) ( Table 4) with higher tipburn incidence in plants grown under low humidity (p ≤ 0.05), this is further confirmed with the interaction HL × CS (p ≤ 0.01) ( Figure 3).
Generally, nitrate content is not affected by cultivar. 'Cuartana' grown in greenhouses during winter of 2014, accumulated more nitrates than 'Natacha' (p ≤ 0.05) ( Table 5) (Figure 4).   In addition, it was observed that the crop system had a significant effect on nitrate content (2013 p ≤ 0.01; 2014 p ≤ 0.05), except in one season (Spring 2013), which presented higher nitrate content in greenhouse plants (p ≤ 0.05) ( Table 5). Humidity did not significantly affect leaf nitrate concentration for most of the trials, except that low humidity favored nitrate accumulation in plants in the spring of 2014 (p ≤ 0.05) ( Table 5). No interactions between humidity level and cultivar or crop system are found for nitrate content.
Humidity level was significant (p ≤ 0.05) in spring 2013, resulting in higher chlorophyll content in plants under low humidity (p ≤ 0.05) ( Table 6). No interactions are found for cultivar, humidity level, and cropping system ( Table 6).

Discussion
Plant growth is promoted by open-field conditions, with higher marketable biomass compared to the greenhouse system. Warmer greenhouse temperatures do not favor the accumulation of biomass. Wurr and Fellows [25], and Wheeler et al. [26], also reported a negative relationship between temperature and yield in lettuce.
The response of the cultivars to tipburn incidence varies over time depending on environmental conditions and the cultivar; 'Natacha' shows more susceptibility to tipburn disorder than 'Cuartana'. The different susceptibility of cultivars in conditions that favored tipburn incidence was observed in other studies with lettuce. These studies found that the incidence of tipburn disorder is genetically determined but highly influenced by environmental conditions [27,28].
The low humidity treatment (55.37% ± 1.49%) favors tipburn occurrence. On the other hand, the increase of the humidity level artificially (high humidity, 68.49% ± 1.23%), decreases the severity of tipburn and the performance of the two cultivars is similar.
This adverse effect of low humidity on the incidence tipburn has been previously reported since low humidity causes an increase in transpiration [29,30]. These conditions may induce leaf water stress which leads to stomatal closure. Therefore, the water stops flowing from the xylem to leaves, and the consequently calcium transport is reduced, favoring tipburn incidence [31][32][33]. In addition, during the day, when the transpiration increases by the low levels of humidity, the outer leaves transpire more rapidly than inner leaves and can limit the concentration of transported calcium [34]. At night, the low humidity could decrease the turgor of leaves and root pressure, causing a loss of calcium that is ascended to the tissues by guttation [35].
On the other hand, less incidence of tipburn with the increase of the humidity level artificially (high humidity, 68.49% ± 1.23%) occurs as high humidity can promote the transport of calcium (by the xylem) to the leaves during expansion. Similarly, according to Kuronuma et al., [36], total calcium concentrations in six of seven Lisianthus cultivars were significantly higher under the high humidity treatment (70% ± 5%) than under a low humidity treatment (50% ± 5%). Two of seven cultivars showed tipburn under high humidity environments [36].
In addition to humidity, nitrogen and calcium are known to influencing the incidence of tipburn. [37] reported an increase in tipburn when the nitrogen was supplied in excess, and [16,18] agree that calcium deficiency is the cause of tipburn. Different studies have shown that calcium is closely associated with nitrogen metabolism [38,39]. Calcium is essential for the reduction of nitrate to nitrite and plants grown without calcium accumulated nitrate in leaves [38,39]. The leaf calcium concentration was closely related to nitrogen levels in plants, and both concentrations were affected by nutrient solution composition [40]. Our results indicate higher nitrate concentration in plants with higher incidence of tipburn grown in greenhouses systems.
The values of nitrate obtained in endive plants never exceeded the standards set by the European Union for the lettuce [6]. Nitrate accumulation has been shown to differ among cultivars of spinach, rocket salad, and lettuce [13,41,42]. Differences are not observed in this experiment between 'Natacha' and 'Cuartana'.
In general, studies about the accumulation of nitrate in plant tissues have shown that in conditions of low temperature and light (autumn and winter), the activity of the enzyme nitrate reductase (the enzyme involved in the reduction of nitrate) is favored, and the content of this compound in plants increases [43,44]. To the contrary, our results indicate that nitrate accumulation was favored in the crop system with the higher temperatures (greenhouse). These results are consistent with the finding of studies that reported nitrogen concentration in endive was higher in high temperature seasons [45].
In the winter of 2013 and in the two seasons of 2014, the nitrate content was higher under greenhouse conditions; in addition, leaf chlorophyll content was low in all seasons. According to several authors nitrate accumulation is favored in seasons with higher temperatures [46,47]. In lettuce (crop with similar requirements to endive), a decline in photosynthesis was observed, causing a reduction in the concentration of accumulated carbohydrates in the vacuoles that were replaced by nitrates to keep the osmotic potential with temperatures over 22 • C [10,48,49]. Additionally, differences in the nitrogen absorption have been observed as a function of temperature. Therefore, the low temperatures in open-field system in comparison to the greenhouse could limit its absorption, especially in nitric form [50].

Conclusions
Our experiment demonstrated that crop systems with low humidity levels (plants cultivated at greenhouse without an extra supply of humidity) favored the occurrence of tipburn in endives. In that condition we observed 'Natacha' plants had greater susceptibility to this disorder in comparison to 'Cuartana'. These results suggest that tipburn incidence in endives depends on the interaction of genetic and climatic factors that affect the transpiration and root pressure. Related to the nitrate accumulation in leaves, few differences were found between cultivars that could indicate the possibility of reducing the nitrate content in endives with the selection of a cultivar. Leaf nitrate content was favored by high air temperature conditions, possibly because the temperatures reached in the experiment caused a decrease in the chlorophyll content.