Bristly Oxtongue (Helminthotheca echioides (L.) Holub) Responses to Sowing Date, Fertilization Scheme, and Chitosan Application

Abstract

Bristly oxtongue (Helminthotheca echioides (L.) Holub) is a broad-leaved weed species that is commonly found in cereal crops. However, it is also an edible species whose leaves are consumed at the rosette growth stage of the plant. Three pot experiments were conducted to evaluate different cultivation protocol suggestions for this underutilized wild leafy vegetable. In the first experiment, early sowing (14 October) increased the rosette diameter and fresh biomass of bristly oxtongue compared with late sowing (13 November). In the second experiment, the application of calcium ammonium nitrate (26-0-0) at a rate of 100 kg ha⁻¹ (N10 treatment) increased the relative chlorophyll content in the leaves compared with the control treatment. Similarly, the highest rosette diameter, leaf number, and fresh biomass values were recorded in the N10 treatment, while chitosan application had no impact on growth of plants. In the third experiment, increased root dry biomass was obtained with top dressing application of calcium ammonium nitrate at a rate of 100 kg N ha⁻¹ compared with 0 or 50 kg N ha⁻¹, regardless of the basal fertilization (15-5-20) rate applied. Moreover, the highest rosette diameter, relative chlorophyll content, and fresh biomass values were recorded in the treatment where the highest top dressing rate of calcium ammonium nitrate was applied. In conclusion, our results reveal that the appropriate selection of the sowing date, as well as the combination of the basal fertilizer (15-5-20) at a rate of 250 kg ha⁻¹ with 100 kg N ha⁻¹ calcium ammonium nitrate, can maximize the growth and yield of bristly oxtongue.

1. Introduction

Weeds are mainly studied for their negative effects on crop growth and productivity. However, some weed species are edible or have medicinal value; therefore, upscaling their cultivation to the commercial level could result in a highly profitable agri-business [1]. Important weed species that are widely cultivated are milk thistle (Silybum marianum (L.) Gaertn.) and common purslane (Portulaca oleracea L.) [2,3]. Milk thistle is mainly grown for its pharmaceutical compound silymarin, which is contained in its fruits [3,4,5,6], while common purslane is cultivated for its edible leaves and stems [2,7]. Several other weeds of the Asteraceae family, such as common sowthistle (Sonchus oleraceus L.), dandelion (Taraxacum officinale (L.) Weber ex F.H. Wigg.), prickly lettuce (Lactuca serriola L.), and bristly oxtongue (Helminthotheca echioides (L.) Holub; synonym Picris echioides L.) are edible (mainly the leaves) underutilized species and are usually collected from the wild (uncultivated) [8,9,10].

Among those edible species mentioned above, bristly oxtongue is of great interest for cultivation as a new crop. It is an annual or biennial broad-leaved species, has stiff hairs in the leaves [11,12,13,14] and form rosettes [12,15] during the winter and early spring. Usually, the young rosette, the edible part of the plant [15], is of high nutritional value due to the various bioactive compounds it contains. Petropoulos et al. [16] identified 10 phenolic compounds (e.g., luteolin-O-glucuronide, luteolin-7-O-rutinoside, apigenin-O-glucuronide, and apigenin-7-O-glucoside) in the leaves of the rosettes, of which the flavones luteolin-O-glucuronide and luteolin-7-O-rutinoside were the highest in quantity (3.65–23.1 mg 100 g⁻¹ fresh weight (fw)). The organic compound a-tocopherol was also identified in lower amounts (1.26–2.64 mg 100 g⁻¹ fw). In another study, Sergio et al. [17] identified the bioactive compounds chlorogenic acid, chicoric acid, 3,5-Dicaffeoylquinic acid, and luteolin-7-glucoside. Among these compounds, chicoric acid and luteolin-7-glucoside were detected in higher amounts (115–301 mg 100 g⁻¹ dry matter). Compared with other wild edible greens, such as common chicory (Cichorium intybus L.) and Mediterranean hartwort (Tordylium apulum L.), bristly oxtongue showed a greater antioxidant ability [18].

Bristly oxtongue can be cultivated as an annual crop, while it is propagated with seeds that exhibit a high germination rate. Thus, the establishment of this crop can be carried out either by direct sowing in the field or by the transplanting of the seedlings. Moreover, it is important to point out that there is no information in the literature about the cultivation practices that should be applied to this crop in order to achieve high yields. Thus, two basic cultivation practices that should be examined in this regard are the sowing date and fertilization method. In the Mediterranean region, as with other autumn weeds, bristly oxtongue germination mainly occurs in late autumn to early winter, while a second germination “wave” occurs in late winter as the temperatures rise. An important factor in determining the appropriate sowing time is the base temperature for seed germination, which is very low (5.2 °C) according to Guillemin et al. [19], while at 20 °C, a high percentage of seeds germinate (92%) [19]. Based on these data, the suitable sowing period seems to be from autumn to the beginning of winter. Therefore, it is important to study the effects of different sowing dates on bristly oxtongue growth and yield as well as the effect of low temperatures on plant growth in the initial period after germination. With late sowing in autumn, the risk of frost damage to plants must also be assessed. Sowing in the late winter should be avoided since, in other Asteraceae species that also form rosettes, such as spiny chicory (Cichorium spinosum L.), a decrease in productivity occurs under such conditions due to the early bolting and development of flowering stems [20]. Regarding fertilization, weeds have low to moderate nutrient requirements and usually grow sufficiently in soils with low fertility. However, when grown as productive plants, the fertilization of these species increases their yields, as has been observed in other studies. Nitrogen fertilization (calcium ammonium nitrate) at 125 kg ha⁻¹ increased the fruit yield of milk thistle by 27.1 to 30.1% compared with plants given a control treatment [3]. In another study, Chen et al. [21] reported that shoot biomass and height of green amaranth (Amaranthus viridis L.) in the high nitrogen fertilization rate (240 kg N ha⁻¹) was higher by 36.5% and 12.1%, respectively, compared to the low fertilization rate (120 kg N ha⁻¹). Thus, those results mentioned above reveal the importance of optimizing the fertilization of underutilized crops in order to maximize their productivity.

Recently, the study of biostimulant effects on growth and yield of crops has attracted the research interest of scientists. Biostimulants have several applications in agriculture and the main categories are amino acids, chitosan, humic and fulvic acids, seaweed extracts, nitrogen-fixing bacteria (e.g., Azotobacter spp., Rhizobium spp.), and phosphorus solubilizing micro-organisms (e.g., Bacillus subtilis) [22,23,24,25,26,27]. Chitosan is a polysaccharide that promotes the growth of plants [28], improves the crop productivity [29], alleviates the negative effects of abiotic stress on plant growth [30,31], and induces defense mechanisms against pathogens such as Sclerotinia sclerotiorum and the cucumber mosaic virus [32,33]. Based on the above, it is of great importance to study the effect of chitosan on the growth and productivity of underutilized leafy greens with high nutritional value.

Thus, the aim of this study was to evaluate the effects of various cultivation practices on the growth and yield of bristly oxtongue. For this purpose, three pot experiments were conducted during two consecutive growing seasons with the aim being to evaluate the impacts of: (a) the sowing date (early, mid, and late); (b) the nitrogen fertilization rate combined or not with the biostimulant chitosan; and (c) the combination of basal with top dressing fertilization at different rates on the growth (e.g., rosette diameter, leaf number, root biomass), relative chlorophyll content, and the yield (fresh biomass) of bristly oxtongue.

2. Materials and Methods

2.1. Description Site and Biological Material

Three pot experiments were carried out in two consecutive growing seasons (2020/21 and 2021/22) at the School of Agricultural Sciences in Volos. This region is characterized by mild winters and a low annual precipitation level (<500 mm). The seeds of bristly oxtongue (Helminthotheca echioides (L.) Holub) were collected in late August 2020 from plants grown naturally on a field (39.0354 N, 22.3380 E) in the Domokos region (Central Greece). This species is a cereal weed that is commonly found in this region. The mean monthly air temperatures and precipitation during the bristly oxtongue growth period (October to April) are presented in Figure 1. Moreover, the annual air temperature and precipitation (average values for 2021 and 2022) were 18.3 °C and 693 mm, respectively.

2.2. Experiment 1: Sowing Date

The first experiment was conducted using a randomized complete block design with three treatments (early sowing, mid sowing, and late sowing) and five replications per treatment. Early, mid, and late sowing was performed on 14 October, 30 October, and 13 November 2020, respectively. Forty-five plastic pots (3 L, 15 pots per treatment) were used in this experiment, and fifty seeds were sown in each pot. Thinning of plants was performed at the two to four leaf stage, resulting in five plants per pot. Sandy clay loam soil (texture: clay 26%, sand 38%, and silt 36%) with a pH of 7.4, was used as the substrate. Regarding fertilization, calcium ammonium nitrate (100 kg ha⁻¹) was applied to all pots on 1 February 2021, 108 days after the first sowing.

2.3. Experiment 2: Fertilization and Chitosan

The second experiment was carried out following a randomized complete block design with three treatments, as described in Table 1. Forty-five plastic pots (3 L, 15 pots per treatment) were used in this experiment. Fifty seeds were sown in each pot on 30 October 2020. For the substrate, we used the same soil as in the first experiment. Moreover, the thinning of plants at the two to four leaf stage resulted in six plants per pot.

2.4. Experiment 3: Fertilization

In 2021/22, a pot experiment was designed following a randomized complete block design with five treatments (Table 2). As the substrate, we used sandy clay loam soil (see experiment 1). Seventy-five plastic pots (2 L) were used, and the sowing of fifty seeds was performed on 4 November 2021. After thinning of plants at the two to four leaf stage, four plants remained in each pot.

2.5. Measurements and Sampling in All Experiments

The rosette diameter, fresh above-ground biomass, dry above-ground biomass, relative chlorophyll content, and the number of fully formed leaves were measured throughout the growing period (22 February, 11 March, 1 April, and 15 April 2021—experiments 1 and 2; 21 February, 8 March, 29 March, and 12 April 2022—experiment 3). The last sampling was performed when the bristly oxtongue plants were at the rosette complete growth stage and before the stem elongation. Usually, after this stage, bristly oxtongue plants cannot be consumed due to the hardening of the leaf texture and leaf bitterness. At each sampling date, the parameters mentioned above were measured on three plants per pot and a total of three pots per treatment. The dry biomass was determined after the drying of samples at 60 °C for 96 h, while the relative chlorophyll content was measured using a SPAD-502 chlorophyll meter (Konica Minolta Optics, Osaka, Japan). In the third experiment, the root dry biomass was also measured in the middle of April. Finally, the roots were separated from the soil substrate and then placed in an oven for drying at 60 °C for 72 h.

In all three experiments, irrigation was carried out at regular intervals depending on the weather conditions (precipitation and temperature) and the plant’s irrigation requirements.

2.6. Statistical Analysis

The data collected in the three experiments were analyzed using the statistical package SigmaPlot 12. In the first stage of the statistical analysis, a Two-Way ANOVA was applied, and then the Fisher’s least significant difference (LSD) test was used as the criterion for comparing the means for all measured parameters at a significance level of p = 0.05. Using the same software, Pearson correlation coefficients between the main parameters were also determined.

3. Results

3.1. Experiment 1

3.1.1. Relative Chlorophyll Content

The SPAD values of bristly oxtongue in the first experiment ranged from 28.13 to 44.60. For the first two measurements (130 and 147 DAS), no differences among the early, mid, and late sowing treatments were obtained (130 DAS: Fvalue = 0.222, p = 0.810; 147 DAS: Fvalue = 1.834, p = 0.272). However, at 167 and 181 DAS, early sowing resulted in increased SPAD values compared with late sowing. Comparing the four measurements, the greatest values of this parameter were recorded at 181 DAS followed by at 130 DAS (Figure 2).

3.1.2. Leaf Number and Rosette Diameter

The rosette diameter of bristly oxtongue plants was significantly affected by the sowing date, with the highest values recorded in the early sowing treatment. In the late sowing, the rosette diameter decreased by 33.2%, 24.1%, 25.7%, and 25.9% at 130 DAS, 147 DAS, 167 DAS, and 181 DAS, respectively, compared with the early sowing treatment. Moreover, no significant differences were found between mid and late sowing dates (Figure 3).

The leaf number of bristly oxtongue plants ranged from 6.7 to 13.8. At the first and final sampling dates (130 and 181 DAS), the highest values were recorded in the early sowing date, while there were insignificant differences between the mid and late sowing dates. For the other two DAS, no significant differences were observed among the three sowing dates (147 DAS: Fvalue = 0.795, p = 0.512; 167 DAS: Fvalue = 0.373, p = 0.710). When comparing the four measurements, the greatest values of this growth indicator were found to be recorded at 147 DAS.

3.1.3. Fresh and Dry Biomass

Both fresh and dry biomass were affected by the sowing date. More specifically, late sowing decreased the fresh biomass by 68.6%, 38%, 61.3%, and 44.1% at 130 DAS, 147 DAS, 167 DAS, and 181 DAS, respectively, compared with early sowing. Similarly, late sowing resulted in a decrease in fresh biomass by 64.6%, 34.5%, 49.9%, and 47.2% at 130 DAS, 147 DAS, 167 DAS, and 181 DAS, respectively, compared with the early sowing date (Figure 4). It is also important to point out that there were no statistically significant differences between the late and the mid sowing dates for either parameter. Finally, the biomass data revealed that, regardless of the sowing time, bristly oxtongue plants exhibit a slow growth rate until mid-March.

3.2. Experiment 2

3.2.1. Relative Chlorophyll Content

Nitrogen fertilization had a significant impact on the relative chlorophyll content in the leaves of bristly oxtongue (114 DAS: Fvalue = 7.296, p = 0.046; 131 DAS: Fvalue = 9.532, p = 0.03; 151 DAS: Fvalue = 27.214, p = 0.005; 165 DAS: Fvalue = 29.872, p = 0.004). At 151 and 165 DAS, the SPAD values in the N10 treatment were 29.7 to 35.7% higher compared with those in the control treatment, while there were no significant differences between the N10 and N10 + chitosan treatments (Figure 5).

3.2.2. Leaf Number and Rosette Diameter

Regarding the leaf number of bristly oxtongue plants, no significant differences were found among the treatments at 114 and 131 DAS. For the third measurement, the lowest number of leaves was recorded in the control treatment, while there were insignificant differences between the N10 and N10 + chitosan treatments. Similarly, no differences were noted between the N10 and N10 + chitosan treatments at the final measurement date. Moreover, a gradual decrease in the number of leaves was recorded over time in the control treatment after the first sampling. The rosette diameter of bristly oxtongue was found to be positively affected by nitrogen fertilization (114 DAS: Fvalue = 30.553, p = 0.004; 131 DAS: Fvalue = 35.443, p = 0.003; 151 DAS: Fvalue = 125.837, p = <0.001; 165 DAS: Fvalue = 20.673, p = 0.008), since its lowest values were recorded in the control treatment. However, no differences were recorded between the N10 and N10 + chitosan treatments, clearly indicating that the application of chitosan had no impact on bristly oxtongue growth (Figure 6).

3.2.3. Fresh and Dry Biomass

Nitrogen fertilization has a noticeable impact on both dry and fresh biomass of bristly oxtongue plants. At all sampling dates, the lowest dry and fresh biomass values were recorded in the control treatment compared with all other treatments. At 114 and 131 DAS, the fresh biomass content was 61–77.6% higher than in the control treatment, while no differences were found between the N10 and N10 + chitosan treatments for either fresh or dry biomass throughout the experiment (Figure 7). Finally, at 151 and 165 DAS, no significant differences were recorded between the N10 and N10 + chitosan treatments. As for experiment 1, the growth of bristly oxtongue plants was slow until mid-March.

3.3. Experiment 3

3.3.1. Relative Chlorophyll Content

The application of 100 kg N ha⁻¹ of calcium ammonium nitrate as the top dressing combined with either 250 kg ha⁻¹ or 500 kg ha⁻¹ of the inorganic fertilizer 15-5-20 as the basal dressing (Fert B and Fert D) resulted in an increased relative chlorophyll content in the leaves of bristly oxtongue throughout the experiment (Figure 8). This was followed by the application of 50 kg N ha⁻¹ of calcium ammonium nitrate as the top dressing combined with either 250 kg ha⁻¹ or 500 kg ha⁻¹ of the inorganic fertilizer 15-5-20 as the basal dressing (Fert A and Fert C) and the control. Significant differences among the control and the Fert A and Fert C schemes appeared only at 155 DAS and 169 DAS.

3.3.2. Root Biomass

Our results showed that the fertilization scheme had a noticeable impact on the root growth of bristly oxtongue (Fvalue = 17.896, p < 0.001). Specifically, the application of 100 kg N ha⁻¹ of calcium ammonium nitrate as top dressing combined with either 250 kg ha⁻¹ or 500 kg ha⁻¹ of the inorganic fertilizer 15-5-20 as the basal dressing (Fert B and Fert D) resulted in an increased root dry biomass followed by the application of 50 kg N ha⁻¹ of calcium ammonium nitrate as the top dressing combined with either 250 kg ha⁻¹ or 500 kg ha⁻¹ of the inorganic fertilizer 15-5-20 as the basal dressing (Fert A and Fert C) and the control. No significant differences were found between treatments Fert A and Fert C or between Fert B and Fert D (Figure 9).

3.3.3. Leaf Number and Rosette Diameter

Concerning the leaf number of bristly oxtongue plants, our results reveal that at 109 DAS, 124 DAS, and 155 DAS, there were insignificant differences among the control, Fert A, and Fert C treatments. The greatest number of leaves was recorded in the Fert B and Fert D treatments where a double rate of calcium ammonium nitrate was applied. In the last measurement period, the leaf number of the plants supplied with Fert B and Fert D were 50.6% and 47.7% higher than the control treatment. Similar results were recorded for the rosette diameter of bristly oxtongue plants (Figure 10). For example, at 169 DAS, the rosette diameters of plants supplied with Fert B and Fert D were 54.7% and 56.5% higher than the control treatment, thereby revealing the positive effects of both basal and top dressing fertilizer application on the growth of bristly oxtongue plants.

3.3.4. Above-Ground Biomass and Root:Shoot Ratio

The fresh and dry biomass data revealed useful information about the effects of basal and top dressing fertilization on the growth of bristly oxtongue plants. For both parameters, at 109 DAS and 124 DAS, there were no significant differences between the control and the treatments Fert A and Fert C in which calcium ammonium nitrate was applied at a rate of 50 kg N ha⁻¹. However, the application of a double rate of calcium ammonium nitrate significantly increased the biomass of the tested plants compared with the other three treatments. In the last measurement period, the lowest fresh and dry biomass values were recorded for the control treatment, while the application of the maximum rate of calcium ammonium nitrate led to a 47.3–78.1% increase in the biomass of bristly oxtongue (Figure 11). Regarding the root:shoot ratio, significant differences were found among the five treaments (Fvalue = 6.092, p = 0.015), while the lowest values (1.01) were recorded in the control treatment. Τhe data mentioned above show that bristly oxtongue plants develop a dense root system (Figure 12 and Figure 13).

4. Discussion

The diversification of cropping systems by introducing underutilized and/or new crops is an agricultural adaptation strategy that aims to mitigate the negative impact of climate change on crop growth and productivity [34,35]. For underutilized crops such as bristly oxtongue, the ideal sowing period should be evaluated as a priority. To the best of our knowledge, there have been no studies on the effects of sowing time on the growth and yield of bristly oxtongue grown outdoors during winter. Our study reveals that, regardless of the sowing time, no frost damage was observed in the bristly oxtongue plants during the winter. During the growing season (October to April) of the first experiment, January and February were the coldest months with mean temperatures of 11.0 and 11.1 °C, respectively, as well as the months with the lowest temperatures recorded (−2.2 °C and −1.9 °C, respectively) throughout the experiment. Moreover, germination was not affected by the sowing date. This can be ascribed to the fact that the base temperature for the seed germination of bristly oxtongue is very low (5.2 °C) [19]. At the experimental site, the mean monthly, high, and low temperatures in November were 13.9 °C, 21.9 °C, and 6.1 °C, respectively.

Plant growth was enhanced by the early sowing time since the highest leaf number, rosette diameter, and biomass (fresh and dry) values were recorded in this treatment. These results indicate that greater initial plant growth before the first frosts in winter favors rapid plant growth during March when the temperature rises. The effect of planting time has been studied in other native species belonging to the Asteraceae family, such as the spiny chicory. In a pot experiment conducted in an unheated greenhouse, Petropoulos et al. [20] reported that the yield (fresh biomass of leaves) of the spiny chicory was 29.7–66.1% higher in the early planting stage (transplanting in early December) compared with that in the late planting stage (transplanting in the middle of February). On both planting dates, the harvest was conducted once in the growing period. The decrease in the fresh yield of spiny chicory in the late planting stage was due to early bolting (development of flowering stem).

Chitosan is a biostimulant that promotes the growth of plants and increases the yield and quality of vegetables [22,23,26]. The results of the current study show that the foliar application of chitosan hydrochloride had no impact on the growth of bristly oxtongue plants since there were no significant differences in the rosette diameter, leaf number, or biomass (dry and fresh) of plants between the N10 and N10 + chitosan treatments. To the best of our knowledge, no studies related to the effects of chitosan on the growth of bristly oxtongue have been conducted. However, the effect of chitosan has been studied in other leafy vegetables. Zhang et. al. [30] reported that the application of chitosan 7 days after transplanting alleviated the negative effects of NaCl stress on lettuce (Lactuca sativa L.) since its application led to increases of 58.5% and 48.7% in the total leaf area and dry shoot biomass of plants grown under salinity stress (100 mM NaCl) compared with values in plants grown under salinity stress but without chitosan application. Moreover, the application of this biostimulant in the soil increased the shoot biomass of Indian mustard (Brassica juncea (L.) Czern.) plants grown under various soil moisture levels by 35.8 to 61.6% compared with the control [36]. Similarly, in a pot experiment, the application of chitosan oligosaccharide lactate promoted the growth of red perilla (Perilla frutescens var. frutescens f. purpurea) plants [26].

The positive effects of chitosan on crop growth are probably due to the increase in the rate of photosynthesis and the chlorophyll content. Xu and Mou [37] reported that when this biostimulant was applied in the soil at a dose of 0.3%, the photosynthetic rate of lettuce plants increased by 44% compared with the control. In another study, chitosan applied in the soil had a positive effect on the chlorophyll content in the leaves of Indian mustard plants, as the SPAD values increased by 12.7 to 40.6% compared with those in the control treatment plants, while the increase in SPAD values was higher when this biostimulant was applied in combination with an organic fertilizer [36]. In contrast, our results revealed that there were no significant differences between the N10 and N10 + chitosan treatments for the chlorophyll content in the leaves of bristly oxtongue plants. Zhang et al. [30] also observed that the foliar application of chitosan did not increase the total chlorophyll content in the leaves of lettuce plants, while it led to an increase in the chlorophyll a content of 6% compared with that the control treatment. In conclusion, the effects of chitosan on the growth and yield of bristly oxtongue need to be further evaluated, and the effect of chitosan applied as a soil drench on the yield of this crop should also be examined.

Crop fertilization with the required amount of nutrients is important to increase productivity. Fertilization is a basic crop management practice that affects the growth and yield of several leafy vegetables, such as lettuce and common chicory [38,39]. However, the application of fertilizers, mainly nitrogen, at high rates can lead to environmental pollution due to the presence of nitrates in groundwater [40]. Therefore, the application of nitrogen fertilizers at the optimum rates is important not only for plants to achieve high yields but also to reduce the nitrate pollution of groundwater [41,42]. For underutilized leafy greens, the determination of nutritional needs for optimum plant growth is also important since these crops are usually grown in harsh saline environments and soils with poor vegetation and a low nitrogen content [43,44]. To date, no studies have been conducted on the impact of fertilization on bristly oxtongue crops. Our results indicate that nitrogen fertilization at the rosette stage enhances the growth of bristly oxtongue, since the highest leaf number, rosette diameter, and biomass values (fresh and dry) of plants were recorded in the N10 treatment, regardless of the application of chitosan. In particular, calcium ammonium nitrate applied at a rate of 100 kg N ha⁻¹ increased the yield (fresh biomass of plants) by 85.9% compared with the control treatment. In lettuce crops grown in either summer or winter, the application of urea at rates of 60 and 90 kg ha⁻¹ led to an increase in yield (fresh biomass) of up to 36.9% compared with that in the control treatment, while the application of urea at higher rates (120, 150, and 180 kg N ha⁻¹) did not cause a greater increase in the yield [39]. In contrast, the highest yield of chicory was recorded at the high N fertilization rate of 180 kg N ha⁻¹ [38]. Thus, the data mentioned above show that the bristly oxtongue crop has similar or lower N requirements to other leafy vegetables belonging to the Asteraceae family. The increases in both the rosette diameter and fresh biomass due to the nitrogen fertilization treatments can be ascribed to the increase in the relative chlorophyll content, since the application of calcium ammonium nitrate at 100 kg N ha⁻¹ increased the SPAD values in leaves of bristly oxtongue at harvest time by 35.7% compared with the control treatment. The statistical analysis also showed a positive and significant correlation between the relative chlorophyll content and fresh biomass (r = 0.976, p = 0.001) or the rosette diameter (r = 0.969, p = 0.001) since, an increase in chlorophyll content improves the photosynthesis process and, consequently, the plant growth.

The results of the third experiment revealed that the application of inorganic fertilizer 15-5-20 at a rate of 250 kg ha⁻¹ before sowing in combination with calcium ammonium nitrate applied in the rosette stage increased the rosette number and biomass yield of bristly oxtongue, while the application of this fertilizer at the double rate did not cause further increases in the values of these parameters. The combination of basal fertilization at a rate of 250 kg ha⁻¹ with the top dressing fertilization of calcium ammonium nitrate at a rate of 100 N kg ha⁻¹ increased the rosette diameter and the fresh biomass yield of bristly oxtongue by 54.7% and 79.9%, respectively. In another study, Guarise et al. [45] reported that the fresh biomass yield of hedge mustard (Sisymbrium officinale (L.) Scop.), a wild leafy vegetable, increased up to 21% at the 100% fertilization level (4 g of inorganic fertilizer 14-7-17 per pot) compared to that at the 50% fertilization level. Similarly, the starter NP fertilization (27 kg N ha⁻¹ and 69 kg P₂O₅ ha⁻¹) at sowing increased the grain yield of maize crops by 6.5 to 12.6% in different soil types compared with a control treatment [46].

5. Conclusions

The results of our study show that the examined agronomic management practices affect the growth and biomass yield of bristly oxtongue. Plants showed frost tolerance during the winter, while the growth and yield of plants were higher in the early sowing compared with plants sowed at other dates. Although positive effects of chitosan application on plant growth, yield, and physiological parameters (e.g., photosynthetic rate and chlorophyll content) have been reported in other crops, the present results indicate that the growth of bristly oxtongue plants was not affected by the foliar application of this biostimulant. Regarding the effects of fertilization, our results indicate that nitrogen fertilization at the rosette stage enhances the growth of bristly oxtongue, since the highest leaf number, rosette diameter, and biomass values (fresh and dry) of plants were recorded in the N10 treatment, regardless of the basal fertilization (15-5-20) rate applied. In conclusion, the early sowing of bristly oxtongue during October, the application of basal fertilizer (15-5-20) at a rate of 250 kg N ha⁻¹, and the top dressing fertilization (100 kg N ha⁻¹) in early February are recommended to optimize the growth and yield of this underutilized wild leafy green.

Author Contributions

Conceptualization, A.K.; formal analysis, G.N. and A.K.; investigation, A.K., G.T., E.N. and V.M.; methodology, G.N. and A.K.; supervision, A.K.; writing—original draft preparation, G.N. and A.K.; writing—review and editing, G.N. and A.K. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in this article.

Conflicts of Interest

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures and Tables

Figure 1. Monthly precipitation and mean air temperatures from October to April in 2020/21 and 2021/22 in Volos.

Figure 2. Relative chlorophyll content (SPAD values) in the leaves of bristly oxtongue as affected by sowing date (early, mid, and late). For each sampling date, bars followed by the same letters were not found to significantly differ with Fisher’s least significant difference (LSD) test. Errors bars show the Standard Error values. Final measurement (source of variation: treatments): Fvalue = 9.139, p = 0.032, DF (degrees of freedom) = 2.

Figure 3. Leaf number (no plant−1) and rosette diameter (cm) of bristly oxtongue as affected by the sowing date (early, mid, and late). For each sampling date, bars followed by the same letters were not shown to significantly differ with Fisher’s least significant difference (LSD) test. Error bars show the Standard Error values. Final measurement (source of variation: treatments): Leaf number: Fvalue = 31.155, p = 0.004, DF (degrees of freedom) = 2 and Rosette diameter: Fvalue = 9.305, p = 0.031, DF = 2.

Figure 4. Fresh and dry biomass (g plant−1) of bristly oxtongue as affected by the sowing date (early, mid, and late). For each sampling date, bars followed by the same letters were not shown to significantly differ with Fisher’s least significant difference (LSD) test. Errors bars show the Standard Error values. Final measurement (source of variation: treatments): Fresh biomass: Fvalue = 9.105, p = 0.032, DF (degrees of freedom) = 2 and Dry biomass: Fvalue = 7.382, p = 0.045, DF = 2.

Figure 5. Relative chlorophyll content (SPAD values) of bristly oxtongue as affected by nitrogen fertilization and chitosan application. For each sampling date, bars followed by the same letters were not shown to significantly differ with Fisher’s least significant difference (LSD) test. The error bars show the Standard Error values. Final measurement (source of variation: treatments): Fvalue = 29.872, p = 0.004, DF (degrees of freedom) = 2.

Figure 6. Leaf number (no plant−1) and rosette diameter (cm) of bristly oxtongue as affected by nitrogen fertilization and chitosan application. For each sampling date, bars followed by the same letters were not shown to significantly differ with the Fisher’s least significant difference (LSD) test. Errors bars show the Standard Error values. Final measurement (source of variation: treatments): Leaf number: Fvalue = 12.049, p = 0.020, DF (degrees of freedom) = 2 and Rosette diameter: Fvalue = 20.673, p = 0.008, DF = 2.

Figure 7. Fresh and dry biomass (g plant−1) of bristly oxtongue as affected by nitrogen fertilization and chitosan application. For each sampling date, bars followed by the same letters were not shown to significantly differ with Fisher’s least significant difference (LSD) test. Errors bars show the Standard Error values. Final measurement (source of variation: treatments): Fresh biomass: Fvalue = 12.787, p = 0.018, DF (degrees of freedom) = 2 and Dry biomass: Fvalue = 12.453, p = 0.019, DF = 2.

Figure 8. Relative chlorophyll content (SPAD values) in the leaves of bristly oxtongue plants as affected by various fertilization regimes. For each sampling date, bars followed by the same letters were not found to significantly differ with Fisher’s least significant difference (LSD) test. Errors bars show the Standard Error values. Final measurement (source of variation: treatments): Fvalue = 52.242, p < 0.001, DF (degrees of freedom) = 4.

Figure 9. Root dry biomass (g plant−1) of bristly oxtongue plants as affected by various fertilization regimes. For each sampling date, bars followed by the same letters were not shown to significantly differ with Fisher’s least significant difference (LSD) test. The error bars show the Standard Error values. Final measurement (source of variation: treatments): Fvalue = 17.896, p < 0.001, DF (degrees of freedom) = 4.

Figure 10. Leaf number (no plant−1) and rosette diameter (cm) of bristly oxtongue as affected by various fertilization regimes. For each sampling date, bars followed by the same letters were not shown to significantly differ with Fisher’s least significant difference (LSD) test. The error bars show the Standard Error values. Final measurement (source of variation: treatments): Leaf number: Fvalue = 34.642, p <0.001, DF (degrees of freedom) = 4 and Rosette diameter: Fvalue = 20.279, p < 0.001, DF = 4.

Figure 11. Fresh and dry biomass (g plant−1) of bristly oxtongue as affected by various fertilization regimes. For each sampling date, bars followed by the same letters were not shown to significantly differ with Fisher’s least significant difference (LSD) test. The error bars show the Standard Error values. Final measurement (source of variation: treatments): Fresh biomass: Fvalue = 21.578, p < 0.001, DF (degrees of freedom) = 4 and Rosette diameter: Fvalue = 18.995, p < 0.001, DF = 4.

Figure 12. Root:shoot ratio of bristly oxtongue as affected by various fertilization regimes. For each sampling date, bars followed by the same letters were not shown to significantly differ with Fisher’s least significant difference (LSD) test. The error bars show the Standard Error values. Final measurement (source of variation: treatments): Fvalue = 6.092, p = 0.015, DF (degrees of freedom) = 4.

Figure 13. Root growth of bristly oxtongue plants in the control (II.), Fert C (III.), and Fert D (IV.) treatments (12 April 2022). Bristly oxtongue plants at the rosette growth stage on 12 April 2022 (I.).

Table 1

Fertilization and chitosan treatments conducted in experiment 2.

Treatments	Nitrogen Fertilization 1	Chitosan Hydrochloride 2
Control	0 kg ha−1	No
N10	100 kg ha−1	No
N10 + chitosan	100 kg ha−1	Yes

¹ Calcium ammonium nitrate was applied on 1 February 2022, 92 days after sowing (DAS). ² Chitosan hydrochloride (PROJECT ONE, chitosan hydrochloride 3%) was applied on 2 February (93 DAS), 23 February (115 DAS), 12 March (132 DAS), and 2 April (153 DAS), 2021. A rate of 5 L ha⁻¹ was used, while the spray volume was 300 L ha⁻¹.

Table 2

Fertilization treatments conducted in experiment 3.

Treatments	Basal Fertilization (15-5-20 (+2+8); N-P2O5-K2O (MgO + S) 1	Top Dressing Fertilization 2
Control	0 kg ha−1	0 kg N ha−1
Fert A	250 kg ha−1	50 kg N ha−1
Fert B	250 kg ha−1	100 kg N ha−1
Fert C	500 kg ha−1	50 kg N ha−1
Fert D	500 kg ha−1	100 kg N ha−1

¹ The inorganic fertilizer 15-5-20 was applied before sowing. ² Calcium ammonium nitrate was applied in two equal rates on 2 February 2022 and 8 March 2022.

References

1. Voutsinos-Frantzis, O.; Ntatsi, G.; Karavidas, I.; Neofytou, I.; Deriziotis, K.; Ropokis, A.; Consentino, B.B.; Sabatino, L.; Savvas, D. Exploring the Simultaneous Effect of Total Ion Concentration and K:Ca:Mg Ratio of the Nutrient Solution on the Growth and Nutritional Value of Hydroponically Grown Cichorium spinosum L. Agronomy; 2022; 12, 2214. [DOI: https://dx.doi.org/10.3390/agronomy12092214]

2. Karkanis, A.C.; Petropoulos, S.A. Physiological and Growth Responses of Several Genotypes of Common Purslane (Portulaca oleracea L.) under Mediterranean Semi-arid Conditions. Not. Bot. Horti Agrobot.; 2017; 45, pp. 569-575. [DOI: https://dx.doi.org/10.15835/nbha45210903]

3. Liava, V.; Karkanis, A.; Tsiropoulos, N. Yield and Silymarin Content in Milk Thistle (Silybum marianum (L.) Gaertn.) Fruits Affected by the Nitrogen Fertilizers. Ind. Crops Prod.; 2021; 171, 113955. [DOI: https://dx.doi.org/10.1016/j.indcrop.2021.113955]

4. Arampatzis, D.A.; Karkanis, A.C.; Tsiropoulos, N.G. Silymarin Content and Antioxidant Activity of Seeds of Wild Silybum marianum Populations Growing in Greece. Ann. Appl. Biol.; 2019; 174, pp. 61-73. [DOI: https://dx.doi.org/10.1111/aab.12470]

5. Zahra, N.; Wahid, A.; Hafeez, M.B.; Alyemeni, M.N.; Shah, T.; Ahmad, P. Plant Growth Promoters Mediated Quality and Yield Attributes of Milk Thistle (Silybum marianum L.) Ecotypes under Salinity stress. Sci. Rep.; 2021; 11, 23200. [DOI: https://dx.doi.org/10.1038/s41598-021-02435-4]

6. Marceddu, R.; Dinolfo, L.; Carrubba, A.; Sarno, M.; Di Miceli, G. Milk Thistle (Silybum Marianum L.) as a Novel Multipurpose Crop for Agriculture in Marginal Environments: A Review. Agronomy; 2022; 12, 729. [DOI: https://dx.doi.org/10.3390/agronomy12030729]

7. Nemzer, B.; Al-Taher, F.; Abshiru, N. Phytochemical Composition and Nutritional Value of Different Plant Parts in two Cultivated and Wild Purslane (Portulaca oleracea L.) Genotypes. Food Chem.; 2020; 320, 126621. [DOI: https://dx.doi.org/10.1016/j.foodchem.2020.126621]

8. Mallor, C.; Carravedo, M.; Montaner, C. Field Collection and Conservation of Non-Cultivated Edible Species in the Germplasm Bank of Zaragoza (SPAIN). Acta Hortic.; 2011; 918, pp. 59-64. [DOI: https://dx.doi.org/10.17660/ActaHortic.2011.918.5]

9. Alexopoulos, A.A.; Assimakopoulou, A.; Panagopoulos, P.; Bakea, M.; Vidalis, N.; Karapanos, I.C.; Petropoulos, S.A. Impact of Salinity on the Growth and Chemical Composition of Two Underutilized Wild Edible Greens: Taraxacum officinale and Reichardia picroides. Horticulturae; 2021; 7, 160. [DOI: https://dx.doi.org/10.3390/horticulturae7070160]

10. Freitas, J.A.; Ccana-Ccapatinta, G.V.; Da Costa, F.B. LC-MS Metabolic Profiling Comparison of Domesticated Crops and Wild Edible Species from the Family Asteraceae Growing in a Region of São Paulo State, Brazil. Phytochem. Lett.; 2021; 42, pp. 45-51. [DOI: https://dx.doi.org/10.1016/j.phytol.2021.02.004]

11. Andersen, M.C. An Analysis of Variability in Seed Settling Velocities of Several Wind-Dispersed Asteraceae. Am. J. Bot.; 1992; 79, pp. 1087-1091. [DOI: https://dx.doi.org/10.1002/j.1537-2197.1992.tb13702.x] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30139130]

12. Ronel, M.; Khateeb, S.; Lev-Yadun, S. Protective Spiny Modules in Thistles of the Asteraceae in Israel. J. Torrey Bot. Soc.; 2009; 136, pp. 46-56. [DOI: https://dx.doi.org/10.3159/08-RA-043R.1]

13. Al-Balawi, S.M.; Alwahibi, M.S.; Al-Harbi, N.A. Diversity and Floristic Study of Weeds Species in Tabuk Region Farms in Saudi Arabia. Int. J. Bot. Stud.; 2021; 6, pp. 1560-1566.

14. Vurro, M.; Townley, H.E.; Morrison, R.; Boari, A.; Masi, M.; Evidente, A. Augmented Phytotoxic Effect of Nanoencapsulated Ophiobolin A. Nat. Prod. Res.; 2022; 36, pp. 1143-1150. [DOI: https://dx.doi.org/10.1080/14786419.2020.1860975]

15. Pieroni, A.; Sulaiman, N.; Sõukand, R. Chorta (Wild Greens) in Central Crete: The Bio-Cultural Heritage of a Hidden and Resilient Ingredient of the Mediterranean Diet. Biology; 2022; 11, 673. [DOI: https://dx.doi.org/10.3390/biology11050673]

16. Petropoulos, S.A.; Fernandes, Â.; Tzortzakis, N.; Sokovic, M.; Ciric, A.; Barros, L.; Ferreira, I.C.F.R. Bioactive Compounds Content and Antimicrobial Activities of Wild Edible Asteraceae Species of the Mediterranean Flora under Commercial Cultivation Conditions. Food Res. Int.; 2019; 119, pp. 859-868. [DOI: https://dx.doi.org/10.1016/j.foodres.2018.10.069]

17. Sergio, L.; Boari, F.; Pieralice, M.; Linsalata, V.; Cantore, V.; Di Venere, D. Bioactive Phenolics and Antioxidant Capacity of Some Wild Edible Greens as Affected by Different Cooking Treatments. Foods; 2020; 9, 1320. [DOI: https://dx.doi.org/10.3390/foods9091320]

18. Savo, V.; Salomone, F.; Mattoni, E.; Tofani, D.; Caneva, G. Traditional Salads and Soups with Wild Plants as a Source of Antioxidants: A Comparative Chemical Analysis of Five Species Growing in Central Italy. Evid. Based Complement. Altern. Med.; 2019; 2019, 6782472. [DOI: https://dx.doi.org/10.1155/2019/6782472]

19. Guillemin, J.P.; Gardarin, A.; Granger, S.; Reibel, C.; Munier-Jolain, N.; Colbach, N. Assessing Potential Germination Period of Weeds with Base Temperatures and Base Water Potentials. Weed Res.; 2013; 53, pp. 76-87. [DOI: https://dx.doi.org/10.1111/wre.12000]

20. Petropoulos, S.; Fernandes, A.; Karkanis, A.; Antoniadis, V.; Barros, L.; Ferreira, I.C.F.R. Nutrient Solution Composition and Growing Season Affect Yield and Chemical Composition of Cichorium spinosum plants. Sci. Hortic.; 2018; 231, pp. 97-107. [DOI: https://dx.doi.org/10.1016/j.scienta.2017.12.022]

21. Chen, C.-C.; Huang, M.-Y.; Lin, K.-H.; Hsueh, M.-T. The Effects of Nitrogen Application on the Growth, Photosynthesis, and Antioxidant Activity of Amaranthus viridis. Photosynthetica; 2022; 60, pp. 420-429. [DOI: https://dx.doi.org/10.32615/ps.2022.034]

22. Pichyangkura, R.; Chadchawan, S. Biostimulant Activity of Chitosan in Horticulture. Sci. Hortic.; 2015; 196, pp. 49-65. [DOI: https://dx.doi.org/10.1016/j.scienta.2015.09.031]

23. Mukhtar Ahmed, K.B.; Khan, M.M.A.; Siddiqui, H.; Jahan, A. Chitosan and its Oligosaccharides, a Promising Option for Sustainable Crop Production—A Review. Carbohydr. Polym.; 2020; 227, 115331. [DOI: https://dx.doi.org/10.1016/j.carbpol.2019.115331] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31590878]

24. Sible, C.N.; Seebauer, J.R.; Below, F.E. Plant Biostimulants: A Categorical Review, Their Implications for Row Crop Production, and Relation to Soil Health Indicators. Agronomy; 2021; 11, 1297. [DOI: https://dx.doi.org/10.3390/agronomy11071297]

25. Deolu-Ajayi, A.O.; van der Meer, I.M.; van der Werf, A.; Karlova, R. The Power of Seaweeds as Plant Biostimulants to Boost Crop Production under Abiotic Stress. Plant Cell Environ.; 2022; 45, pp. 2537-2553. [DOI: https://dx.doi.org/10.1111/pce.14391]

26. Salachna, P.; Łopusiewicz, Ł. Chitosan Oligosaccharide Lactate Increases Productivity and Quality of Baby Leaf Red Perilla. Agronomy; 2022; 12, 1182. [DOI: https://dx.doi.org/10.3390/agronomy12051182]

27. Shahrajabian, M.H.; Cheng, Q.; Sun, W. The Effects of Amino Acids, Phenols and Protein Hydrolysates as Biostimulants on Sustainable Crop Production and Alleviated Stress. Recent Pat. Biotechnol.; 2022; 16, pp. 319-328.

28. Stasińska-Jakubas, M.; Hawrylak-Nowak, B. Protective, Biostimulating, and Eliciting Effects of Chitosan and Its Derivatives on Crop Plants. Molecules; 2022; 27, 2801. [DOI: https://dx.doi.org/10.3390/molecules27092801]

29. Fouda, S.E.E.; El-Saadony, F.M.A.; Saad, A.M.; Sayed, S.M.; El-Sharnouby, M.; El-Tahan, A.M.; El-Saadony, M.T. Improving Growth and Productivity of Faba Bean (Vicia faba L.) Using Chitosan, Tryptophan, and Potassium Silicate Anti-Transpirants under Different Irrigation Regimes. Saudi J. Biol. Sci.; 2022; 29, pp. 955-962. [DOI: https://dx.doi.org/10.1016/j.sjbs.2021.10.007]

30. Zhang, G.; Wang, Y.; Wu, K.; Zhang, Q.; Feng, Y.; Miao, Y.; Yan, Z. Exogenous Application of Chitosan Alleviate Salinity Stress in Lettuce (Lactuca sativa L.). Horticulturae; 2021; 7, 342. [DOI: https://dx.doi.org/10.3390/horticulturae7100342]

31. Khodadadi, F.; Ahmadi, F.S.; Talebi, M.; Moshtaghic, N.; Matkowski, A.; Szumny, A.; Rahimmalek, M. Essential Oil Composition, Physiological and Morphological Variation in Salvia abrotanoides and S. yangii under Drought Stress and Chitosan Treatments. Ind. Crops Prod.; 2022; 187, 115429. [DOI: https://dx.doi.org/10.1016/j.indcrop.2022.115429]

32. Wang, Q.; Zuo, J.-H.; Wang, Q.; Na, Y.; Gao, L.-P. Inhibitory Effect of Chitosan on Growth of the Fungal Phytopathogen, Sclerotinia sclerotiorum, and Sclerotinia Rot of Carrot. J. Integr. Agric.; 2015; 14, pp. 691-697. [DOI: https://dx.doi.org/10.1016/S2095-3119(14)60800-5]

33. Rendina, N.; Nuzzaci, M.; Scopa, A.; Cuypers, A.; Sofo, A. Chitosan-Elicited Defense Responses in Cucumber Mosaic Virus (CMV)-Infected Tomato Plants. J. Plant Physiol.; 2019; 234–235, pp. 9-17. [DOI: https://dx.doi.org/10.1016/j.jplph.2019.01.003] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30640158]

34. Mustafa, M.A.; Mayes, S.; Massawe, F. Crop diversification through a wider use of underutilised crops: A strategy to ensure food and nutrition security in the face of climate change. Sustainable Solutions for Food Security; Sarkar, A.; Sensarma, S.; vanLoon, G. Springer: Cham, Switzerland, 2019; pp. 125-149.

35. Zhao, J.; Bindi, M.; Eitzinger, J.; Ferrise, R.; Gaile, Z.; Gobin, A.; Holzkämper, A.; Kersebaum, K.-C.; Kozyra, J.; Kriaučiūnienė, Z. et al. Priority for Climate Adaptation Measures in European Crop Production Systems. Eur. J. Agron.; 2022; 138, 126516. [DOI: https://dx.doi.org/10.1016/j.eja.2022.126516]

36. Geremew, A.; Carson, L.; Woldesenbet, S.; Carpenter, C.; Peace, E.; Weerasooriya, A. Interactive Effects of Organic Fertilizers and Drought Stress on Growth and Nutrient Content of Brassica juncea at Vegetative Stage. Sustainability; 2021; 13, 13948. [DOI: https://dx.doi.org/10.3390/su132413948]

37. Xu, C.; Mou, B. Chitosan as Soil Amendment Affects Lettuce Growth, Photochemical Efficiency, and Gas Exchange. HortTechnology; 2018; 28, pp. 476-480. [DOI: https://dx.doi.org/10.21273/HORTTECH04032-18]

38. Collins, M.; McCoy, J.E. Chicory Productivity, Forage Quality, and Response to Nitrogen Fertilization. Agron. J.; 1997; 89, pp. 232-238. [DOI: https://dx.doi.org/10.2134/agronj1997.00021962008900020013x]

39. Sylvestre, T.D.B.; Braos, L.B.; Batistella Filho, F.; Cruz, M.C.P.D.; Ferreira, M.E. Mineral Nitrogen Fertilization Effects on Lettuce Crop Yield and Nitrogen Leaching. Sci. Hortic.; 2019; 255, pp. 153-160. [DOI: https://dx.doi.org/10.1016/j.scienta.2019.05.032]

40. Semenov, M.A.; Jamieson, P.D.; Martre, P. Deconvoluting Nitrogen Use Efficiency in Wheat: A Simulation Study. Eur. J. Agron.; 2007; 26, pp. 283-294. [DOI: https://dx.doi.org/10.1016/j.eja.2006.10.009]

41. Woli, P.; Hoogenboom, G.; Alva, A. Simulation of Potato Yield, Nitrate Leaching, and Profit Margins as Influenced by Irrigation and Nitrogen Management in Different Soils and Production Regions. Agric. Water Manag.; 2016; 171, pp. 120-130. [DOI: https://dx.doi.org/10.1016/j.agwat.2016.04.003]

42. Ren, K.; Xu, M.; Li, R.; Zheng, L.; Liu, S.; Reis, S.; Wang, H.; Lu, C.; Zhang, W.; Gao, H. et al. Optimizing Nitrogen Fertilizer Use for More Grain and Less Pollution. J. Clean. Prod.; 2022; 360, 132180. [DOI: https://dx.doi.org/10.1016/j.jclepro.2022.132180]

43. Chatzigianni, M.; Alkhaled, B.; Livieratos, I.; Stamatakis, A.; Ntatsi, G.; Savvas, D. Impact of Nitrogen Source and Supply Level on Growth, Yield and Nutritional Value of two Contrasting Ecotypes of Cichorium spinosum L. Grown Hydroponically. J. Sci. Food Agric.; 2018; 98, pp. 1615-1624. [DOI: https://dx.doi.org/10.1002/jsfa.8636] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28842916]

44. Chatzigianni, M.; Ntatsi, G.; Theodorou, M.; Stamatakis, A.; Livieratos, I.; Rouphael, Y.; Savvas, D. Functional Quality, Mineral Composition and Biomass Production in Hydroponic Spiny Chicory (Cichorium spinosum L.) are Modulated Interactively by Ecotype, Salinity and Nitrogen Supply. Front. Plant Sci.; 2019; 10, 1040. [DOI: https://dx.doi.org/10.3389/fpls.2019.01040] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31555310]

45. Guarise, M.; Borgonovo, G.; Bassoli, A.; Ferrante, A. Effect of Fertilization on Yield and Quality of Sisymbrium officinale (L.) Scop. Grown as Leafy Vegetable Crop. Agronomy; 2019; 9, 401. [DOI: https://dx.doi.org/10.3390/agronomy9070401]

46. Blandino, M.; Battisti, M.; Vanara, F.; Reyneri, A. The Synergistic Effect of Nitrogen and Phosphorus Starter Fertilization Sub-surface Banded at Sowing on the Early Vigor, Grain Yield and Quality of Maize. Eur. J. Agron.; 2022; 137, 126509. [DOI: https://dx.doi.org/10.1016/j.eja.2022.126509]