Performance and Stress Tolerance of Poppy (Papaver somniferum L.) in Response to Biostimulant Treatments

Abstract

The goal of the study was to identify the effects of fulvic acid (FULV) and methyl jasmonate (MEJA) in increasing the yield, alkaloid content and drought-resistance of poppy (Papaver somniferum L.). The trials were carried out in both field and controlled conditions; in the latter, with two water supply regimes (50 and 75% soil water capacity). The treatments were applied by exogenous foliar spraying. In the field experiment, we observed a 22.4% increase in yield (capsules with seeds) under of FULV and a 44.2% increase due to MEJA. The treatments could not intensify the concentration of alkaloids. Under controlled conditions, MEJA decreased total biomass but capsule yield was not lower, its proportion even increased. Antioxidant capacity (AC) and total phenolic content (TPC) increased (11 and 22%, respectively) together with proline concentration (by 134%) under dry conditions. In addition, biostimulant sprayings stimulated the AC (by 6.6% MEJA and by 11.5% FULV). FULV was effective also in graising the TPC (by 14.5%) and producing a 417% rise in the concentration of soluble sugars. Our results may contribute to the protection of poppy under drought as well as to a more detailed understanding of its stress responses.

1. Introduction

Poppy (Papaver somniferum L.) is a widely known and highly valued medicinal plant that has been cultivated and utilized since ancient times [1,2]. It is a dual-purpose crop, the seeds of which are consumed as food, while various medicines are prepared from its capsules [3]. The deseeded and mechanically crushed capsules—also called poppy straw -, are processed by the modern pharmaceutical industry because of the valuable alkaloids, among which the most important ones are the so-called benzylisoquinoline alkaloids: morphine, codeine and thebaine [4]. Salts of morphine and codeine are active ingredients in valuable analgesic and spasmolytic medicines, while thebaine is primarily used as a basis for semi-synthetic opioid pharmaceuticals [5]. Due to the risk of narcotic abuse, poppy cultivation is internationally regulated by the International Narcotics Control Board (Vienna, Austria) [6]. In addition, poppy is cultivated as a food crop in numerous countries, where manufacturers aim to produce the nutrient-rich poppy seed and/or the delicious edible oil extracted from the seeds [7].

In recent years, one of the biggest obstacles to crop production has been increasing drought in many poppy-growing regions, including Hungary and Slovakia [8,9,10]. When the water supply is inadequate, germination and emergence are critical. If a severe drought occurs during the growth phase, the root system of the poppy plant develops poorly. Only a small leaf rosette is formed, the stem becomes weak, and as a result, the development of the generative organs is also insufficient [11,12]. In the agricultural practice, farmers can mitigate the negative effects of drought on poppy plants using several methods, including irrigation, primarily through irrigation, but this is not possible everywhere [13]. Therefore, the possibilities of agrotechnical adaptation to critical weather factors in traditional poppy-growing regions are an active area of study [14].

Abiotic stress factors are responsible for the largest proportion of yield reductions not only in poppy but also in crop production worldwide [15]. Drought is a common, multidimensional stress factor, the primary cause of which is the lack of precipitation, which is most often combined with high temperatures [16]. As a result of the stress effect, the accumulation of reactive oxygen species (ROS) results in harmful oxidative processes [17,18]. In consequence, the stability of the cell membrane and photosynthetic activity decreases, protein synthesis slows down, and aging processes accelerate [19,20]. In this situation, several protective plant responses are known, such as activation of the antioxidant enzyme system or accumulation of osmoprotective molecules, most often proline or monosaccharides [21,22].

Nowadays, intensive research is being conducted on expanding the natural resistance of plants. Lately, biostimulants have become popular. Biostimulants can be described as products that improve a plant’s nutrient uptake and utilization, abiotic stress tolerance, or crop quality regardless of nutrient content [23]. In the cultivation practice, products containing humic substances, plant hormones, or amino acids are common [24]. Several studies have already reported on the beneficial effects of different biostimulants, especially in relation to drought and salt stresses. However, further studies are needed to expand knowledge of these substances and to better understand how they behave [25].

Popular ingredients of biostimulants are humic substances, including fulvic acid (FULV) [26]. Fulvic acid is characterized by its stability even under strongly acidic conditions. It has good solubility in water, is high in phenolic compounds and carboxylic acid groups, and contains high cation exchange capacity [27]. The literature has no reliable data on the effect of fulvic acid on poppy plants, although it has been used successfully in many species. Among others, Anjum et al. [28] observed the yield-increasing effect of fulvic acid in maize both under dry conditions and with abundant water supply. Bayat et al. [29] treated yarrow with different dosages of FULV. They observed an improvement in growth parameters and total phenolic and flavonoid contents, as well as in the antioxidant activity of yarrow after treatment with different dosages of FULV. The stress-relieving effect of FULV under heavy metal stress was also demonstrated by Wang et al. [30] on lettuce plants.

Methyl jasmonate (MEJA) is a plant hormone derived from jasmonic acid, which plays an active role in plant stress responses. MEJA is able to activate the antioxidant system and initiate the synthesis of defense compounds. Although its use in practical agriculture is not yet widespread, the application has been studied intensively for many crops. In the experiment of Ahmadi et al. [31], MEJA was able to reduce the harmful effects of salt stress in Brassica napus, by increasing the sugar accumulation of the treated plants. In the case of some medicinal plants, MEJA treatment increases the concentrations of active ingredients (essential oil, polyphenols) [32]. In the root system of a related species of poppy, Papaver bracteatum, Esfahnani et al. [33] observed an increase in thebaine content after MEJA treatment. Unlike poppy, the main alkaloid of P. bracteatum can be said to be thebaine. That is because P. bracteatum accumulates the largest amount of thebaine, primarily in its roots.

In view of the above-mentioned knowledge gaps, we investigated the effect of exogenous FULV and MEJA treatments on Papaver somniferum plants in two stages. Our aim was to determine whether these natural compounds are able to increase the resistance of poppy plants to drought stress in vivo. In open field conditions, we explored the effect of the treatments on yield, proportion of plant organs, and accumulation of active ingredients—parameters that determine a prosperous cultivation of industrial poppy. As a second stage of the experiment, the trials were continued under controlled conditions. In addition to observations on growth parameters, some biochemical marker compounds were also measured in order to detect the background of the plant reactions. Furthermore, our goal was to provide a scientifically viable recommendation for the practical use of these compounds as biostimulants for poppy.

2. Materials and Methods

2.1. Open Field Experiment

The open field experiment was carried out in Borsod-Abaúj-Zemplén County, Hungary, on a large-scale poppy production field. The soil was a meadow soil with medium consistency, 3.81% organic matter content, with a pH (KCl) value of 6.1. It was well-supplied with NPK ((NO₂₋ + NO₃₋)-N: 14.2 mg/kg, P₂O₅: 417 mg/kg, K₂O: 634 mg/kg) and microelements. During the vegetation period, the average daily temperature was 14.18 °C, the total amount of precipitation was 238 mm, and no irrigation was applied. We used the variety ‘Meara’, which is characterized by a short growing season, white flower color with purple petal spots, high yield, and alkaloid content. The variety has been widely used in Hungarian production in recent years; its main alkaloid is morphine, while it also accumulates codeine and thebaine in smaller proportions. Agrotechnology for the experiment was consistent with methods typical of the region.

After autumn plowing, in March, we prepared the fine-crumb seedbed with a combination cultivator. During seedbed preparation, 300 kg/ha of 8-16-24 NPK fertilizer is applied to the soil. Sowing was performed on 3 March 2023 as follows: 2 kg/ha to a row spacing of 30 cm and depth of 1.5 cm. For each treatment, 5 m² plots were formed in three replications, in a randomized block design. To keep the experimental area clean, herbicides containing mesotrione (SC formulation, 144 g/ha a.i.), tembotrione (OD formulation, 88 g/ha a.i.), and fluazyfop-P-butyl (EC formulation, 150 g/ha a.i.) were applied in the leaf-rosette phenophase. No other plant protection procedures were necessary. At the end of the rosette-phenophase, 200 kg/ha of lime ammonium nitrate was applied as fertilizer to provide nitrogen.

The first treatment was applied on 22 May at the initial stage of stem formation, while the second was carried out immediately after petal abscission, on June 14. For the treatments, we used MEJA (Sigma Aldrich/Merck, Darmstadt, Germany) in 4.2 mM (pre-dissolved in ethanol) and 4.5 g/L of FULV (Adler Agro SL, Albuixech, Spain). These quantities were determined from our prior experiments [34]. In both cases, the solvent was water mixed with an adhesion-enhancing additive (0.25 mL/L 60% dioctyl sulfosuccinate Na salt). These solutions were sprayed onto the leaves of the plants at a rate of 150 mL per plot (=300 L/ha) during both application times. Untreated control plants were designated with the code KONT. We sprayed these plants with the same amount of solution, which did not contain a biostimulant but did contain the adhesion enhancer.

The plots were harvested by hand when capsules were fully ripe, on 15 July. The yields were determined for each square meter within the plots in order to obtain the most reliable results. Thus, we worked with 15 replications (5 m × 1 m × 3 plot replicates) per treatment in terms of yield data. The fruits of the poppy were broken open, and the capsule and seed fractions were measured separately. From the capsule fraction for each plot, bulk samples were obtained and ground to powder (<0.2 mm) to obtain the laboratory samples necessary for alkaloid determination. The quantitative and qualitative analysis of the main alkaloids—morphine, thebaine, and codeine—was performed using the HPLC method, which we described in detail in our previous work [35]. In the remainder of this paper, we refer to the sum of these three main alkaloids as the total alkaloid content.

2.2. Phytotron Experiment

The second phase of the experiment was performed in climatic chambers to ensure controlled conditions for a more detailed study. Two phytotrons (Fitotron SGC120, Weiss Gallenkampf Ltd., Loughborough, Leicestershire, UK) were used to simulate a drought stress condition (hereafter drought chamber) in parallel with a well-watered environment (hereafter control chamber). Identical models of the two chambers were run in the same room, thus eliminating possible chamber effects. The growth medium was a commercially available soil mixture (Florasca Bio „B”, Florasca Kft., Osli, Hungary) consisting of 10% sand, 65% peat, and 25% cattle manure compost. All pots had an equal weight of 950 g DW. Soil water capacity (SWC) was determined using the gravimetric method [36]. Irrigation was carried out 3 times per week by measuring pot weight and filling the pots to a standard weight. During the germination and rosette formation phenophases, we applied a uniformly good water supply (75% SWC) in both chambers. From day 76 onwards, the water supply differed between the two chambers. The weight of the drought stress treatment represented 50% saturation of SWC, and for the control, it was a weight representing 75% SWC. The environmental conditions during the plant development are given in

Table 1. Plant phenophases and the corresponding environmental circumstances. Except for the water supply, the environmental parameters were similar in both chambers.

Time (Day)	Phenophase	Temperature Day/Night, (°C)	Light Cycle Day/Night (Hour)	SWC Saturation (%)		Relative Humidity (%)	Light Intensity (lux)
				Control	Drought
16 (day 1–16)	germination	13/8	10/14	75		60 ± 2	16,000
59 (day 17–75)	rosette-formation	20/12	12/12	75		60 ± 2	16,000
27 (day 76–102)	stem growth	22/12	14/10	75	50	50 ± 2	16,000
7 (day 103–109)	flowering	26/15	14/10	75	50	50 ± 2	16,000
14 (day 110–124)	capsule growth	26/15	14/10	75	50	50 ± 2	16,000
12 (day 125–136)	maturation	26/15	14/10	75	50	50 ± 2	16,000

.

The seeds of the previously mentioned variety ‘Meara’ were sown on 20 September 2023 at a depth of 1 cm in the pots. We prepared 6 pots of each treatment-water regime combination, and allowed 3 plants to develop in each pot. The plants emerged in 16 days. Fungal diseases appearing during the germination period were eliminated by irrigation with fosetyl and propamocarb fungicides. The nutrient supply of the plants came from general horticultural fertilizers, with particular attention to the supply of boron and calcium.

The first treatment was applied on day 75, when stem initiation began, while the second treatment was applied on day 110, immediately after flowering. The concentration of the applied biostimulants was the same as that used in the field experiment, while the dosage was 1 mL solution per plant. On day 124, representative bulk samples were prepared from the upper leaves of the plants, which were still completely green. The antioxidant capacity, total polyphenol content, proline content, and antioxidant enzyme activity of the samples were measured in 5 replicates from the fresh biomass as described below. The sugar content was measured from dried material, also in 5 replicates. Drying was by means of a drying cabinet at 70 °C. On day 136, the experimental poppy plants were harvested in the early stage of ripening. At this time, we measured the height of each plant, assessed the degree of hairiness on the stem, and determined the characteristic leaf position (stem-enclosing or non-stem-enclosing) for each individual. The hairiness was assessed visually on a scale from 1 (least hairy) to 5 (most hairy) by observing the stem sector between the uppermost leaf and the fruit. After that, the plants were removed from the soil and cleaned. Separate measurements were taken for (1) the fresh weights of the roots, (2) the vegetative above-ground organs (stem + leaves), and (3) the fruits themselves. The samples were dried to a fixed weight and re-measured in order to determine the dry weight. The difference between the two values revealed the moisture content of the samples.

2.3. Analytical Testing Methods

The activity of catalase and ascorbate peroxidase enzymes in the collected leaf samples was measured according to the method of Ádám et al. [37]. Activity of glutathione reductase was measured according to the method of Smith et al. [38], and the activity of the glutathione-S-transferase was determined according to the method of Mannervik and Guthenberg [39]. The total phenolic content (TPC) was determined by the modified method of Singleton and Rossi [40]. The ferric reducing antioxidant power (FRAP) assay was performed according to the procedure of Benzie and Strain [41] with slight modifications. The sugar content of the leaf samples was determined by the method of Molnár et al. [42]. The proline content of the samples was measured according to the method of Bates et al. [43].

2.4. Statistical Methods

For the field experiment statistical evaluation, we performed a one-way ANOVA. For the phytotron experiment, we used a two-way ANOVA to assess the effects of biostimulant treatments and water supply, as well as the effects of their interaction. The results were considered significant at p < 0.05. In the case of significant interaction, the effect of the treatment was evaluated separately on the two water supply levels. If the ANOVA result was significant, we used the Tukey HSD post hoc test. The normalization of the data was checked with the Shapiro–Wilk test, and the homogeneity of variance was established with the Levene test.

3. Results

3.1. Yield and Active Ingredient Accumulation in the Field Experiment

During our field experiment, the production of poppy plants increased significantly as a result of the treatments. When treated with MEJA, the total yield (capsules with seeds, DW) was 188.50 g/m² as a mean, while it was 160.08 g/m² with the FULV treatment. Both values exceeded the yield of the control plots (130.75 g/m²) significantly (p < 0.001). The yields of both capsule and seed followed this tendency. The capsule yield on the treated plots (both MEJA and FULV) gave significantly (p < 0.001) higher values (75.08–79.20 g/m²) than the yield of the control plots (57.50 g/m²). The seed yield of the control was 73.25 g/m², which was significantly lower than both FULV and MEJA (85.00–109.30 g/m²) treatments, and in this case, these treatments were also statistically different from each other (p < 0.001). The ratio of the yield components also changed. The total yield of the control and that of the MEJA-treated plants was 43.98 and 42.02% capsule, with 56.02 and 57.98% seeds, respectively. In contrast, the FULV treatment resulted in significantly higher (p < 0.001) capsule (46.90%) and lower seed (53.10%) mass ratios compared to the control. A summary of the yield data is provided in

Table 2. Yields and characteristics of yield components measured during the field experiment (mean ± SE). Different lowercase letters indicate significant differences between treatments in each row (p < 0.001).

	KONT	FULV	MEJA
Seed yield (g/m2)	73.25 ± 2.26 a	85.00 ± 2.24 b	109.3 ± 4.53 c
Capsule yield (g/m2)	57.50 ± 2.07 a	75.08 ± 2.27 b	79.20 ± 2.77 b
Total yield (g/m2)	130.75 ± 4.03 a	160.08 ± 3.97 b	188.50 ± 6.96 c
Seed ratio (% of total mass)	56.02 ± 0.01 b	53.10 ± 0.01 a	57.98 ± 0.01 b
Capsule ratio (% of total mass)	43.98 ± 0.01 a	46.90 ± 0.01 b	42.02 0.01 a
Morphine yield (g/m2)	1.15 ± 0.05 a	1.31 ± 0.04 a	1.67 ± 0.09 b
Total alkaloid yield (g/m2)	1.25 ± 0.05 a	1.39 ± 0.04 a	1.79 ± 0.10 b

The morphine and total alkaloid yields were calculated as the product of the capsule yield (Table 2) and the respective alkaloid content (Figure 1) data.

.

Changes were also observed in the accumulation level and composition of alkaloids. In the control samples, the concentration of the major alkaloid morphine was 2.003%, while the minor alkaloids, codeine and thebaine, were present in lower concentrations (0.157 and 0.023%, respectively), resulting in a total alkaloid level of 2.182%. In the MEJA treatment samples, codeine content was 0.132%, significantly lower than the control (p < 0.001). However, the morphine (2.090%), thebaine (0.018%), and total alkaloid concentrations (2.240%) were similar to the control. As a result of the higher capsule yield and unchanged alkaloid concentrations, the alkaloid yield, calculated as the product of these two characteristics, was increased by MEJA treatment. In contrast, FULV treatment resulted in a significantly lower (p < 0.001) accumulation of all alkaloids (Figure 1), and because of that, in spite of higher capsule yields, it could not ensure a significantly higher yield of alkaloids compared to the control.

3.2. Morpho-Phenological Characteristics of Poppy Plants in the Phytotron Experiment

The height of the plants was significantly influenced by each of the treatments (p < 0.005), the water supply, and their interaction (p < 0.001). The drought reduced the plant height by 31.5% compared to the plants in the control chamber (

Table 3. Morphological parameters of plants under different biostimulant treatments and water supply (mean ± SE). The lowercase letters indicate significant (p < 0.005) differences among biostimulant treatments at the two water supply levels. The uppercase letters indicate significant (p < 0.001) differences between the means of two water supply levels.

Water Supply	Control Chamber				Drought Chamber
Treatment	KONT	FULV	MEJA	Mean	KONT	FULV	MEJA	Mean
Plant height (cm)	80.50 ± 2.73 b	82.18 ± 1.27 b	69.88 ± 2.69 a	77.52 ± 2.20 B	51.57 ± 0.94 a	53.75 ± 1.23 a	53.94 ± 0.92 a	53.09 ± 1.02 A
Root length (cm)	20.13 ± 1.15 a	19.95 ± 0.66 a	19.47 ± 0.82 a	19.85 ± 0.90 A	18.50 ± 1.11 a	23.06 ± 2.30 a	22.16 ± 1.42 a	21.24 ± 1.60 A
Position of stem leaves	non stem-enclosing				stem-enclosing

). Examining the effect of biostimulant treatments at the two water supply levels separately, no difference was found in the control chamber between the non-treated control (KONT) and FULV-treated plants (80.50–82.18 cm). On the other hand, the plant height decreased by 13.2–15.0% as a result of the MEJA compared to the well-watered KONT and FULV plants. In the drought chamber, the height was between 51.57 and 53.94 cm, not influenced by any of the treatments. We did not observe significant differences in root length due to either water supply (p = 0.220) or biostimulant treatments (p = 0.294).

Morphological observations showed that in the drought chamber, the flower stalks only rose slightly above the leaves (Figure A1) and the leaves adopted a characteristic stem-enclosing position. In the control chamber, the stem between the upper leaves and the flowers was elongated, and the leaf form was not stem-enclosing.

The hairiness of the plants was significantly influenced by both the water supply (p < 0.005) and the treatments (p < 0.001), with no interaction effect between these two factors (p = 0.396). The mean hairiness of the plants in the drought chamber (4.17) exceeded the value in the normal chamber by 25% (3.33). As for the treatments, the hairiness in FULV-treated plants reached 4.54 as a mean, while that of MEJA reached 4.09, and thus, both exceeded the value of the control (2.63) significantly (<0.001).

3.3. Development and Plant Mass in the Phytotron Experiment

Based on the results of two-way ANOVA, water supply had a strongly significant effect (p < 0.001) on all components of plant mass. On the other hand, the biostimulant treatments had significant effects on the following yield components: fresh and dry masses of the vegetative above-ground parts (p < 0.05), the total fresh mass of the plant, and the dry matter content of the capsule (p < 0.005). The interaction between the two factors was not significant for any of the plant mass indicators.

The total plant fresh weight under well-irrigated (control) conditions was 40.19 g/plant on average, while it was 27.52 g/plant under drought stress (Figure 2). Both the fresh and dry masses of all organs were significantly different due to the different water supply. However, their ratios remained practically unchanged. The proportion of stems and leaves within the green mass was 85% and almost the same, 84%, under well-irrigated and drought conditions, respectively. Similarly, the proportion of roots was 8% in the fresh mass and 6% in the dry mass under both water supply regimes. The proportion of capsule masses differed only in dry mass. There was a proportion of 8% fresh mass in both chambers, but the ratio of capsules in the total mass was more than double in the drought chamber if we calculate it from the dry mass (p < 0.005). These differences are related to the dry matter content of the plants: under control conditions, it was 43% at harvest, while under drought stress, it was 50%.

Examining the effect of the treatments on the mean of the two water supply levels, it can be established that the treatments did not change either the fresh (2.29–2.60 g/plant) or dry mass (0.56–0.62 g/plant) of the roots. However, they significantly influenced the fresh (p < 0.001) and dry (p < 0.05) masses of the above-ground vegetative organs: stems and leaves. In the MEJA treatment, their fresh weight was 19% lower (24.18 g/plant) than in the FULV treatment (29.65 g/plant) and 26% lower than the KONT (32.69 g/plant). The dry weight of the stems and leaves was also the lowest in the MEJA group: 6.99 g/plant, compared to the value of the FULV plants, which was significant (p < 0.05), about 13% higher (8.35 g/plant). The value of KONT plants was 8.00 g/plant, which is not statistically different from any of the treated groups (Figure 3).

The total fresh weight followed the trend of stem and leaf: KONT and FULV plants significantly exceeded the MEJA treatment (p < 0.005). However, this difference disappears when examining the total dry weight, and the values of the groups (8.89–10.30 g/plant) become statistically equal. This difference is due to the different dry matter content of the plants. Data-based calculations show that the dry matter content of the total plant mass is 25.3% in the case of KONT plants, while that of the treated groups is higher, 29.5–30.5%. In the case of the capsules, this tendency is even more obvious: the fresh mass of the capsules was statistically different only at a lower probability level (p = 0.080) in the three groups (2.71–2.74 g/plant); however, the treatments had a significant effect (p < 0.005) on the dry matter content of the capsules. It was 39.1% in the KONT, 48.9% in the FULV treatment, and 48.3% in the case of MEJA capsules. Calculated from these, the treatments, especially MEJA, elevated the proportion of capsules in the total biomass (28% higher than KONT) and also in the dry, harvested plant mass (36% higher than KONT). It was also observed that the treatments resulted in a higher proportion of healthy capsules in the treated plants even under dry conditions, while in the case of the control, abnormal capsules also occurred due to stress (Figure A2).

3.4. Stress Markers and Antioxidant Enzymes in Phytotron

The accumulation of soluble sugars in the leaf samples was significantly (p < 0.001) influenced by water supply, biostimulants, and the interaction of these two factors. In the well-watered (control) chamber, there was no detectable difference in the sugar content of samples originating from the two biostimulant treatments. The accumulation of glucose was between 6.24 and 6.84 mg/g DW (p = 0.884), and that of the fructose between 7.04 and 9.10 mg/g DW (p = 0.184), so the total amount of these soluble sugars lay between 13.48 and 15.34 mg/g DW (p = 0.675) (Figure 4). Under dry conditions, however, the leaf samples of MEJA and FULV treatments resulted in significantly higher (p < 0.001) concentrations of the osmotically active monosaccharides. The control plants contained 4.54 mg/g DW glucose and 6.84 mg/DW fructose. The MEJA treatment elevated the glucose content by 115% and the fructose content by 90% while the FULV treatment showed extremely high values, an almost 6.5 fold increase for glucose and a 4.3 fold one for fructose (Figure 4).

The AC and TPC measured in leaves were significantly (p < 0.001) affected by the two treatment factors and their interaction (

Table 4. Characterization of different stress marker molecules in the leaf samples of poppy in consequence of the biostimulant treatments under two irrigation regimes (mean ± SE). The lowercase letters indicate significant (* p < 0.05, *** p < 0.001) differences among the treatments at the two different water supply levels. The uppercase letters indicate significant differences between the means of two chambers.

Water Supply	Control Chamber				Drought Chamber
Treatment	KONT	FULV	MEJA	Mean	KONT	FULV	MEJA	Mean
AC (mgAAE/mL) ***	0.1585 ± 0.0010 a	0.1777 ± 0.0013 b	0.1725 ± 0.0027 b	0.1696 ± 0.0022 A	0.1776 ± 0.0011 a	0.1849 ± 0.0022 b	0.1981 ± 0.0015 c	0.1884 ± 0.0022 B
TPC (mgGAE/mL) ***	0.1420 ± 0.0090 a	0.1900 ± 0.0038 c	0.1728 ± 0.0069 b	0.1683 ± 0.0060 A	0.2009 ± 0.0011 b	0.2300 ± 0.0033 c	0.1836 ± 0.0021 a	0.2048 ± 0.0049 B
proline (nmol/gFW)	92.41 ± 6.40 a	89.05 ± 13.85 a	78.10 ± 3.52 a	86.52 ± 4.78 A	169.02 ± 28.96 a	282.89 ± 87.17 a	155.63 ± 18.03 a	202.51 ± 30.89 B
GR (nkatal/gFW) ***	16.93 ± 1.31 a	12.87 ± 0.20 a	12.48 ± 0.88 a	14.09 ± 1.11 A	17.11 ± 1.96 b	17.13 ± 2.11 b	9.02 ± 1.24 a	14.42 ± 1.20 A
GST (nkatal/gFW)	3.31 ± 0.49 a	3.28 ± 0.28 a	3.12 ± 0.31 a	3.24 ± 0.18 B	2.94 ± 0.34 a	2.39 ± 0.39 a	2.08 ± 0.22 a	2.47 ± 0.22 A
APX (nkatal/gFW) *	45.43 ± 2.73 a	54.04 ± 3.51 a	44.73 ± 0.77 a	48.07 ± 1.93 A	45.74 ± 1.67 ab	51.48 ± 3.57 b	36.80 ± 3.91 a	44.67 ± 2.47 A
CAT (nkatal/gFW)	1296 ± 585 a	1158 ± 629 a	1452 ± 454 a	1302 ± 302 A	1076 ± 483 a	1479 ± 281 a	2085 ± 443 a	1547 ± 456 A
GPX (nkatal/gFW)	209.81 ± 93.13 a	93.47 ± 37.9 a	183.14 ± 95.47 a	162.14 ± 44.81 B	20.91 ± 8.06 a	30.17 ± 3.71 a	25.47 ± 0.80 a	25.51 ± 3.08 A

AC: antioxidant capacity, AAE: ascorbic acid equivalent, TPC: total polyphenol content, GAE: gallic acid equivalent, GR: glutathione reductase, GST: glutathione S-transferase, APX: ascorbate peroxidase, CAT: catalase, GPX: guaiacol peroxidase. Significance level: * p < 0.05, *** p < 0.001.

). The mean concentrations of AC and TPC determined in leaf samples were higher by 11.1% (AC) and 21.7% (TPC) in the drought chamber, compared to well-watered conditions. These values could also have been elevated by both biostimulant treatments. Under well-watered conditions, the AC measured in control plants was 0.1585 mg AAE/mL, which increased to 0.1777 under FULV treatment and to 0.1725 mg AAE/mL under MEJA treatment. Similar results were observed under dry conditions: the value of the control samples was 0.1776 mg AAE/mL. However, we observed activities that were higher by 11 and 12% for the FULV and MEJA treatments. The TPC of the leaf samples in the control plants from the control chamber was 0.1420 mg GAE/mL, which was exceeded by 22 and 34% as a result of the MEJA and FULV treatments, in that order (Table 4). A different trend was observed under the influence of drought: the TPC in the MEJA-treated samples (0.184 mg GAE/mL) was significantly lower (p < 0.001) than the value of the controls (0.201 mg GAE/mL), while the FULV treatment increased it (0.230 mg GAE/mL). Only the water supply was significantly affected by this (p < 0.005): the drought treatment resulted in 134% higher values as a mean, compared to the control chamber.

The activities of the examined stress enzymes showed variable results (Table 4). The activities of GST and GPX were determined by the water supply (p < 0.05), each showing higher activities under well-watered conditions by 31 and 535%, respectively. Otherwise, the activities of GR and APX were influenced by the biostimulant treatments (p < 0.001), but only under the drought stress condition. In the drought chamber, MEJA decreased activities, although it was only significant for GR. FULV treatment did not cause a significant change in either activity compared to the non-treated control. The CAT enzyme did not react to either of the studied factors (water supply and biostimulants). Their interaction did not have a significant effect on any of the analyzed enzymes, either.

4. Discussion

The alkaloid yield of poppy is primarily determined by the dry matter production, especially that of the capsules, and by the concentration of the alkaloids. On the other hand, water supply affects alkaloid yield mainly by influencing dry matter production and the ratio of plant organs [11]. In our field experiment, exogenous foliar treatments with the biostimulants increased the yield of capsules per unit area by 30.5 and 37.7% and the seed yield by 16.0 and 48.8% due to FULV and MEJA, respectively. Our results are consistent with several studies, like Sheteawi [44], that observed a similar yield increase in soybean plants treated with jasmonic acid under salt stress conditions. Fulvic acid treatment increased resistance and yield in corn [28] and in lettuce [30]. Nevertheless, according to our knowledge, our data are the first ones for poppy.

In our field experiment, we demonstrated an 18% decrease in the accumulation of alkaloids in the FULV-treated plants, although the ratio of morphine increased as well. Due to the above-mentioned increased capsule yield, however, this concentration decrease did not significantly influence the total yield of alkaloids. In fact, we measured a 14% increase in the morphine yield and an 11% increase in the total alkaloid yield per unit area. The potential of FULV and other humic acid compounds in enhancing the accumulation of several secondary compounds in numerous plant species has been demonstrated [45], but results on alkaloids are sporadic [46,47]. In this study, MEJA did not influence the accumulation level of alkaloids. However, due to the significant increase in capsule production, the alkaloid yield also rose significantly in the case of this treatment. We determined a 45% rise in morphine and 43% in the total alkaloid yield with MEJA spraying. Inducing MEJA increased production of secondary metabolites in numerous plant species [32,48] as well as even stimulating alkaloid concentration in vitro [49], but data on elicitation of alkaloid biosynthesis in vivo are limited. Esfahnani et al. [33] observed increased root yield and thebaine concentrations in a related species, Papaver bracteatum, under MEJA treatment.

In the controlled environment under well-irrigated (control) conditions, there was no significant difference in capsule weight between the untreated control and treated plants; capsule development was undisturbed. However, under a poor water supply, the non-treated plants could not develop main capsules on the primary branches. Instead, they tried to develop secondary branches. However, the secondary capsules remained small and their development and maturation were delayed (Figure A2). In contrast, both the FULV and MEJA treatments were able to stimulate the plants to develop the primary capsules normally, a process that most likely played a fundamental role in increasing capsule production of the treated individuals.

As expected, under dry conditions, plant height and biomass decreased compared to the better water supply. But unexpectedly, these parameters were further reduced by the application of FULV and MEJA. However, with the decreased fresh mass, the dry masses did not show differences between the two chambers. Therefore, we conclude that the loss of the biomass that we mentioned may be the result of lower water content of the tissues. These data suggest that the treatments -especially MEJA- stimulated the plant to mature and complete its life cycle faster, which is even more characteristic under unfavorable conditions, such as the shortage of water here. This can be beneficial in the cultivation practice. Another advantage is that both treatments demonstrated a tendency to increase the yield of the most important plant organ, the capsule, and its ratio in the total biomass. It suggests resource reallocation, which is worth investigating further under field conditions.

In the phytotron experiment, we could establish that the measured stress marker molecules reacted differently and specifically to both the water supply and biostimulant treatments. Shortage of water decreased the activities of GST and GPX enzymes while increasing the AC, TPC, and proline content. Biostimulants also had significant effects on the AC and TPC, along with the soluble sugar concentration and the GR and APX enzyme activities. Among them, the sugar concentration and the activity of GR and APX were influenced by the sprayings only under drought conditions.

Presumably as a result of these beneficial effects, plant growth and capsule formation were less damaged by drought, and the ripening process could proceed normally. Compared to FULV, which elevated both AC and TPC, MEJA increased AC; however, surprisingly, it decreased TPC. This confirms that MEJA may influence the stress responses of poppy to drought by various mechanisms, as in the case of many other plant species [50]. Besides the defensive role of MEJA, the growth inhibitory and morphological regulatory function might contribute greatly to its complex effect [32,51]. In our study, the MEJA treatment altered the mass proportions of plant organs and thus enhanced the formation of capsules.

The largest modification was determined in the sugar content, especially after FULV treatment, which was able to increase fivefold the concentration of the osmoprotectant monosaccharides in the leaves. The findings show that the accumulation level of soluble sugars may play an important role in the stress tolerance of poppy. Compared to that, the proline content could not be influenced by the applied concentration of the biostimulants, although its level is significantly elevated under drought conditions. Changes caused by drought stress in the studied biochemical markers, such as proline, osmolites, and phenolics, have hardly been studied before in poppy. According to Jászberényi et al. [52], frost tolerance of poppy accessions is associated with an increase in the concentrations of proline and monosaccharides. By accumulating osmoprotectants, the plant is able to defend itself against frost, which is physiologically in close connection to drought stress. As for other species, Ahmadi et al. [31] reported an increase in sugar content in Brassica napus plants treated with MEJA under salt stress conditions. Also under salt stress conditions, Chen et al. [53] found increased contents in proline, glycine betaine, and soluble sugars in MEJA-treated Jatropha curcas plants. Zhang et al. [54] observed an increase in soluble sugars and proline levels in Poncirus trifoliata following a treatment with potassium fulvic acid under salt stress conditions. As a result, the plants avoided scorching of leaves and increased the chlorophyll and carotenoid contents of the seedlings. Similarly, He et al. [55] observed a significant increase in sugar content in lemons due to fulvic acid treatments. Also, TPC has been elevated after elicitation with jasmonates in different horticultural species; however, the effective dosages depend very much on the circumstances [32].

In the case of numerous species, FULV and MEJA were able to significantly influence the activity of antioxidant enzymes. Among others, increased activity of the antioxidant enzymes APX, SOD, and CAT was observed in rice and soybean, but other enzymes like GPX remained unchanged in the same species [56,57]. Under salt-stressed conditions, Zhang et al. [54] experienced an increase in activities of SOD, POD, and CAT enzymes due to treatment with fulvic acid. Considering the novelty of these substances, the dose, concentration, and number of treatments are difficult to determine. In the literature, the application parameters of FULV and MEJA vary widely. However, it can be generally said that in most cases they are applied in slightly lower doses than what we investigated and in very diverse numbers of repetitions (1–4).

In contrast to these results, the activities of the studied five antioxidant enzymes were only affected by our treatments in a few cases, and no clear tendencies could be established. Only the decreased activity of GR due to MEJA treatment may serve as an exception. Glutathione reductase (GR) is responsible for maintaining the glutathion pool in the cell and regulating the antioxidant status. In addition, it has been proven that GR has an important role in plant development [58]; therefore, the interaction of jasmonates and glutathion might be a very complex one and a target of further studies, especially in poppy.

5. Conclusions

We demonstrated that exogenous application of FULV and MEJA is able to increase capsule and seed yields of poppy. Under poor water supply, these biostimulants promote normal capsule formation. Regarding industrial poppy cultivation, FULV may decrease the concentration of alkaloids; however, in our trial, the increase in biomass could compensate for the yield of alkaloids per unit area.

In the phytotron experiment, we established that the protective role of the treatments under drought conditions is primarily manifested by elevating the concentration of soluble sugars (glucose and fructose) and stimulation of the antioxidant capacity. In these responses, FULV is especially effective. On the other hand, it seems that the antioxidant enzymes (APX, CAT, GPX, GR, GST) have a less significant role in drought stress tolerance of poppy under our treatments and conditions. The role of the non-enzymatic antioxidants is reflected in the elevated concentration of the total phenolic content. Also, the role of proline may be important, but it could not be influenced by the applied concentrations of either FULV or MEJA. However, as a morphological marker, a significant increase in the hairiness of the shoots was also evident under both treatments. This could contribute to the defense against access transmission.

6. Patents

This section is not mandatory, but may be added if there are patents resulting from the work reported in this manuscript.