The influence of mineral nutrition and humic acids on the intensity of photosynthesis, as well as the yield and quality of seeds, roots, and aboveground phytomass of milk thistle Silybum marianum (L.) Gaertn. in marginal growing conditions

A field experiment with the vegetation of Silybum marianum (L.) Gaertn. was established under the operating conditions of the processing company Agrokarpaty in the village of Plavnica, with plantations in the foothills of northeastern Slovakia with a mildly cold and humid climate. An experiment covering an overall area of 4 ha was concentrated on a plot with medium heavy clay soil, at an altitude of 540 m (above sea level) within close proximity to the Poprad river. In Table 1 we present the chemical properties of the site, determined by the analysis of soil samples from layers of topsoil of 0–30 cm taken in May 2020 before the start of the experiment.

The MIREL variety (a variety characterised by its origins in the Czech Republic and by both high silychristin and silybinin content, chemotype A) was sown on May 20, 2020, at a depth of 3 cm and a sowing rate of 350,000 seedlings per ha, and commercial seeds were used. Basic tillage to a depth of 24 cm was done in the fall, during the spring preparation prior to sowing; NPK nutrition, and humic preparation HUMAC Agro were incorporated into the soil at a depth of 15 cm. Differentiated nutrition (Table 2) for a dose of 200 kg/ha NPK consisted of 30 kg/ha N, 30 kg/ha P₂O₅, and 30 kg/ha K₂O. After emergence, the stand was treated against weeds with Pendigan (pendimethalin, 400 g/l) at a dose of 3.0 l/ha.

The intensity of plant photosynthesis was measured in-situ on 21 July 2021 with the LCPro-SD instrument in the phenophase of full thistle flowering (BBCH 69). On each variant, a fully vegetating leaf was measured (flag leaf -3) for a duration of 112 minutes within a sequence of 24 three-minute steps with a gradual change in the intensity of the photosynthetically active FAR light (at a stable temperature) and subsequent temperature (at a constant FAR intensity). Overview of accompanying parameters of the tested range of temperature and light sequence is part of the evaluation (Table 11, Figures 1 and 2).

Figure 1

Figure 2

Collection of plant and soil samples for biometric and laboratory analysis was carried out on the day of harvest, 27 August 2020; the stand was not desiccated before harvest. Samples were analysed in the laboratory NPPCVUA Michalovce, where biometric measurements were also performed on plant samples. The qualitative content of the components of the silimarin complex silychristin (SC), silydianin (SD), silybinin A (SB A), silybinin B (SB B), isosilybinin A (ISB A), and isosilybinin B (ISB B) as flavonolignans (SC + SD + SB A + SB B + ISB A + ISB B) with relative retention times (RRT) in comparison with silybin B are listed in order of the components 0.61, 0.65, 0.96, 1.00, 1.04 and 1.07 of seeds and roots, as determined in the laboratory of Moravol s.r.o. by HPLC. The RRTs stated are for guidance only and depend on the current state of the column and mobile phase. The measuring device is a liquid chromatograph operating in gradient elution mode, ie, Knauer assembly, equipped with a column thermostat with Peltier cells, a UV/VIS detector and a manual dispenser. The system also includes Clarity control and evaluation software running on the Windows operating system. Column: Type RP C18 Nucleosil C18; column temperature: 30° C; flow: 0.5 ml/min; detection: 288 nm.

Acid-detergent fiber (ADF), neutral-detergent fiber (NDF), acid-detergent lignin, crude cellulose (CrC), and hemicellulose (HeC) were determined according to the regulation MP SR no. 2,136 / 2,004-100 by extraction systems: Fiber extractor – Fibertest, Model F-6 and Cold Extraction Unit, Model EF-6. Combustion heat and calorific value were determined in the IKA C 5,000 calorimetric system, in accordance with the STN standard ISO 1928.

Table 3 presents data on the course of weather conditions for the vegetation period of milk thistle. Data were obtained from a meteorological observation station located near the experiment site and are part of the SHMU (Slovak Hydrometeorological Institute) network with guaranteed record quality. In the pre-vegetation period of 2020, the average daily temperature and the total precipitation for January, February, March, and April were as follows: −2.6° C and 16.6 mm; 1.3° C and 60.6 mm; 2.6° C and 16.8 mm; and 6.7° C and 33.9 mm.

Harvest of milk thistle seeds and roots, as well as the values of the main biometric parameters monitored, growth and indicators of seed quality, were significantly differentiated according to the established treatments and variants (Tables 4, 5, 6, 7, and 8). The highest seed yield was achieved with the variant NPK, for which, in terms of dry matter, the yield was 0.534 t/ha; in comparison with the NPK variant (100%), seed yield was 0.254 t/ ha with the HUMAC Agro variant (47.6%), resp. 16.3% at 0.087 t/ ha, and seed yield for the untreated control.

Differences in the yield of milk thistle seeds according to treatment variants, order of variants (i) NPK, (ii) HUMAC Agro, and (iii) untreated control correlated with the biometric parameters of the stand, with differences and with the order of variants for the monitored stand parameters. Plant height, the number of plants per area, and coverage were highest with the NPK variant and lowest with the untreated control; similarly, the weight of heads at harvest per m², the weight of seeds at harvest per m² and per plant, as well as aboveground crop phytomass without buds exhibited the same characteristics (Table 4).

The seeds were characterised by a markedly different moisture content at the time of harvest, which was highest with the HUMAC Agro variant with a value of 32% and lowest with the untreated control with a value of 12.6%. With variant NPK, the moisture content of the seeds was 20.3%. For the bulk density of the seeds and the weight of a thousand seeds, the highest values were observed with the HUMAC Agro variant (665.9 g/l; 21.4 g), lower for the NPK variant (664.3 g/l; 21.1 g), and the lowest in untreated control (653.1 g/l; 20.5 g). The proportion of fully developed mature seeds was highest with the NPK variant (83.8 %), slightly lower with the HUMAC Agro (82.5 %), and the lowest with the untreated control (78.2 %). Immature seeds, red achenes had markedly low values of bulk density (461 g/l) and weight per thousand tons (16.8 g). According to Giuliani et al. (2018) the dark colour of fully ripe fruits is due to the accumulation of condensed tannins in the pericarp of the subepidermal cell layer. The asynchronous flowering and spontaneous release, according to Alemardan et al. (2013), are among the main problems in the cultivation of milk thistle. During the harvest period, the plants have inflorescences at all stages of development results in nonuniform seed ripening. Agronomic measures, as well as breeding efforts, should therefore focus on obtaining plants with unified flowering and reduced seed loss. According to Grest and colleagues (2006), seeds in secondary inflorescence are markedly lower in weight (−56%) compared with the primary inflorescence. Similarly, the weight of the plants and the weight of the main branch are in a positive relationship and are directly or indirectly linked to the yield of seeds. In the definition of the ideotype of plants, ie, stand density of milk thistle, it is therefore appropriate to focus on the high proportion of the main inflorescence and the main branch.

Carrier et al. (2003) found that the average milk thistle plant grown in dry conditions produced 2.0 g, 2.9 g, and 3.4 g of dry seeds per inflorescence of the plant, which corresponded to the harvest in phenophase in the middle of flowering, late flowering, and spontaneous opening of inflorescence, with the highest yield of silymarin detected in the late flowering and spontaneous inflorescence during fruit ripening at the beginning of September.

According to Qavami et al. (2013) The content of silymarin in milk thistle seeds depends on the variety and the geographical and climatic conditions. Also, according to Arampatzis et al. (2019), the impact of climatic conditions on the crop and its quality is demonstrable, whilst the lowest values of silymarin content and harvest of seeds were recorded in a dry year in the season from March to May. The results of Arampatzis et al. (2019) also show that plant density has significant impact on the growth and yield of seeds. The main indicators of growth, such as height, yield of above-ground phytomass, and seeds were highest when plant density was highest.

In the ideal agroecological conditions of the warm lowlands of Slovakia, Haban et al. (2009) studied the impact of intermediate crops, mineral nutrition, and the incorporation of pre-crop post-crop residues into the yield and quality of milk thistle seeds. The seed yields recorded ranged from 1.43 to 1.83 t/ha, and silymarin yields were in the range of 16.5 to 24.6 kg/ha. The content of silymarin complex in the seeds was in the range of 1.5% to 2.0%. Haban et al. (2016), in a similar experiment on an identical site from a later period, state that milk thistle yields ranged from 0.30 to 0.75 t/ha, which is probably closely related to the decline of the genetic value of the farm seed used.

In the agroecological conditions north of Slovakia, humidity and thermal conditions during three years of research, according to Anrdzejewska et al. (2011) caused the yield of seeds to range from 0.55 to 1.68 t/ha and silymarin yield from 13.3 to 35.4 kg/ha, the average silymarin content in the crops was 2.18%. With comparable conditions Cwalina-Ambroziak et al. (2012) state that the milk thistle yield is increased in response to increasing nitrogen utilisation, tested in the range of 0–120 kg N/ha.

Estaji et al. (2016) did not show any demonstrable effect on silybin content based on the tested doses of nitrogen 0–100 kg N/ha. According to Rahimi and Kamali (2012), the delayed sowing time apparently significantly reduces inflorescence for all methods of fertilisation. They concluded that the highest yield of seeds and essential oil was obtained with the earliest sowing date, whilst a declining trend was observed with a sowing delay.

According to Karkanis et al. (2011) milk thistle is considered drought resistant and average rainfall is sufficient even in the semi-arid conditions of Greece. In a Mediterranean environment, in strong drought conditions, milk thistle should be irrigated during seed growth and filling. In the experiments of Afshar et al. (2014, 2015) with declining irrigation, where a mild to severe decline of moisture resulted in a 7% and 27% reduction of seed yield compared with the full irrigation dose. Regardless of the irrigation regime, the application of poultry manure and vermicompost increased the yield of milk thistle. Application of these organic materials to soil had no significant effect on the silymarin and oil content of the seeds, but the silymarin and oil increased owing to the beneficial effect on seed yield.

In view of the findings of Omidbaigi et al. (2003) with the most suitable plant density of 6.7 units per m² (at 50 × 30 cm, this is 1.500 cm² per plant) it is clear that the organisation of milk thistle growth is strongly influenced by agroecological conditions. This is also confirmed by the conclusions of the same study on the suitability of the sowing date, which states that the best time to sow milk thistle is in the autumn, especially in September. In trials, Omidbaigi studied the effect of sowing dates September 11 and October 17 as autumn sowing and March 10 and April 12 as spring sowing. Within plant density, they monitored growth, seed yield, and active ingredients at 8.0, 6.7, and 5 plants per m². According to Haban et al. (2009), in the central European climatic conditions of Slovakia, milk thistle does not hibernate during the winter, and therefore only spring sowing is possible.

Similar to seed yield, the yield of aboveground phytomass of milk thistle is significantly different based on the treated variants (Table 4) as well as the diameter and other monitored parameters of the main root (Table 6). The highest dry matter yield of aboveground phytomass without buds was 2.86 with the NPK variant t/ha (100%), significantly lower with HUMAC Agro 1.35 t/ha (47.2%), and lowest with the untreated control 0.28 t/ha (9.8%). The highest weight of the main round root was in the NPK variant 0.272 t/ha (100%), lower with HUMAC Agro 0.171 t/ha (62.9%), and lowest with the untreated control 0.072 t/ha (26.5%). The average thickness of the main round root and its weight were highest for NPK variant (1.19 cm and 1.59 g, respectively 100% and 100%), lower for HUMAC Agro (1.15 cm and 1.50 g, 96.6% and 94.3%, respectively) and lowest for the untreated control (0.84 cm and 0.75 g, and 70.6% and 47.2%, respectively). The moisture of the root at harvest was the highest in the NPK variant 44.3% (relatively 100%), lower for the HUMAC Agro variant 42.3% (relatively 95.5%), and the lowest for the untreated control 27.5% (relatively 62.1%).

Qualitative indicators of above-ground phytomass without buds, which are important indicators for the use of phytomass of milk thistle for various energy purposes, are listed in Table 9. ADF content (56.24%–60.39%), ADL (7.74%–10.25%), cellulose (48.49%–52.05%), hemicellulose (4.55%–4.88%), and NDF (61.13%–65.17%) were not significantly differentiated in the phytomass above ground level, according to the variants monitored, similarly to the calorific value (16.06–16.14 kJ/g at comparable humidity, 7.20%–6.62%) and combustion heat (16.23–16.31 kJ/g). According to the yield of of milk thistle phytomass above ground level (without buds) the energy yield was highest in the variant NPK 46.649 GJ/ha. However, with significantly differentiated yields of phytomass above ground level, the energy yield was significantly different depending on the variants monitored. The highest phytomass energy was found in the NPK variant 46.649 GJ/ha (100%), significantly less with HUMAC Agro 21.894 GJ/ha (49.9%), and even less for the untreated control 4.504 GJ/ha (9.7%).

Milk thistle provides suitable above-ground phytomass for various energy uses, however, in the somewhat cold and humid foothill conditions of northern Slovakia, and the yield is not quite satisfactory. It is necessary to take into account the losses during harvest of phytomass, which may be approximately 20%–30% and more. In the conditions of southern Slovakia with warmer lowlands, the yield potential of the phytomass of milk thistle above ground level is higher, if we take into account our own findings and literature data, especially owing to the earlier onset of spring and therefore the possibility of earlier sowing. Southern locations provide the advantage of a higher input of solar radiation, while northern locations provide the advantage of more sufficient moisture. Higher daily temperatures alone during the second half of the milk thistle growing season are not a clear advantage when growing milk thistle.

In the semi-arid conditions of Greece, Tsiaousi et al. (2019) found that milk thistle growth produced 15.49 to 20.89 t/ ha of dry matter of phytomass above ground level, in which the yield was reduced by the encroachment of weeds onto the plantation and the gross calorific value of milk thistle phytomass ranged from 15.5 to 16.1 MJ/kg. At an average net energy gain (energy yield reduced by energy deposits in cultivation) in the range of 80.13 GJ/ha to 111.87 GJ/ha, in the conditions of the Mediterranean climate, milk thistle provides suitable phytomass of satisfactory quality for energy use.

According to Martinelli (2020), an analysis of the composition of milk thistle phytomass shows that the contents of substances that are possible to extract, such as ash, lignin, and cellulose, is comparable with other herbal energy crops with these traits show only limited variability. With milk thistle, only xylans form the hemicellulose fraction, and its content, therefore, appears on average lower compared with the phytomass of other herbs.

Milk thistle is sown in the second half of May and harvested in late August. In terms of weather conditions, the vegetation period in the given locality is characterised by sufficient to abundant precipitation with a total of 379.4 mm and an average daily temperature of 15.4° C, (Table 3). Planting did not suffer from a lack of moisture; in May it was even exposed to an excess amount of moisture. The sowing of the stand was postponed by two weeks in comparison with the optimal sowing date for the given area, owing to the delayed onset of spring.

The chemical properties of the soil monitored, as well as the condition and categorisation before the start of the experiment are provided in Table 1. The condition and categorisation after the end of the experiment are provided in Table 10. The soil on the plot before the establishment of the experiment was characterised by low P (29.7 mg/kg) and K (80.2 mg/kg) as well as low humus content (1.897%). The use of mineral nutrients on the NPK variant partially eliminated (with P sufficient and K insufficient) the effect of the low content of PK nutrients in the soil on the growth and yield of milk thistle, whereas with variants HUMAC Agro and untreated control no dose of industrial nutrients was used, and growth and yield of milk thistle were limited differentially. Compared with the state before sowing, higher content of P and K in the soil after the end of the experiment on each variant treatment (Table 10) can be associated mainly with the date of collection (the effect of higher mineralisation of nutrients in warm August rather than in cold April), while to a much lesser extent we also assume it results from making nutrients available through the root secretions of milk thistle itself (for the main nutrients P and K it shows a different final effect). In the case of large-scale, partly operational experiments, to some extent the surface heterogeneity of the plot plays a role, which is associated with the different humus content according to the treatment variants, where the final state may be distorted more than the nutrient content.

The intensity of milk thistle photosynthesis was significantly different among the variants monitored, similarly to the sequence of temperature changes as well as the sequence of changes in the intensity of photosynthetically active radiation (Table 11, Figure 1 and 2). In both sequences, the highest intensity of photosynthesis was in the untreated control (20.115, 12.386 mmol/m²/s¹, 100%–100%), significantly lower for HUMAC Agro (16.386 and 9.653 mmol/m²/s¹, 81.5%–77.9%) and lowest at NPK (10.933 and 7.813 mmol/m²/s¹, 54.4%–63.1%). The polynomial trend line of the temperature sequence also shows that the intensity of photosynthesis in the untreated control is characterised by a sharper decrease due to increasing temperatures compared with both treated variants.

The performance of photosynthesis is a parameter that is measured and related to a unit of leaf area. Increased milk thistle yield in both treated variants compared to the untreated control is in strong negative correlation with the decrease in photosynthesis performance, ie the size of the leaf is decisive in the formation of the crop area per unit area of land. Although mineral nutrition and the use of humic preparations in the soil reduce the photosynthetic performance of leaves, in a wide range of their actions they provide an opportunity (ideally if they are used in combination) to optimise the growth and yield process of milk thistle.

According to Geneva et al. (2008), increasing the yield and quality of milk thistle seeds using plant growth regulators in combination with soil or foliar mineral fertilisers is associated with the regulation of growth and development, and the aim should be to prolong the reproductive stage and increase the yield of seeds with a high silymarin content. They found that treatment of milk thistle with foliar fertilisers and growth regulators resulted in an increase in mature inflorescences.