4.1. Milk Thistle Varieties
Variety significantly affected plants growth, since the rosette diameter and height of plants in Spata were higher by up to 13.57% and 15.40%, respectively, compared with Palaionterveno. The impact of genetic material in plants growth parameters was observed in previous studies as Ram et al. [
15], Gresta et al. [
26], Shokrpour et al. [
22], and Sulas et al. [
27] recorded plant height from 78 cm to 207 cm in various milk thistle genotypes. Moreover, varieties significantly affected the number of fruits per inflorescence as in Palaionterveno this number was higher by up to 32.38% than that in Spata. The number of fruits per inflorescence in these two varieties of Greek origin ranged from 58.9 to 147.5 and was similar to that observed in previous studies. Stancheva et al. [
28] reported values from 119.4 to 185.5, while Shokrpour et al. [
22] recorded fewer fruits per inflorescence, ranging from 51.6 to 101.4. In contrast, 1000-fruit weight was not affected by variety. With regard to fruit yield, in 2021, the highest values were recorded in the variety Palaionterveno, while in 2020, there were no differences between the two varieties. In 2020, in Palaionterveno, a reduction in the seed germination was observed and as a consequence, the plant density was reduced by 20% resulting in a lower fruit yield than that in 2021. According to Arampatzis et al. [
18], a high plant density can lead to the greatest seed yield. In another study, Shokrpour et al. [
22] assessed various ecotypes and recorded yields from 889 to 2416 kg ha
−1. The above results show that the selection of a productive variety is important in order to maximize crop yield.
The oil content was ranged from 22.1 to 25.1% in the two varieties. In other studies conducted in Italy and Greece, Martinelli et al. [
23] and Arampatzis et al. [
25] observed that oil content in fruits of several milk thistle genotypes had higher values that ranged from 26.7 to 31.7% and from 24.7 to 31.1%, respectively. Compared with other species, the oil content of milk thistle is similar to hemp (
Cannabis sativa L.) in which is ranging from 25.5 to 28.2% [
29], but less than that in sesame (
Sesamum indicum L.) and sunflower (
Helianthus annuus L.) seeds that oil content is ranging from 34.2 to 36.5% and from 37.9 to 51%, respectively [
30]. In 2021, the oil content in both varieties was higher compared with that in 2020, probably due to the wetter weather conditions that prevailed during April and May in 2020. Similarly, in Iran, water stress increased oil accumulation in the fruits of the plants [
10].
Silymarin content in the fruits of the two varieties ranged from 2.4 to 4.4%, and variety Spata had significantly higher silymarin content than Palaionterveno. This finding is in agreement with Arampatzis et al. [
25] as Spata had the highest silymarin content between thirty genotypes. In previous studies conducted in Greece, Italy, and India, the silymarin content in the fruits of several milk thistle genotypes ranged from 2.0 to 7.72% [
15,
23,
25]. Moreover, the accumulation of silymarin active components was different, and Spata had a high content of silybin B, while silybin A and B constituted 35–39% of silymarin in Spata and 12% in Palaionterveno. In 2021, the silymarin accumulation was higher by 4.38–8.18% compared with 2020, probably due to the dryer weather conditions that prevailed during the second growing season. However, it is well documented in other studies that water stress causes an increase in silymarin accumulation in the fruits [
10,
18].
4.2. Fertilization Regimes
The application of sheep manure significantly increased rosette diameter and above-ground biomass in both years compared with the untreated control, while plant height was affected only in the first growing season. Similarly, Saad-Allah et al. [
20], reported that the use of poultry manure increased plant height and dry biomass of milk thistle, while in rice (
Oryza sativa L.), the application of manure increased plant height and the effect was more obvious when the manure was combined with urea [
31]. However, the plant growth was greater when the calcium ammonium nitrate was applied compared to the use of sheep manure. The application of the sheep manure was not sufficient to fully meet plants nitrogen requirements due to the slow nitrogen mineralization from this organic fertilizer. This result can be explained since milk thistle is characterized by rapid growth in the period of mid-March to early May and as a consequence, the requirements are more intense during this period. Similarly, Popin et al. [
32] reported that the use of urea or manure increased the height of maize (
Zea mays L.) compared with control, and the effect of urea was more intense. It is also important to point out that the application of calcium ammonium nitrate at a high dose led to the maximum rosette diameter, aboveground biomass, and height of plants. Previously, an increase in nitrogen fertilizer dose increased plants height or biomass of different milk thistle genotypes [
17,
33]. Moreover, the use of sheep manure or calcium ammonium nitrate significantly influenced the number of fruits per inflorescence. Similarly, Afshar et al. [
16] observed that poultry manure increased this trait. In contrast, 1000-fruit weight was not affected by fertilization.
The use of sheep manure beneficially affected fruit yield. This finding is in agreement with previous experiments as Saad-Allah et al. [
20] observed that the application of chicken manure led to higher fruit yield compared to control, and an augmentation in the rate of the applied manure further increased the fruit yield. In general, the application of manure increased yield in other crops such as winter wheat (
Triticum aestivum L.), maize, rice, and the effect was more obvious when the manure was combined with chemical fertilizers [
31,
34,
35]. However, milk thistle plants show rapid growth and produce high aboveground biomass, and thus the nitrogen needs cannot be completely covered by sheep manure due to the slow nitrogen mineralization. As a result, the application of calcium ammonium nitrate led to a greater yield than manure. In a previous study, nitrogen fertilization significantly increased plants productivity, especially the application of a high dose [
17]. In 2021, there was an interaction effect on fruit yield between the two factors, since sheep manure influenced the fruit yield only in Palaionterveno. Similarly, in rice [
36] and bread wheat [
37], there was an interaction between genotype and nitrogen fertilization on grain yield.
Moreover, fertilization had no impact on oil content. Similarly, Afshar et al. [
16] observed that manure application had no impact on oil content in milk thistle fruits, while Li et al. [
38] reported that the application of nitrogen fertilizers did not affect the oil content in sunflower seeds. These results show that the oil content mainly depends on the genetic material. Moreover, oil yield ranged from 181.2 to 377.4 kg ha
−1 similar to previous studies that recorded 217.5–376.1 kg ha
−1 [
16] and 353–591 kg ha
−1 [
18] in different genotypes. However, sunflower shows greater oil productivity (669 to 1210 kg ha
−1) [
30] compared to milk thistle crop. In our study, the application of calcium ammonium nitrate led to higher oil yield compared with manure; consistent with the maximum oil yield in canola crop that was observed in the plots where the manure was applied in [
39]. Moreover, the calcium ammonium nitrate increased the oil yield of both varieties, while sheep manure affected this parameter only in the variety Palaionterveno. Similarly, in milk thistle [
16], canola (
Brassica napus L.) [
39], and hemp [
29] crops, the application of manure increased oil yield compared to control.
Silymarin content was not influenced by sheep manure and calcium ammonium nitrate application. In other studies, Afshar et al. [
16] observed that the use of manure did not affect the silymarin content, while Saad-Allah et al. [
20] mentioned that manure application led to a higher silymarin accumulation than that in control. Regarding the effects of fertilization on the content of silymarin constituents, our results indicated no significant differences between sheep manure and calcium ammonium nitrate, while Geneva et al. [
21] and Stancheva et al. [
28] reported that the soil and foliar fertilization (NPK) decreased the content of silybins A and B, silychristin, silydianin, and taxifolin. However, both varieties showed silymarin yield (27.1–50 kg ha
−1) comparable to previous studies that recorded 13.3–63.3 kg ha
−1 [
6,
18], while fertilization positively affected silymarin yield. Sheep manure induced an increase only in the first year of the experiment, while calcium ammonium nitrate, especially in a high dose, influenced silymarin yield of the two varieties in both years. These findings are in agreement with that of Geneva et al. [
21], Afshar et al. [
16], and Liava et al. [
17] in different genotypes, as they observed that the application of manure or inorganic fertilizers enhanced silymarin yield owing to higher fruit yield.