An Appraisal of Calcium Cyanamide as Alternative N Source for Spring-Summer and Fall Season Curly Endive Crops: Effects on Crop Performance, NUE and Functional Quality Components

A two-year study was conducted in both spring-summer and fall seasons to evaluate calcium cyanamide (CaCN2) as an alternative nitrogen (N) source for curly endive (Cichorium endivia L. var. crispum) grown in a Mediterranean environment. Four types of N applications were administered: (i) pre-transplanting base application of 100 kg N ha−1 corresponding to 100% of the supplied N (100CC), (ii) pre-transplanting base application of 50 kg N ha−1 corresponding to 50% of the supplied N (50CC) complemented with 50 kg N ha−1 as ammonium nitrate (50AN) supplied through fertigation, (iii) standard application of 100 kg N ha−1 as ammonium nitrate (100AN) supplied entirely through fertigation, and (iv) a N-deprived control (0 kg N ha−1) used as base reference to calculate the N use efficiency indices (NUE). Fall season increased head fresh weight, head height, stem diameter and plant visual quality, compared with the spring-summer season. The CaCN2 and standard fertigation N applications were equally effective in increasing head fresh weight and other physical parameters such as, head height, stem diameter, visual quality, number of leaves and head dry matter when compared to the unfertilized control. However, in spring-summer season, CaCN2, especially when applied straight at 100 kg N ha−1, effectively increased ascorbic acid and total phenolic content, whereas, in fall season, an increase in TSS and ascorbic acid was recorded. In both, spring-summer and fall seasons, CaCN2 significantly decreased N content and nitrogen accumulation (Nacc). Furthermore, CaCN2 pre-transplant application improved NUE indices both in terms of N fertilizer recovery efficiency and in terms of physiological efficiency of applied N. Our results finally demonstrated that NUE indices increased in the fall season as compared to the spring-summer season.


Introduction
Curly endive (Cichorium endivia L. var. crispum) is a leafy green vegetable belonging to the Asteraceae family, native to the eastern Mediterranean region and probably originated from the cross between C. intybus and C. pumilum [1,2]. Curly endive is an extensively grown vegetable in Europe and is praised for its typical crunchy texture and slightly bitter taste, rendering it suitable for direct consumption or as an ingredient of mixed ready-to-eat salads. Besides, curly endive is attractive due to its significant amount of nutritional and nutraceutical compounds such as minerals, ascorbic acid, phenolics, glucosinolates and sesquiterpene lactones [3][4][5]. Nitrogen above-optimal fertilization of vegetable crops represents a potential risk for human health. In particular, due to its accumulation in the edible part of leafy vegetables, NO 3 − is considered an anti-nutrient [6,7] and therefore a cut back of its dietary consumption is suggested as a precautionary health care action. We assume that curly endive, although less cultivated than other leafy vegetables, is equally prone to NO 3 − accumulation in the leaves when subjected to above-optimal nitrogen fertilization. Various authors reported that calcium cyanamide (CaCN 2 ) might be used as a soil fumigant for managing soil-borne diseases [8], pests and viruses in the soil [9], or as a tool to impede soil acidification, and expand yield and the quality of fruits [10,11]. However, CaCN 2 is also a well-recognized nitrogen fertilizer, particularly in the vegetable crop sector, and it may represent a valuable alternative to traditional soluble fertilizers. It may also improve the crop NUE [12] through a better modulation of N supply [13]. Several researches have demonstrated that nitrogen use efficiency (NUE) and NO 3 − accumulation are affected by numerous interrelated factors such as fertilization rate and source, irrigation type, and growing season [14][15][16][17][18][19][20]. CaCN 2 application represents an easy, efficient and practical technique for leafy green vegetables fertilization. However, to our knowledge, there are no reports concerning the relationship between growing season and CaCN 2 fertilization doses for curly endive. Thus, the scope of our research was to assess the effects of various CaCN 2 pre-transplanting applications on yield, NUE, nutritional and functional traits of curly endive grown in spring-summer and fall seasons.

Plant Material, Growing Conditions and Treatments
The experiments were conducted in open field in two successive cropping seasons, spring-summer and autumn 2018 and repeated in 2019 at the Experimental Farm of the Department of Agricultural, Food and Forest Sciences of Palermo (SAAF), Sicily (Italy). Meteorological data were collected by the meteorological station of SAAF. Maximal and minimal daily temperature and rainfall in the course of the plant growth cycles were registered (Figures 1 and 2).
On 3 May and on 24 September 2018, curly endive (Cichorium endivia L., var. crispum Hegi) (var. Trusty, HM Clause, France) plug plants at the four-five true leaves stage were transplanted at a density of 10 plants m −2 . The soil of the experimental field was a Typic Rhodoxeralf soil made of sand, silt and clay with a percentage of 46.5, 22.3 and 31.2, respectively, and a pH of 7.2, highly rich in exchangeable K 2 O, phosphorous, total nitrogen and organic matter with the following concentrations of 660 ppm, 68 ppm, 2% and 3.1%, respectively. Two doses of CaCN 2 at 100 kg N ha −1 (100 CC ) or at 50 kg N ha −1 (50 CC ) were applied by broadcasting on the whole plot surface and subsequently incorporating into the soil (10 cm depth) 505.0 kg ha −1 or 252.5 kg ha −1 , respectively of granular CaCN 2 (19.8% of N) (Perlka; AlzChem, Trostberg, Germany). CaCN 2 was applied 15 days before transplant. The pre-transplant implementation treatment of 100 kg N ha −1 (100 CC ) (100% of the supplied nitrogen) was compared with the treatment including a pre-transplant implementation of 50 kg N ha −1 (50 CC ) (50% of the supplied nitrogen) by adding to the latter an implementation of 50 kg N ha −1 (50 AN ) supplied in the form of NH 4 NO 3 (26% of N) by fertigation (192.3 kg ha −1 NH 4 NO 3 ). These two treatments were compared with a treatment including a conventional implementation of 100 kg N ha −1 (100 AN ) supplied in the form of NH 4 NO 3 (26% of N) by fertigation (384.6 kg ha −1 NH 4 NO 3 ). Drip irrigation was applied to all plots as per standard grower practice and in order to avoid plant water stress. Two growing seasons (spring-summer vs. autumn) were combined with the four fertilization rates (Table 1) in a two factorial experimental design accounting for a total of eight treatments. Each treatment was made of three replicates with 10 plants per replicate and 240 plants overall. The experiment was repeated in 2019 in the same growing period.
Agronomy 2020, 10, x FOR PEER REVIEW 3 of 12 treatment was made of three replicates with 10 plants per replicate and 240 plants overall. The experiment was repeated in 2019 in the same growing period.

Growth, Yield and Plant Visual Quality
Seventy-four days after transplanting curly endive plants were harvested. Afterwards, the following measurements were carried out for all the plants: head fresh weight and height, stem diameter and leaf number. At harvest, the visual quality of plants was also assessed based on a continuous score ranging from one to nine. Where one indicated a seriously damaged plant, three indicated a condition where the plant produced a usable but not saleable head, five indicated a fair condition where the head is at a marketability limit, seven indicated a good condition and nine to an excellent appearance. Five randomly selected plants from each replicate were sampled and placed in a thermo-ventilated oven (Memmert, Serie standard, Venice, Italy) to determine dry matter content. The first two days the oven was set at 80 • C and then at 105 • C until the samples reached constant dry weight.

Nutritional and Functional Traits
The qualitative analysis samples were collected as stated by Sabatino et al. [5]. Thus, all samples were collected immediately after harvest. A sample of 200 g from each replicates was placed in a commercial juicer to extract its juice that was filtered later on. Then the content of soluble solids (SSC) was assessed by a digital refractometer (MTD-045nD, Three-In-161 One Enterprises Co. Ltd., New Taipei, Taiwan). Afterwards, through potentiometric titration with NaOH (0.1 M) up to a pH of 8.1, titratable acidity (TA) was evaluated. For this purpose, 15 mL of the extracted juice was used and the results were expressed in malic acid percentage equivalent [21]. A RQflex* 10 m reflectometer (Merck, Darmstadt, Germany) and Reflectoquant Ascorbic Acid Test Strips (Merck, Darmstadt, Germany) were used to measure ascorbic acid content. Distilled water was used to dissolve the leaf juice sample (1 g), where water was added and mixed until reaching a final volume of 10 mL. Then the test strips were immersed in the prepared samples and placed into the reflectometer. The obtained results were expressed as mg of ascorbic acid per kg fresh weight. Five grams of leaf sample extracted in methanol, were used to measure total phenolic content that was assayed quantitatively by A765 and expressed as mg of caffeic acid g −1 fresh weight, according to the Folin-Ciocalteu method [22] with slight modifications [23]. Nitrogen (N) content was determined from the Kjeldahl method. Particularly, a sample rate was subjected to acid-catalyzed mineralization to convert the organic nitrogen into ammoniacal nitrogen. The ammoniacal nitrogen was then distilled in an alkaline pH. The ammonia created during this distillation was collected in a boric acid solution and determined via titrimetric dosage.

Nitrogen Accumulation and Nitrogen Efficiency Indices
Total N accumulation (N acc ) was determined by multiplying head dry matter, expressed as kg ha-1, by its percentage content of N (N acc = head dry matter × Ncontent). Nitrogen efficiency indices were calculated immediately after harvest, as reported by Greenwood et al. [24]. The partial factor productivity of applied N (PFP N ) represents the harvested product (kg) per the amount of applied N (kg), where the calculation was done as follows: where NF is the used N fertilizer (kg ha −1 ), and YF is the obtained crop yield (kg ha −1 ) with the implementation of a noted NF rate (kg ha −1 ). The agronomic efficiency of applied N (AE N ) represents the increased yield (kg) per the amount of applied N (kg), where the calculation was done as follows: The apparent N fertilizer recovery efficiency (REC N ) by the crop represents the increased N acc (kg) per the amount of applied N (kg), where the calculation was done as follows: The physiological efficiency of applied N (PE N ) represents the increased yield (kg) per the increased N acc (kg) from the applied fertilizer, where the calculation was done as follows: where YF is the crop yield (kg ha −1 ) acquired with the implementation of a noted NF rate (kg ha −1 ), Y0 is the crop yield acquired without the implementation of N fertilizer, AF is plant total N acc (kg ha −1 ) at maturity in the aerial biomass when N fertilization occur, and A0 is the corresponding plant total N acc (kg ha −1 ) at maturity in the aerial biomass when no N fertilization occur.

Experimental Design and Statistical Analysis
The SPSS software package version 14.0 (StatSoft, Inc., Chicago, IL, USA) was used to analyze the data, which were subjected to one-way analysis of variance. Tukey Honestly Significant Difference (HSD) test (p < 0.05) was used to separate the means when the nitrogen source was significant, and to separate the means of the treatment in case a significance was demonstrated for a particular measured characteristic. In order to evaluate the effect of the year, a preliminary two-way analysis of variance (nitrogen source × year) was performed.

Results
The experiment was repeated a second year using the same experimental scheme and obtaining similar results (Table S1). Therefore, data from 2018 are presented.

Crop Performance
In spring-summer season, nitrogen-unfertilized plants had the lowest head fresh weight ( Table 2). The latter was not significantly affected by the type of supplied nitrogen, as it ranged from 363.87 g in plants grown with 100 AN to 375.40 g in plants supplied with 50 AN -50 CC . Data collected on head height, stem diameter and visual quality, number of leaves and head dry matter supported the trend established for the head fresh weight (Table 2). Although, plants cultivated in the fall season gave higher values in terms of head fresh weight, head height, stem diameter and visual quality, number of leaves and head dry matter, tendencies recorded in fall season were perfectly in accord with those recorded in the spring-summer season. Significance *** NS * ** ** *** *** * ** *** *** *** Data within a column followed by the same letter are not significantly different at p ≤ 0.05 according to Tukey Honestly Significant Difference (HSD) Test. The significance is designated by asterisks as follows: *** statistically significant differences at p-value below 0.001; ** statistically significant differences at p-value below 0.01; * statistically significant differences at p-value below 0.05; NS, not significant. 0, no nitrogen; 100 AN , fertilized with ammonium nitrate at 100 kg N ha −1 ; 50 AN -50 CC , fertilized with ammonium nitrate at 50 kg N ha −1 and with calcium cyanamide at 50 kg N ha −1 ;100 CC fertilized with calcium cyanamide at 100 kg N ha −1 .

Nutritional Traits, Functional Properties and N Accumulation
In spring-summer season, untreated plants and 100 AN treated plants showed the highest values in terms of TA (Table 3). Treating endive plants with 100 CC and with 50 AN -50 CC decreased TA by 32.0% and 16.0%, respectively, compared to plants supplied with 100 AN . In terms of TA, plants grown in the fall season showed higher values than plants cultivate in the spring-summer season, however, data recorded in the fall season supported the trend established in the spring-summer season (Table 3). Nitrogen source markedly affected ascorbic acid content. Treating endive plants with 100 CC or with 50 AN -50 CC increased ascorbic acid by 17.6% and 8.2%, respectively, compared to plants grown with 100 AN . The lowest ascorbic acid content was recorded in plants grown in absence of nitrogen. In respect to ascorbic acid, data recorded in the fall season supported the trend established in spring-summer season ( Table 3).
Plants cultivated in the spring-summer season and supplied with 100 CC , 50 AN -50 CC or 100 AN had a significantly higher TSS than those unfertilized. Whereas, in fall season, plants treated with 100 AN showed no significant statistical differences compared to the untreated ones (Table 3). Moreover, in both seasons, unfertilized plants gave the lowest TSS value. Plants from the spring-summer season supplied with 100 CC gave the highest total phenolic content (0.90 mg of caffeic acid g −1 fw) followed by those supplied with 50 AN -50 CC . Unfertilized plants and plant fertilized with 100 AN gave the lowest total phenolic values, whereas, in fall season, the lowest total phenolic levels were recorded in plants from control plots. Remarkably, in fall season, plants grown on plots treated with 100 CC , 50 AN -50 CC or 100 AN did not show significant differences in terms of total phenolic content.
As regards N content, plants grown in both seasons with 100 AN gave the highest N content followed by those supplied with 50 AN -50 CC . In fall season, significantly lower N values were detected in plants supplied with 100 CC . Whereas, in spring-summer season, ANOVA and mean separation did not highlight significant differences between plants treated with 50 AN -50 CC and those treated with 100 CC . Finally, data on Nacc supported the trend established for leaves N content (Table 4). Table 3. Effect of nitrogen source on titratable acidity (TA) and ascorbic acid, total soluble solid (TSS) and total phenolic of curly endive grown in spring-summer and fall seasons.  Significance *** *** *** *** Data within a column followed by the same letter are not significantly different at p ≤ 0.05 according to Tukey Honestly Significant Difference (HSD) Test. The significance is designated by asterisks as follows: *** statistically significant differences at p-value below 0.001. 0, no nitrogen; 100 AN , fertilized with ammonium nitrate at 100 kg N ha −1 ; 50 AN -50 CC , fertilized with ammonium nitrate at 50 kg N ha −1 and with calcium cyanamide at 50 kg N ha −1 ; 100 CC fertilized with calcium cyanamide at 100 kg N ha −1 .

Nitrogen Efficiency Indices
In both, spring-summer and fall seasons, nitrogen source did not significantly affect PFP N and AE N ( Table 5). Data within a column followed by the same letter are not significantly different at p ≤ 0.05 according to Tukey Honestly Significant Difference (HSD) Test. The significance is designated by asterisks as follows: *** statistically significant differences at p-value below 0.001; * statistically significant differences at p-value below 0.05; NS, not significant. 0, no nitrogen; 100 AN , fertilized with ammonium nitrate at 100 kg N ha −1 ; 50 AN -50 CC , fertilized with ammonium nitrate at 50 kg N ha −1 and with calcium cyanamide at 50 kg N ha −1 ; 100 CC fertilized with calcium cyanamide at 100 kg N ha −1 The effects of tested treatment on PE N are presented in Table 5. Plants from the spring-summer season supplied with 100 CC and with 50 AN -50 CC increased PE N by 75.4% and 65.9%, respectively compared to the plants supplied without 100 AN . Whereas, PE N in plants from the fall season and supplied with 100 CC or 50 AN -50 CC increased by 132.1% and 61.3%, respectively compared to the plants supplied with 100 AN . Plants both from fall and spring-summer season and fertilized with 100 AN had the lowest PE N values.
The effects of the nitrogen source on REC N are presented in Table 5. In spring-summer season, REC N was the highest in plants cultivated with 100 AN and the lowest in plants cultivated with 50 AN -50 CC or 100 CC . In fall season, the highest REC N values were observed in 100 AN treatment, whereas, the lowest values were recorded from plants from 100 CC treatment. Data collected in fall season supported the trend established in spring-summer season. Overall, growing endive plants during the spring-summer season substantially reduced REC N as compared to growing plant in the autumn season. Furthermore, increasing the amount of N provided by CaCN 2 from 0 to 100% resulted in a REC N decrease, although, in respect to the spring-summer season, ANOVA analysis did not show significant differences between plants treated with 50 AN -50 CC and those treated with 100 CC .

Discussion
In this work, we evaluated the potential use of CaCN 2 as source of nitrogen to enhance production and quality of curly endive grown in two different seasons. Our outcomes are consistent with those obtained by Adamczewska-Sowińska and Uklańska [25] who, by investigating the effects of different type and doses of nitrogen fertilization on endive, found that marketable yield increased with increasing doses of nitrogen as compared to unfertilized control. However, in our study the types of nitrogen source tested (CaCN 2 and standard fertigation) were equally effective in increasing head fresh weight and other parameters such as head height, stem diameter, visual quality, number of leaves and head dry matter when compared to unfertilized control. These results agree with those observed in lettuce, another member of the Asteraceae family, by Montemurro et al. [26] who, by comparing dicyandiamide, a nitrification inhibitor (NI), with urea did not find significant differences in terms of yield. Our results are also consistent with those of Di Gioia et al. [13] who found the same leaf area index, fresh yield and dry weight when comparing two lettuce types either fertilized with calcium cyanimide or with a standard split application of soluble nitrogen.
There are some reports demonstrating that applying CaCN 2 or other stabilized fertilizers that contain NIs is able to raise the yield in potato [27], soybean [28], maize [29], and wheat [30]. However, as hypothesized by Frye [31], Di Gioia et al. [13] and Sabatino et al. [11] the application of NIs may cause an increase in yield traits only at low N application rates or when N is lost by leaching and/or denitrification, causing a shortage in N level quite strong to reduce crop yield in absence of NI.
Our findings demonstrated that autumn season positively affected yield traits and plant visual quality. Currently, there is a mounting proof indicating the additive and synergetic effects of natural bioactive compounds on human health, especially from plant sources since they are able to diminish the risk of several dysfunctions linked to oxidative stress [32][33][34]. Our findings demonstrated that, compared with standard fertigation, a CaCN 2 pre-transplant treatment, in spring-summer season, can improve the ascorbic acid and polyphenol contents in curly endive. Whereas, the CaCN 2 pre-transplant treatment in fall season, positively effects ascorbic acid and TSS content. Our findings are coherent with those acquired by Mercelle [35], Stefanelli et al. [36] and Sabatino et al. [11], who indicated negative effects of high N administrations on nutritional and functional fruit traits. Consequently, we may hypothesize that curly endive plants in plots treated with the highest dose of CaCN 2 had higher TSS, ascorbic acid and total phenolic contents, caused by a slow nitrification process defined by CaCN 2 application and the remains of ammonium-N in the soil which can take part in reducing nutritional stresses procured by the risks of excesses or N deficiency in specific phases of the growing cycle. Moreover, our data revealed that, except for 100 AN treatment, spring-summer season positively influenced total phenolic content in curly endive plants. Thus, since stress conditions prompt phenolics accumulation [37][38][39][40][41] and considering that the optimum growth temperature for curly endive is about 15-18 • C [42], we may hypothesize that the higher total phenolic content observed in plants from the spring-summer cycle could be due to the high temperatures (thermal stress), which are usual in the Mediterranean climate regions during spring-summer time.
Our findings showed that, in the fall season, N acc decreased as the percentage of nitrogen supplied by CaCN 2 increased. This is in line with the results of Pleysier et al. [43] who, by inquiring the consequences of the application of CaCN 2 four weeks before maize and rice plantation, found that N acc decreased with increasing doses of nitrogen supplied via CaCN 2 . This response was attributed by these authors to N loss by volatilization between application and planting time. Besides, our results demonstrated that autumn growing season positively affected N acc compared to the spring-summer season. This could be related to the fact that autumn temperatures in the Mediterranean region, differently from the tropics, do not reach quite high levels to provoke considerable losses of ammonium-N by volatilization. Furthermore, our results are, also, in accord with those of Colonna et al. [44], who by investigating on the nutritional traits of ten leafy green vegetables harvested at two light intensities, found lower protein and NO 3 -N content when plants are grown at lower PAR level. Our results concerning NUE indices, either in terms of REC N or in terms of NUE expressed as PE N , demonstrated that both growing seasons and nitrogen supplied via CaCN 2 play a major role. As regards the NUE expressed as PFP N and AE N , the application of CaCN 2 did not result in any improvement. However, PFP N and AE N indices were considerably higher in autumn season than in the spring-summer season. Our findings are in line with those of Chen et al. [12] and with those of Pleysier et al. [43], who reported that CaCN 2 or other NI can potentially enhance NUE. Nevertheless, our results are partially coherent with those of Di Gioia et al. [13], who found that applying CaCN 2 improved NUE neither in terms of REC N nor in terms of NUE expressed as PFP N , AE N , and PE N . Since, the experimental fertilization schemes proposed in this study improved imperative features in curly endive, they seem to be feasible for curly endive commercial production in both spring-summer and fall seasons.

Conclusions
Our study suggests that a pre-transplant application of CaCN 2 can be an alternative and valuable source of N for curly endive production. Overall, CaCN 2 did not improve crop yield. Nevertheless, CaCN 2 , especially when used at 100 kg N ha −1 (100 CC ), effectively increased ascorbic acid and total phenolic in spring-summer season and ascorbic acid and TSS in fall season. Simultaneously, CaCN 2 pre-transplant treatment reduced N content and N acc . Furthermore, CaCN 2 improved NUE indices both in terms of REC N and PE N . Our results also showed that autumn season increased yield traits, plant visual quality, ascorbic acid content and NUE indices as compared to the spring-summer season.