1. Introduction
The selenium (Se) content of soils varies greatly worldwide, from very low to toxic levels even within the same country [
1,
2]. Its total concentration in the soil is relatively low, generally between 0.01 and 2 mg kg
−1 [
3], with a global average of 0.4 mg kg
−1 [
4].
However, in addition to the total Se content of the soil, the availability of Se to plants is also an important aspect which is greatly influenced by the chemical form of the element.
The presence and distribution of different Se forms in soil is a function of the interaction between soil factors. The more mobile selenate (Se
6+) occurs primarily in alkaline and well-aerated soils, whereas selenite (Se
4+) is less mobile and more common in neutral or acidic soils and under less oxic conditions. Selenide (Se
2-) is relatively immobile, is formed under acidic conditions and is strongly bound to mineral or organic compounds. Under reducing conditions, selenium forms may also undergo precipitation [
5]. The solubility and plant availability of Se increase with increasing soil pH [
6,
7]. Selenite sorption correlates well with the Fe and Al contents, so Fe oxides may also play a significant role as Se sorption sites [
8]. However, Coppin et al. [
9] found that in a grassland soil, Se was bound largely to the organic fraction, with only a minor part bound to mineral components.
The essential role of Se has not been proven for plants, but in certain circumstances, it may have beneficial effects [
10], such as mitigating the negative effect of certain abiotic stress factors like heavy metals, drought, salinity and temperature [
11,
12,
13]. The application of Se in certain doses may result in yield increases [
14]. A low concentration of supplementary selenium in the growth medium may improve physiological processes, like the efficiency of photosynthesis [
12,
15,
16,
17], especially under abiotic stress conditions. Based on their ability to absorb Se, plant species can be classified as hyperaccumulators (>1000 mg kg
−1 Se), secondary-accumulators (100–1000 mg kg
−1 Se) and non-accumulators (<100 mg kg
−1 Se) [
15].
The Se concentration of the soil in a specific area determines the Se content of wild and cultivated plants and is thus closely related to the daily Se intake of the people and animals living in that area. The ability of plants to absorb Se is essential for human health since Se is known to be an essential nutrient for humans and animals. It plays a significant role as an antioxidant and has diverse effects on health [
18]. In areas with a low Se supply, additional Se should be provided [
19]. A more direct way to compensate for low Se intake in humans is to produce Se-enriched functional foods, a process known as biofortification [
20]. In Finland, the low dietary intake of Se was improved from the 1980s onwards by mixing Na-selenate (Na
2SeO
4) with fertilizer [
21]. Effective measures have also been taken in the UK to increase the Se content of foods [
22]. In Hungary, both the Se supply in soils and the Se concentration in the blood of the population are low, so biofortification with Se should be a priority [
23].
The enrichment of irrigation water, i.e., fertigation, is one way of producing functional foods in order to enhance the intake of certain elements [
24,
25].
The biofortification of various plant species with Se has been extensively studied [
20,
26,
27,
28]. Tomato, potato and cabbage have been given considerable attention in this respect, while less research has been reported about beans, although this is the most important legume crop for direct human consumption [
29]. According to the literature, Se has been investigated in roughly equal proportions in hydroponic experiments and in a soil medium (either in the field or in pots). When soil was used as the growth medium, Se was typically applied in one or several doses, either by adding a solution to the soil or in the form of leaf spraying [
30]. Selenium fertigation, usually applied to the soil throughout the whole vegetation period, has so far only been investigated to a limited extent for melon, tomato [
24,
25,
31], broccoli and canola [
32]. These studies were largely conducted in the San Joaquin Valley, California, where it is the high Se content of the irrigation water that is causing the problem [
33].
However, Se fortification may have contradictory effects on the element composition of Se-treated plants. The antagonistic or synergistic effect of Se has been studied primarily in human or animal nutrition, with far less research on plant nutrition. Antagonism has been detected between Se and sulphur [
34], Se and mercury [
35] and Se and molybdenum [
36]. The relationship of Se with arsenic species [
37,
38] and cadmium [
39,
40] was also studied and found to be either synergistic or antagonistic.
The interaction between Se and other elements may also depend on whether the studies were performed in soil or in hydroponic cultures. Feng et al. [
41] investigated Chinese brake fern (
Pteris vittata) in both hydroponic and soil culture. In plants grown in soil, Se suppressed the uptake of most of the elements tested, including magnesium (Mg), potassium (K), phosphorus (P), iron (Fe), copper (Cu) and zinc (Zn). In nutrient solution, the uptake of most essential elements was suppressed at lower levels of Se, but higher Se doses had a synergistic effect and stimulated the uptake of Ca, Mg and K. This shows that only the results obtained in soil may be relevant if we want to investigate the expected effect of Se in the field.
To the best of our knowledge, this is the first experiment to use Se-enriched irrigation water for the continuous irrigation of green beans, cabbage, potatoes and tomatoes on three different soil types in order to investigate its effects on biomass production and on the concentration of Se and other elements.
The aim of the experiment was to model the effects of elevated Se levels in the irrigation water and of soil texture on the yield and elemental composition of potato, cabbage, tomato and green bean plants. The recent work of Newman et al. [
42] also drew attention to the human health implications of examining such data. In addition to the elemental composition, the fitness of the plants was examined by measuring the photosynthetic parameters: chlorophyll content index (CCI) and chlorophyll fluorescence (Fv/Fm), which are good indicators of the physiological effect of Se treatment. Based on the above, the following hypotheses were investigated:
(H1) Dissolved Se applied with irrigation water can theoretically easily reach the roots and be absorbed by the plant. Nevertheless, the uptake of Se may be influenced by soil factors, so it is important to examine the extent of this effect. For this reason, three field soils differing significantly in pH, clay and organic matter (OM) content were used in the pot experiment so that the effect of different soil properties on the fate of selenium could also be investigated. Selenium was expected to be more mobile and available for the test plants on soil with lower clay and OM contents but with a higher pH. (H2) It was expected that the Se treatment up to 500 µg L−1 Se concentration in irrigation water would not have a significant negative effect on plant biomass production but that the Se content of the plant tissues would increase. (H3) Se can be enriched in edible plant parts without a significant negative effect on the concentration of other elements, and the vegetables grown can be used as functional foods.
4. Materials and Methods
4.1. Experimental Materials and Conditions
The effect of Se-enriched irrigation was studied on green bean (
Phaseolus vulgaris L., cv. Golden Goal), cabbage (
Brassica oleracea L. var.
capitata cv. Zora), tomato (
Solanum lycopersicum L. cv. Mano) and potato (
Solanum tuberosum L. cv. Balatoni rózsa) in an open greenhouse at the Experimental Station of the Center for Agricultural Research in Őrbottyán, Hungary [
85,
86,
87]. A pot experiment was performed using the top 0–20 cm layer of soils from three different locations in Hungary, all having distinct properties: sand (Mollic Umbrisol (Arenic) from Őrbottyán), silty sand (Luvic Calcic Phaeozem from Gödöllő) and silt (Calcic Chernozem from Hatvan) [
88]. The basic characteristics of the air-dry soils are shown in
Table 7, and total and mobilizable element concentrations of the soils are shown in
Supplementary Table S5.
Four holes, each 0.5 cm in diameter, were drilled in the bottom of the 10 L pots to allow leached water to escape. The bottom of each pot was filled with gravel with a diameter of 4–8 mm in a 1 cm layer, which was covered with a fine synthetic fiber fabric and 10 kg of soil.
The cabbage and tomato seeds and the potato tubers were first germinated, then planted and grown for three weeks in a commercial growth medium (Vegasca Bio soil mix; Florasca Hungary Ltd.; mixture of peat and grey cattle manure compost. OM > 50%; N > 0.3%; P2O5 > 0.1%; K2O > 0.1%; pH of 6.8) under controlled climatic conditions in a growth chamber (16/8 h photoperiod, 25–27/15–17 °C temperature and 600 μmol/m2/s photon flux density). Soil-free seedlings were transplanted into plastic pots (1 seedling/pot) after a 6-day acclimatization period in the greenhouse. Germinated seeds of green bean were planted directly into the soils (1 seed/pot).
The Se-enrichment of the irrigation water started three weeks after planting. The experiment was set up in three replicates on three soil types and included three treatment levels: Se-0: control, Se-1: 100 and Se-2: 500 µg Se L
−1 in the form of Na
2SeO
4 in the irrigation water. These concentrations are an order of magnitude higher than the recommended values for irrigation water by FAO [
89]. The water was stored in 0.5 m
3 tanks before the application. Irrigation was carried out with an automatic irrigation system, with individual drip stakes placed in each pot. The daily volume of irrigation water was adjusted according to the requirements of the plants: soil moisture content was monitored at a depth of 10 cm every hour (Sensor: Decagon EC-5). The nutrient requirements of all the treated plants were met during the vegetation period using 200 mL Hoagland solution per pot, applied weekly by hand. The total number of plants included in the experiment was 3 Se levels × 3 soil types × 4 plant species × 3 replicates = 108 plants. Details of the growth period and irrigation are shown in
Table 8.
The plants received natural sunlight in the greenhouse. Climate data were monitored throughout the growth period, as reported in
Table 9. Pesticide (Decis, Bayer) was applied whenever necessary.
4.2. Sample Preparation, Analysis and Measurements
The plants were washed with deionized water after harvest; then, the plant parts were separated (root, tuber, leafy shoot, fruit), and the fresh biomass was weighed. Subsequently, the roots and aerial parts of the plants were dried at 40 °C in a laboratory dryer for two days, except for tomato and green bean fruits and potato tubers, which were milled and freeze-dried at −70 °C, 200 Pa for 72 h, before measuring the dry mass of the plant organs. Dried samples were homogenized with a blending machine, equipped with plastic housing and stainless-steel blade. The dried, homogenized samples were mineralized in a microwave-assisted acid digestion system (TopWave, Analytik Jena, Germany). Plant samples of 400–500 mg were digested in a mixture of 7 mL 67% HNO3 (VWR International, Radnor, PA, USA) and 3 mL 30% H2O2 (VWR International, PA, USA). After digestion, internal standards (Sc, Y, In) were added to the solutions, which were made up to 15 mL with deionized water.
The concentrations of selenium, macro- and micro-elements in the dry weight (DW) of plant samples were measured using an inductively coupled plasma mass spectrometer (ICP-MS) (PlasmaQuant Elite, Analytik Jena, Germany).
Composite soil samples were collected from the soils to analyze their basic parameters, and soil samples from each pot were also analyzed after removing the plant residues. The soil samples were dried and sieved through a 2 mm mesh sieve. The pH was measured according to the Hungarian Standard Method [
90] in a 1:2.5 soil:water suspension 12 h after mixing, and the OM content was measured using the modified Walkley–Black method [
91]. The total N content was measured with the Kjeldahl method [
92], and the NH
4-N and NO
3-N concentrations from KCl extracts were measured according to the Hungarian Standard [
93]. The CaCO
3 content was measured using the Scheibler gas-volumetric method [
90], and the CEC values were measured with the modified method of Mehlich [
94]. Plant-available P and K concentrations were determined after extraction with ammonium acetate-lactate (AL-P
2O
5 and AL-K
2O) [
95]. Plant-available Se and other element contents were measured in 0.5 M NH
4-acetate + 0.02 M EDTA extract according to Lakanen and Erviö [
96] (referred to as LE). The total Se concentrations were determined from the samples using aqua regia in a microwave teflon bomb [
97]. The element contents of the soil samples were analyzed using the ICP-MS instrument.
To detect plant physiological responses to Se treatments, the photochemical activity of the photosystem (PS) II (Fv/Fm) of dark adapted leaves (15 min) was measured as the chlorophyll
a fluorescence intensity (Opti-Sciences OS-30p+ Fluorometer, Hudson, New Hampshire, USA). F
v/F
m values were calculated by the method of Tsimilli–Michael and Strasser [
98]. The chlorophyll content index (CCI) of the leaves was detected with a Chlorophyll Content Meter (CCM-200 plus, Opti-Sciences, Hudson, NH, USA). The F
v/F
m and CCI values were measured on the youngest adult leaves at the harvesting stage for all the plants.
4.3. Statistical Analysis
A two-factor factorial analysis of variance was used to evaluate the plant parameters, one factor being the soil type and the other the Se dose. The level of significance was set to a 95% confidence interval (
p < 0.05). Significantly different groups were determined using Tukey’s HSD post hoc test. Statistica v.13 (StatSoft Inc.) software was used for all the statistical calculations. Data visualization was made with
R statistical software [
99] using a
ggplot2 package [
100].
5. Conclusions
Irrigation water enriched with Se significantly increased the plant-available Se content of the soil, which was lower in sandy soil and higher in silty sand and silt soils. Regarding the Se treatment’s effect on plant parameters, our main findings are the following:
The treatments caused a slight decrease in the biomass production of the edible parts of green bean, cabbage, potato and tomato, while the dry matter content of cabbage heads and tomato fruit increased.
Se had a beneficial effect on potato and tomato CCI values, with a significant increase in this parameter in plants.
Se treatment had the greatest effect on the Se content of the edible parts of the plants, which increased by several orders of magnitude, while P, K, Fe, Mg, Zn and Cu increased or decreased depending on the soil types or plant species. Se treatments decreased the P and K contents in green beans, the Zn and Cu contents in potato and the P, K, Fe and Cu contents in tomato. Se treatments had a positive effect on Zn content in green beans; on K, Mg and Zn contents in cabbage; on Fe content in potato and on Zn content in tomato. In the other cases, the changes were different as a function of soil type, or the Se treatment had no effect.
With respect to the soil types, the Se content of the edible plant parts was the highest in sandy soil for most species and treatments.
Based on the Se content of the fresh edible plant parts and the recommended dietary allowance, 100 µg Se L−1 fertigation can be recommended for the production of functional foods for green beans, potatoes and tomatoes, but as cabbage accumulates more Se, irrigation water with a lower Se content is recommended. In the case of cabbage, a more accurate determination of the Se content, and in the case of foods typically consumed after processing, a more detailed examination of the effect of frying and cooking on the Se content could serve as topics for further research. The use of Se-enriched irrigation water might be a suitable method for Se biofortification without a significant reduction in plant biomass production and without a remarkable modification of other macro- and microelement contents.