Effect of Biostimulants on the Yield and Quality of Selected Herbs

: The aim of this study was to determine the effect of amino acid biopreparations on the yield of summer savory, marjoram, and lemon balm, and the concentrations of selected biochemical and mineral compounds in their herbage. The ﬁrst experimental factor was plant species: summer savory ( Satureja hortensis L.) var. Saturn , marjoram ( Origanum majorana L.) var. Mira˙z , and lemon balm ( Melissa ofﬁcinalis L.). The second experimental factor was the effect exerted by two biopreparations, Calleaf Aminovital and Maximus Amino Protect, on herbage yield and quality. In the control treatment, plants were sprayed with water. The analyzed herb species differed considerably in yield and the concentrations of selected biochemical compounds and minerals. Lemon balm was characterized by the highest yield (1.73 kg m − 2 ). Marjoram var. Mira˙z was characterized by the highest concentrations of reducing sugars (0.89 g 100 g − 1 FM) and L-ascorbic acid (39.7 mg 100 g − 1 FM). Summer savory was most abundant in total N, K, and Ca. The tested biostimulants contributed to a decrease in nitrate concentrations in the studied plants. The interaction between the experimental factors signiﬁcantly affected the content of nitrates and mineral compounds and total N, P, K, and Ca in the herbage of the analyzed plant species.


Introduction
The improvement of human health is a priority for many individuals dealing with food, including herb, vegetable, and fruit growers. Mineral elements play an important role in healthy growth and development, help maintain fitness, and prevent infections [1]. Among crops, herbs are valuable sources of readily absorbable mineral substances [2,3]. Herbs have gained popularity among consumers due to their medicinal properties and unique aroma [4]. As a source of biologically active substances, bioavailable micronutrients, and macronutrients, herbs can also be used in the prevention and treatment of many fungal diseases. The high bioavailability and optimal ratios of nutrients have contributed to the growing demand for plant raw materials [4][5][6][7][8]. The Polish market offers a wide range of herbs and spices. However, despite their medicinal properties, they are used mostly for culinary purposes, to enhance the taste, aroma, color, and appearance of dishes [9]. Summer savory (Satureja hortensis L.), marjoram (Origanum majorana L.), and lemon balm (Melissa officinalis L.) are well-known and highly valued species of the family Lamiaceae. Their herbage is used in both fresh and dried forms. In Poland, these herbs are used mostly to flavor foods and beverages [9]. The leaves of summer savory and marjoram have antiseptic, anti-inflammatory, analgesic, antioxidant, and antimicrobial properties. Lemon balm also exerts calming and antidepressant effects [4,8,10,11].
The chemical composition and nutrient content of the herbage of medicinal plants are determined by genetic and environmental factors [6]. According to Gugała et al. [12], inte-Appl. Sci. 2022, 12, 1500 2 of 11 grated plant production technologies are insufficient to utilize the full biological potential of herb species due to the poor absorption of nutrients by leaves and difficult transport through plant tissues during periods of climatic or biotic stress. The increased consumption of organic herbs and spices in fresh form has contributed to the development of new cultivation methods. These new methods aim to optimize the growth and development of plants [11,[13][14][15][16]. Amino acid biostimulants can counteract adverse environmental effects. Amino acids are the building blocks of proteins, and their supply decreases energy expenditure for nitrogen (N) synthesis [17]. According to Trawczyński [18], Maciejewski et al. [9], and Sawicka et al. [19], biostimulants support biochemical, morphological, and physiological processes in plants. Biostimulants promote the vegetative growth of herbaceous plants. Amino acid biostimulants available on the market enhance enzyme activity in plants and improve their stress tolerance.
Therefore, the research hypothesis postulates that the tested biostimulants may have a positive effect on the morphological traits and yield of herbs. Biostimulants are expected to increase the content of selected compounds as well as macronutrients and micronutrients in herbal raw material. In view of the above, the objective of this study was to determine the effect of amino acid biopreparations on the morphological traits and yield of summer savory var. Saturn, marjoram var. Miraż, and lemon balm, and the concentrations of selected nutrients and chemical elements in their herbage.

Study Site and Experimental Factors
The experiment was conducted in a heated greenhouse equipped with movable flood tables, at the University of Warmia and Mazury in Olsztyn (20 • 29 E, 53 • 45 N; 125 m a.s.l.), between 28-30 April and 30 June 2017-2018. The experiment had a randomized block design with three replications. The first experimental factor was plant species: summer savory (Satureja hortensis L.) var. Saturn, marjoram (Origanum majorana L.) var. Miraż, and lemon balm (Melissa officinalis L.) (Seed Company W. Legutko, Nad Stawem, Poland). The herbs selected for the study are most widely cultivated in Poland. The second experimental factor was the effect exerted by two biopreparations, Calleaf Aminovital (Calfert ® , Warsaw, Poland), and Maximus Amino Protect (Ekoplon, Grabki Duże, Poland). The products were used in diluted form.
Calleaf Aminovital (according to the product label) is an organic foliar fertilizer in liquid form, with an amino acid-balanced composition, which contains large amounts of polypeptides and free amino acids. The product is obtained in the process of the hydrolysis of natural proteins.
Maximus Amino Protect (according to the product label) is a combination of free amino acids, peptides, and potassium phosphate, which has strong biostimulatory and antistress effects. Potassium phosphite stimulates the immune system of plants and prevents fungal diseases. Peptides improve the biological effectiveness of the fertilizer-they support the active uptake and penetration of micronutrients and plant protection products. It is practically advisable to use this fertilizer as an addition to treatments against fungi attacking plants (fungicide treatments), as it significantly improves the effectiveness of these treatments. In the control treatment, plants were sprayed with water.
In 2017-2018, in the last days of April, 10 seeds of each herb species were sown in 0.7 dm 3 pots. Each treatment consisted of 64 pots and had an area of 1 m 2 . The experiment was performed in triplicate. The horticultural substrate used in the experiment was characterized by salt concentration of 1.5 g dm −3 , pH 5.9, and the following chemical composition: N-NO 3 -112 mg dm −3 , P-80 mg dm −3 , K-143 mg dm −3 , Ca-1240 mg dm −3 , and Mg-383 mg dm −3 . Crop protection chemicals were not applied. Yellow sticky traps were placed over the pots to control the number of pests (Trialeurodes vaporariorum and Sciara militaris). Encarsia formosa (three individuals per m 2 ) were used for the biological control of Trialeurodes vaporariorum. Leaves were harvested at maturity.

Greenhouse Microclimate Conditions
Greenhouse microclimate conditions were modified to meet the needs of plants in different growth stages [20]. The microclimate was controlled by a climate computer, and temperature parameters were manually adjusted. The temperature was maintained at 24 • C during seed germination. To increase air humidity, the pots were covered with non-woven fabric. The non-woven fabric was removed after the seeds had germinated. At the same time, the temperature was lowered to 20 • C during the day and 18 • C at night. During the growing season, cultivation measures were limited to plant watering and pruning at 5 weeks after sowing to promote growth.
The biostimulants were applied three times, at 14-day intervals, beginning at 2 weeks after seedling emergence, at a dose of 0.3%. The doses given in amounts per ha were diluted in water at 500 L ha −1 . In order to prepare a smaller amount of working solution, the recommended dose was adjusted proportionally. Plants were supplied with water from the water mains.

Harvest and Yield of Herbs
A once-over harvest of herbage was carried out. The plants were cut manually, 1.5 cm above the ground, in the vegetative growth stage. Plant height and plant weight per pot were determined, and average samples of herbage were collected from marketable yield in each treatment. Plant height was measured using a 1 m ruler. Fresh weight was determined using the Radwag PST 750 R2 digital balance (Radwag, Radom, Poland). The weight and height of fresh herbs were determined separately for each pot. The results were then summed up and averaged. Marketable yield was identical to total yield.

Chemical Analysis of Herbs
Immediately after cutting and weighing the herbs, all plant material was dried. The herbs were dried for 12 h at 45 • C in a Binder Avantgarde Line laboratory dryer with air circulation. Herbage samples were analyzed to determine the content of selected biochemical compounds. The chemical composition of herbage was analyzed by determining the content of air-dry matter and dry matter (DM) by drying the samples at 105 • C to a constant weight [21] in a Pol-Eko Aparatura SLW 535 SD laboratory dryer. To calculate the percentage of DM in air-dried samples, the weight of the sample after drying was divided by the weight of the sample before drying and multiplied by 100. All herb samples were weighed in containers (in duplicate). The containers were placed in an oven at 60 • C for 72 h. The containers were re-weighed and the weight of the samples was determined. Weight loss is an indicator of the amount of water and DM.
Total sugars and reducing sugars were determined as described by Luff-Schoorl [22], L-ascorbic acid was determined by the method proposed by Tillmans and modified by Pijanowski [23], and total acidity [24] and nitrates (V) were determined colorimetrically with the TECHCOMP UV2310II spectrometer, using salicylic acid [25].
The concentrations of macronutrients were determined in dry and wet mineralized plant materials in three replications. Plants were dried for 24 h at 65 • C in a Binder ED400 dryer (Binder GmbH, Tuttlingen, Germany), and were ground in a Grindomix GM300 knife mill (Retsch GmbH, Haan, Germany). To determine macronutrient content, herbage samples were wet mineralized in H 2 SO 4 with the addition of H 2 O 2 as the oxidizing agent, using the SpeedDigester K-439 unit (Büchi Labortechnik AG, Flawil, Switzerland). To determine micronutrient content, herbage samples were wet mineralized in a mixture of HNO 3 + HClO 4 + HCl using a CEM Mars 5 Digestion Oven (CEM Corporation, Matthews, NC, USA).

Statistical Analysis
The results were presented as means for the years of study since only minor differences were noted. A two-way analysis of variance (ANOVA) was performed to determine significant differences between each treatment and the control treatment for both experiments. The differences between treatment means were pooled by Tukey's test at a 0.05 probability level. The analysis was performed using SAS (Statistical Analysis System) software, ver. 12 (TIBCO Software Inc. Statistica, Paolo Alto, CA, USA).

Results and Discussion
Differences in the growth rate and yields of the analyzed herb species are presented in Table 1 and Figure 1. Lemon balm was characterized by the significantly highest fresh herbage weight per pot (27.8 g). At the time of harvest, summer savory plants were the tallest, and marjoram plants were the shortest. The use of the Maximus Amino Protect biostimulant in the cultivation of the tested plant species has a positive effect on the height and weight of plants, increasing them by 3.4% and 1.4%, respectively, on average. When applied in small concentrations, these substances enhance nutrition efficiency, abiotic stress tolerance, and/or crop quality traits, regardless of their nutrient content. Exogenously applied biostimulants have a similar mode of action to that of plant hormones such as auxins, gibberellins, and cytokinins [26]. Similar results can be obtained by cultivating garden savory [10] and bell peppers [27] at different rates of nitrogen-potassium fertilizers. According to El-Gohary et al. [28], NPK and amino acids have a pronounced effect on all tested parameters (fresh and dry herbage of Satureja hortensis L., essential oil content and its constituents).
The use of an appropriate biostimulant can improve the vitality of roots and shoots, thus increasing crop yields. The total fresh herbage yield was 1.07 kg m −2 (Satureja hortensis L.), 1.35 kg m −2 (Origanum majorana L.), and 1.73 kg m −2 (Melissa officinalis L.) on average. Crop yield is significantly affected by the cultivated species. The obtained results corroborate the findings of other authors, indicating that plants characterized by different growth rates of aboveground and underground organs may differ in the yield of edible parts [10,15].
There are differences in the chemical composition of the analyzed herb species (Table 2, Figure 2). According to other authors, DM content is highest in marjoram (20.4%), savory, lemon balm, hyssop, and chervil (16.4% on average), and lowest in basil and tarragon (12.9% on average) [29]. Fresh herb species tested in this experiment had a relatively low DM content ranging from 12.2% in Satureja hortensis L. to 16.3% in Origanum majorana L. Biostimulants had no significant effect on the DM content of the analyzed herbs. Similar DM content was reported by Nurzyńska-Wierdak and Zawiślak [30] with 9.4% on average in sweet basil and lemon balm, Kazimierczak et al. [13,31] with 22.1 to 26.0% in lemon balm, and Nurzyńska-Wierdak et al. [15] with 13.5 to 17.4% in lemon balm and 11.9 to 17.1% in marjoram, depending on the sowing date and pot size. In a study by Telesiński et al. [32], summer savory and marjoram had higher DM contents (21.4% and 30.3%, respectively).  There are differences in the chemical composition of the analyzed herb species (Table 2, Figure 2). According to other authors, DM content is highest in marjoram (20.4%), savory, lemon balm, hyssop, and chervil (16.4% on average), and lowest in basil and tarragon (12.9% on average) [29]. Fresh herb species tested in this experiment had a relatively low DM content ranging from 12.2% in Satureja hortensis L. to 16.3% in Origanum majorana L. Biostimulants had no significant effect on the DM content of the analyzed herbs. Similar DM content was reported by Nurzyńska-Wierdak and Zawiślak [30] with 9.4% on average in sweet basil and lemon balm, Kazimierczak et al. [13,31] with 22.1 to 26.0% in lemon balm, and Nurzyńska-Wierdak et al. [15] with 13.5 to 17.4% in lemon balm and 11.9 to 17.1% in marjoram, depending on the sowing date and pot size. In a study by Telesiński et al. [32], summer savory and marjoram had higher DM contents (21.4% and 30.3%, respectively).
There were no statistically significant differences in the content of total sugars and reducing sugars between the tested herb species; the content of total sugars ranged from 0.52 to 1.61 g 100 g −1 FM, and the content of reducing sugars ranged from 0.31 to 1.01 g 100 g −1 FM. The concentrations of total sugars and reducing sugars were the highest in marjoram herbage and the lowest in summer savory herbage. Nurzyńska-Wierdak et al. [15] reported similar concentrations of total sugars and reducing sugars, i.e., 0.56-1.49 and 0.25-0.32 g 100 g −1 FM, respectively, in lemon balm, and 0.93-1.00 and 0.17-0.42 g 100 g −1 FM, respectively, in marjoram.   There were no statistically significant differences in the content of total sugars and reducing sugars between the tested herb species; the content of total sugars ranged from 0.52 to 1.61 g 100 g −1 FM, and the content of reducing sugars ranged from 0.31 to 1.01 g 100 g −1 FM. The concentrations of total sugars and reducing sugars were the highest in marjoram herbage and the lowest in summer savory herbage. Nurzyńska-Wierdak et al. [15] reported similar concentrations of total sugars and reducing sugars, i.e., 0.56-1.49 and 0.25-0.32 g 100 g −1 FM, respectively, in lemon balm, and 0.93-1.00 and 0.17-0.42 g 100 g −1 FM, respectively, in marjoram. cdm-Dry matter content, cts-total sugar content, crs-reducing sugar content, cO-organic acid content, cL-L-ascorbic acid content, cN-nitrate (V) content. According to Frąszczak et al. [33], the concentration of L-ascorbic acid in herbs is significantly affected by the species and cultivation method. In the present study, marjoram was characterized by the highest concentration of L-ascorbic acid (39.7 mg 100 g −1 FM on average). The L-ascorbic acid content of the herbage was not significantly modified by the biostimulants. The concentration of L-ascorbic acid increased in marjoram treated with Maximus Amino Protect (44.6 mg 100 g −1 FM), whereas it was significantly lower in lemon balm in all treatments and in summer savory in the control treatment and in the treatment with Maximus Amino Protect. Similar L-ascorbic acid concentrations were determined by Kazimierczak et al. [31] in lemon balm (32.1-76.3 mg 100 g −1 FM depending on the cultivation method), and Nurzyńska-Wierdak et al. [15] (9.8-79.4 mg 100 g −1 FM in lemon balm and 23.6-74.5 mg 100 g −1 FM in marjoram). In a study by Frąszczak et al. [33], the L-ascorbic acid content of lemon balm herbage ranged from 4.2 to 5.3 mg 100 g −1 FM.
The concentrations of organic acids in the analyzed herb species were not significantly affected by the experimental factors or their interaction. Summer savory herbage harvested in the control treatment had the highest organic acid content (0.10 g 100 g −1 FM). The concentration of organic acids was the lowest (0.06 g 100 g −1 FM) in summer savory and marjoram treated with Calleaf Aminovital.
According Macronutrients play important functions in the body, including the regulation and maintenance of the acid-base balance. The daily requirement for these ingredients is over 100 mg. The mineral status of plants is important not only for the nutritional value of food, but also for the growth, development, and yielding of crops [1]. Plants are exposed to the influence of adverse environmental factors, which are known as abiotic stresses, excessive soil salinity, and the absence of mineral salts [3]. The concentrations of selected macronutrients in the analyzed herb species are presented in Figure 3. Herb species had a significant effect (Table 3, Figure 3) on total N content (14.4 g kg −1 DM on average). Summer savory herbage was characterized by the significantly highest total N content (18.5 g kg −1 DM on average). Maximus Amino Protect had a positive influence on total N levels in the examined herb species, which reached 16.1 g kg −1 DM on average. However, the observed differences are not statistically significant. The total N content of summer savory treated with Maximus Amino Protect was significantly higher (24.8 g kg −1 DM) than in the remaining treatments. Lower total N concentrations were noted in lemon balm herbage in all treatments, and in summer savory treated with Calleaf Aminovital. Different total N levels were reported by Seidler-Łożykowska et al. [35]-2.5-3.8% in summer savory and 2.0-2.6% in marjoram, depending on the cultivation method. Dzida and Jarosz [36] found that the total N content of summer savory herbage ranged from 2.9 to 4.5% DM, depending on fertilization. In the experiment conducted by Skubij et al. [3], nitrogen rate and plant growth stage had a significant effect of the total nitrogen content of summer savory var. Saturn.
2.6% in marjoram, depending on the cultivation method. Dzida and Jarosz [36] found that the total N content of summer savory herbage ranged from 2.9 to 4.5% DM, depending on fertilization. In the experiment conducted by Skubij et al. [3], nitrogen rate and plant growth stage had a significant effect of the total nitrogen content of summer savory var. Saturn.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.