Effect of shading and nitrogen fertilization on nitrogen metabolism, essential oil content and antimicrobial activity of Achillea millefolium

The Achillea millefolium L. is a perennial herb with important antibacterial, antifungal, antiinflammatory, antitumoral, and antioxidant properties. This research aimed to investigate the effect of shading (75%; black net) and nitrogen fertilization (0, 75 and 150 kg urea ha-1) on the nitrogen metabolism, essential oil yield and antimicrobial activity of A. millefolium at vegetativeand reproductivestage. The evaluated parameters varied depending on the organ and the phenological stage of the plant considered. Overall, our findings indicated that shading decreased nitrogen assimilation. Decreased activities of nitrate reductase and glutamine synthetase were observed on shaded plants during reproductive and vegetative stages, respectively. Nitrate and total amino acid levels increased in shaded plants at the vegetative stage. Regarding nitrogen supply, the improved nitrogen metabolism and essential oil yield values were accompanied by intermediate concentrations of urea (75 kg ha-1). Plants fertilized with 75 kg urea ha-1 produced the highest amino acids concentration (vegetative stage), ammonium concentration (vegetative stage) and essential oil yield (reproductive stage). Shading or nitrogen supply did not influence the microbial activity of A. millefolium essential oil. However, the essential oil of leaves and flowers were highly effective against fungi and bacteria, especially grampositive bacteria. In conclusion, the current study showed that full light and 75 kg urea ha-1 enhanced the nitrogen metabolism of A. millefolium in both vegetative and reproductive stages.

The concentration and composition of the yarrow essential oil can be influenced by its stage of development; environmental conditions, storage period and plant subtype (Verzár-Petri & Cuong, 1977). The use of different types of protection nets in the growth of a range of plants is increasingly becoming popular. Nets are used to improve the microclimate, protect plants from hail, pests, and excessive sunshine, and modify plant morphology and metabolism. The use of nets for shading is a widely used method for growing medicinal plants (Costa, Chagas, Bertolucci, & Pinto, 2014;Russo & Hornermeier, 2017;Ribeiro et al., 2018). Indeed, the content of chloroplastic pigments increased with shading in A. millefolium plants grown under conditions similar to those used in this study (Lima, Amarante, Mariot, & Serpa, 2011). Similarly, greater survival and rooting of A. millefolium seedlings cultivated in vitro with reduced radiation were observed (Alvarenga, Pacheco, Silva, Bertolucci, & Pinto, 2015) Material and methods

Plant material and experimental conditions
Plant cultivation was carried out in the experimental area Conjunto Agrotécnico Visconte da Graça (CAVG) located in Instituto Federal Pelotas,RS (31º42'44.52" S and 52º18'46.70" W). Yarrow plants (A. millefolium) were obtained from a single clump to ensure the uniformity of data (Martins et al., 2003). Seedlings were grown in a greenhouse for four month, from which they were transferred to the experimental area (planosol soil). Phosphorus and potassium content was previously adjusted based on a soil analysis. Throughout the experiment, plants were irrigated using a drip system.
Forty-day-old plants of uniform size were grown using three different concentrations of nitrogen fertilizer (0, 75 and 150 kg ha -1 ) in the form of urea. In addition, in reproductive-stage plants (full flowering), two levels of shading including 0% shading (full light) and 75% shading (covered with a black net) were assessed. After 56 day under these conditions, plants were harvested. Then, the black net was removed, and nitrogen fertilization was performed (as described above). Four months later, the vegetative stage plants were again shaded (0 and 75%) and they were grown under these conditions for 46 day when the second harvest was performed. A herbarium specimen was prepared and deposited at the PEL Herbarium (24.600).

Activity of nitrate reductase and glutamine synthetase
The activity of NR (EC 1.6.6.1) was previously evaluated using plant materials harvested at different periods of the day. Researchers were able to determine that 8:30 am was the best time to collect materials for the analysis of NR activity (data not shown). The activity of NR was assessed in vivo according to Klepper, Flesher, and Hageman (1971) with some modifications. Fresh leaves and roots (0.5 g) were incubated in 0.2 M phosphate buffer (pH 7.5) containing 0.1 M KNO 3 and 1% (v v -1 ) propanol. Afterward, plant material was vacuum-infiltrated three times (300 mmHg for 1 min.) and incubated in a shaking water bath at 30ºC in the dark. Then, aliquots from the samples were collected and incubated with 1% (w v -1 ) sulfanilamide and 0.02% (w v -1 ) naphthyl ethylene-diamine dihydrochloride at 30ºC for 15 min. and posteriorly analyzed at 540 nm. A standard curve with sodium nitrite was performed for determining nitrite formation.
The activity of GS (EC 6.3.1.2) in fresh plants was evaluated according to Cullimore, Lara, Lea, and Miflin (1983). Briefly, 2.0 g of leaves and roots were homogenized with 100 mM Tris-HCl (pH 7.8) containing 1 mM dithiothreitol, 5 mM glutamic acid, 10 mM MgCl 2, 10% (v v -1 ) glycerol and 20% (w w -1 ) polivinilpolipirrolidone. The homogenate was centrifuged at 20,000 × g and 4ºC for 20 min. and the supernatant was desalted using a Sephadex G-25 column. The reaction was initiated by adding resulting supernatants (100 μL) to media containing 100 mM Tris-HCl (pH 7.8), 100 mM glutamic acid, 80 mM hydroxylamine, 80 mM ATP and 160 mM MgCl 2 for 30 min., followed by the addition of stop solution (0.37 FeCl 3 , 0.2 M TCA and 0.67M HCl). After, the contents were centrifuged at 14,000 × g at 25ºC to precipitate proteins. y-glutamyl hydroxamate content within supernatants was determined by measuring fluorescence at 535 nm and quantified using a standard curve.

Determination of total free amino acids, nitrate, and ammonium
Total free amino acids, nitrate, and ammonium were extracted according to Bieleski and Turner (1966). Frozen leaves and roots (1.0 g) were ground into a powder using liquid N 2 and metabolites were extracted for 24 hours with a 10 mL methanol:chloroform:water (12 5 3 -1 ; v v v -1 ) solution. After centrifugation at 600 × g for 10 min., one volume of chloroform and 1.5 volumes of water were added to each of the 4 volumes of supernatants obtained. The aqueous phase was collected after 24 hours and used for the analysis of amino acids, nitrate, and ammonium. Total levels of soluble amino acids were measured using the ninhydrin method of Yemm, Cocking, and Ricketts (1955) and measured via spectrophotometric analyses at 570 nm. Nitrate was determined spectrophotometrically at 410 nm using the salicylic acid method as described by Cataldo, Maroon, Schrader, and Youngs (1975). The ammonium was analyzed using Berthelot reactions at 625 nm (McCullough, 1967).

Essential oil yield and determination of the minimum inhibitory concentration
Shade-dried, powdered, leaf or flower materials (70 g) were hydro-distilled in a Clevenger-type apparatus for 4 hours. The extracted volatile oils were dried over anhydrous sodium sulfate and the yield of the oils was calculated based on dry the weight of plants (Santos, Alves, Figueirêdo, & Neto, 2004).
The antimicrobial activity of the essential oil of A. millefolium was tested against reference strains (American Type Culture Collection) including Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 29212), Candida albicans (ATCC 18804). The same test was performed for the isolates strains Bacilus cereus, Enterobacter aerogenes, Shigella flexneri, Klebsiella sp., Salmonela sp., E. coli, S. aureus, S. epidermidis, and C. albicans. The essential oil was tested in the concentration ranges of 12.5-100 mg L -1 . The minimal inhibitory concentration (MIC) of each sample was determined after incubating 24 hours at 37ºC and adding 20 μL of 0.5% of 2,3,5-triphenyl tetrazolium chloride. Then, a new incubation was performed at 37ºC for 30 min. until the lowest sample concentration prevented a color change in the medium and exhibited complete inhibition of microbial growth.

Experimental design and statistical analysis
Experiments were arranged in a completely randomized block, 3 x 2 factorial design (nitrogen concentrations and shading levels). All analyses were carried out using four repetitions, and each biological sample consisted of five plants. Data were first submitted to a Shapiro-Wilk test to assess the normality of data and to a Levene's test to verify homoscedasticity, followed by ANOVA. The post hoc comparisons were tested using a Tukey or t-test at the significance level of p < 0.05 with the statistical program SAS (2002).

Results
There was no interaction between shading and nitrogen levels when NR and GS were evaluated. Shaded yarrow leaves from reproductive-stage plants exhibited decreased (1.7-fold) NR activity (Table  1). However, NR was not affected by shading in leaves from vegetative-stage plants and roots from plants at both stages (p < 0.05). Regarding the influence of nitrogen fertilization, NR activity increased (1.5-fold) in yarrow roots from vegetative-stage plants treated with 75 kg urea ha -1 , and did not change in leaves of the same stage or in leaves and roots from reproductive-stage plants (Table 2). In plants at the vegetative stage, the activity of GS decreased 2.2-and 1.6-fold in shaded leaves and roots, respectively (Table 3). However, GS activity increased in leaves of plants at the vegetative stage fertilized with 75 kg urea ha -1 . The GS enzyme was not influenced by the concentrations of nitrogen or shading in reproductive-stage plants (Table 4; p < 0.05). There was an interaction between shading and nitrogen levels with respect to levels of total free amino acids in vegetative-stage plants. In reproductive-stage plants, leaf and root amino acid content was unaffected by shading (Table 3; p < 0.05), but decreased 1.3-fold in plants treated with 75 and 150 kg urea ha -1 (Table 5). In contrast, in shaded plants at the vegetative stage, amino acid content increased 1.5-fold with 75 and 150 kg urea ha -1 treatments, respectively, when compared to levels in plants that did not receive urea treatment (Table 6). The free amino acid content in plants grown under full light increased (1.9-fold) post-treatment with 150 kg urea ha -1 . Other concentrations of urea treatment did not significantly affect free amino acid content. The total amino acid content in plants treated with 75 and 150 kg urea ha -1 was greater (2.8-and 1.5-fold, respectively) when plants were shade-grown, rather than grown under full light (Table 6). Overall, in the two studied stages, nitrate content was higher in roots than in leaves of A. millefolium (Table 7, Figure 1). There was an interaction between shading and nitrogen levels with respect to nitrate content in yarrow plants at the reproductive stage (Table 7). In shaded leaves, nitrate content was increased in plants treated with 150 kg urea ha -1 (17.55 μmol g -1 FW) and 0 kg urea ha -1 (14.80 μmol g -1 FW), respectively, compared with plants treated with 75 kg urea ha -1 (11.72 μmol g -1 FW). In the roots of shaded plants, there were no differences observed between nitrogen supplementation. In unshaded plants, the highest levels of nitrate content in leaves occurred in the privation of nitrogen and in roots in the privation of urea and when the plants were treated with 150 kg urea ha -1 (Table 7). When the plants were treated with 150 kg urea ha -1 , the highest levels of nitrate (1.3-fold) in leaves were found in shaded plants compared with non-shaded plants. In roots, shading decreased nitrate content 1.5-, 1.2-and 1.4-fold, respectively when plants were treated with 0, 75 and 150 kg urea ha -1 . On the other hand, the nitrate content in leaves and roots of shaded plants increased (2.6-and 1.8-fold, respectively) when the plants were shaded at the vegetative stage (Figure 1). The nitrogen supply did not affect nitrate content in vegetative-stage plants (p < 0.05; data not shown). There was no interaction between shading or nitrogen dosage on ammonium content in A. millefolium reproductive-stage leaves. In contrast, in the vegetative stage, ammonium content was increased when the plants were shaded and treated with 0 kg urea ha -1 (3.3 fold) and 150 kg urea ha -1 (2.0 fold), respectively, compared with plants under shading and treated with 75 kg urea ha -1 . Under full light conditions, the highest level of ammonium content was found in plants fertilized with 150 kg urea ha -1 . However, in roots and leaves of shaded and unshaded plants, ammonium levels in response to nitrogen fertilization did not change (Table 8). Shading decreased ammonium levels in the leaves and roots of plants fertilized with nitrogen. This reduction was 3.1-and 1.6-fold for leaves and 2.3-and 7.3-fold for roots treated with 75 and 150 kg urea ha -1 respectively (Table 8). Ammonium levels were also decreased (4.8-fold) in the roots of shaded plants that did not receive nitrogen fertilization. The concentration of nitrogen fertilizer applied and the degree of shading had significant effects on the essential oil yields from leaves and flowers, but there was no interaction observed between the factors. Urea supply did alter yields of the essential oils in leaves (data not shown). However, in flowers, fertilization with 75 kg urea ha -1 increased the oil yields 1.2-fold compared to other treatments (Figure 1). In contrast, leaf oil yields decreased 1.2-fold with shading compared to control leaves (Figure 1), while this parameter did not alter flower essential content (data not shown).
Biological activities of essential oils of both yarrow leaves and flowers were similar among treatments (Table 9-10). Thus, the different concentrations of nitrogen and shading levels studied did not affect the inhibitory activity of the essential oil (p < 0.05). The MIC values of essential oils from yarrow leaves ranged from 12.5 to 25.0 mg mL -1 for S. aureus ATCC 25923, B. cereus, S. aureus and S. epidermidis bacterial strains (Table 9). Similarly, C. albicans ATCC 18804 and C. albicans were inhibited by 12.5 mg mL -1 essential oil (Table 9). On the other hand, MIC values for E. coli ATCC 8739, Salmonella sp. and E. coli bacterial strains ranged from 50 to 100 mg mL -1 . The essential oil of yarrow leaves did not inhibit the growth of E. faecalis ATCC19433, P. aeruginosa ATCC 9027, E. aerogenes, S. flexneri, and Klebsiella sp. bacterial strains at any of the concentrations tested.  The antimicrobial activity of the essential oil of flowers was very similar to that of leaves (Table 10). The B. cereus bacterial strain and C. albicans ATCC 18804 and C. albicans fungal strains were inhibited by 12.5 mg mL -1 essential oil. While S. aureus, E. faecalis ATCC 19433, S. aureus and S. epidermidis bacterial strains were sensitive to essential oil at concentrations of 12.5 and 25 mg mL -1 . For strains, E. coli ATCC 8739, Salmonella sp., E. aerogenes, and E. coli, MIC varied from 50 to 100 mg mL -1 . Finally, for P. aeruginosa ATCC 9027, S. flexneri and Klebsiella sp. strains, the essential oil showed no inhibitory activity (Table 10).  submitted to levels of shading (black net -75%; full light -0%) or different concentrations of nitrogen (kg urea ha -1 ). Distinct uppercase letters indicate differences between roots and leaves, lowercase letters indicate differences between shading or among nitrogen supply treatments. Distinct uppercase letters indicate significant differences in the line while distinct lowercase letters indicate significant differences in the column (Tukey; p < 0.05).

Discussion
Nitrogen is an essential element for plants that is available in agricultural soils mainly in the form of nitrate and ammonium (Pinton, Tomasi, & Zanin, 2016). In the leaves of shaded plants treated with the highest concentration of nitrogen, enhanced nitrate accumulation was observed. This increase in nitrate levels is due to the low light intensity required for ion reduction and the increased availability of nitrate supplied by nitrogen fertilizer. Further, the increased levels of nitrate found in the roots of unshaded plants compared to those that were not shaded may be a result of enhanced nitrification of the soil. Under these conditions, decreased humidity and increased temperatures may have favored microbial activity in comparison to shady conditions, thus allowing more nitrate to be accessed via the roots of plants. In contrast, nitrogen supply in soil treated with 75 kg of urea ha -1 for reproductive-stage plants seems to have improved nitrate metabolism, thus reducing its accumulation in the leaves of shaded and unshaded plants and the roots of control plants.
After uptake, nitrate can be stored in vacuoles or metabolized by NR in both the plant root and shoot. Subtropical and perennial plants such as A. millefolium carry out a substantial proportion of their nitrate assimilation in the shoot, which occurs independently of the external concentration of nitrate (Andrews, 1986). In fact, lower concentrations of nitrate were found in leaves than roots, which may have been due to the relatively higher nitrate reduction activity that occurs within leaves. Moreover, NR requires reducing compounds, such as NAD(P)H, which are derived from photosynthesis. Thus, enzyme activity in leaves is favored, since roots and other subterranean organs must translocate and oxidize carbohydrates to provide the reducing power for NR activity (Guo et al., 2017).
Light is also important for the regulation of NR. It is well known that NR is positively regulated by light at the transcriptional level (Hageman & Flesher, 1960;Creighton et al., 2017). Furthermore, NR activity is inhibited by phosphorylation at a conserved serine residue when plants are transferred from light to darkness (Nemie-Feyissa et al., 2013). Indeed, NR activity is enhanced drastically by light exposure in reproductive-stage yarrow plants; and in the vegetative stage, shaded plants accumulate nitrate in leaves and roots. In addition to light, nitrate also regulates NR activity in plants. However, nitrogen fertilization did not alter NR activity in yarrow leaves at either stage of development. These findings may have been a result of the nitrogen source supplied, which provided more ammonium than nitrate. Ammonium can be converted to nitrate in the soil via the nitrification process, which varies depending on soil microbial activity, precipitation, oxygen availability and pH (Pinton et al., 2016;Hernández-Martínez, Prado, Cayetano-Salazar, Bischoff, & Siebe, 2018). Thus, the nitrate may have been supplied in varying amounts to the plants and in concentrations insufficient to modify NR activity. The increase in NR activity in roots was only observed in vegetative-stage plants treated with 75 kg of urea ha -1 . The increase in nitrogen supply in the form of urea (150 kg of urea ha -1 ) did not increase NR activity, probably because increased levels of ammonium, rather than nitrate, were made available to the plants.
Light positively affected the accumulation of ammonium in the leaves and roots of vegetative-stage yarrow. Metabolizing nitrate to amino acids involves the conversion of nitrate to nitrite by NR, followed by the conversion of nitrite to ammonium by nitrite reductase (Krapp, 2015). As NR is more responsive to light (Creighton et al., 2017), the accumulation of ammonium in unshaded plants may be due to nitrate reduction. Nitrate metabolism to ammonium may also have contributed to the accumulation of ammonium in plants treated with nitrogen since only leaves exposed to full light conditions had increased levels of ammonium when treated with both concentrations of urea. In addition, the ammonium produced within leaves by photorespiration may also have contributed to the values of ammonium observed. On the other hand, in shaded plants, ammonium accumulation differed between leaves and roots. In leaves, the lowest levels of ammonium accumulation occurred in plants treated with 75 kg of urea ha -1 , however, in roots the same treatment resulted in enhanced accumulation of ammonium. These effects may have been due to differing availabilities of ammonium, as a result of differences in moisture, pH and microbial activity (Hernández-Martínez et al., 2018). It may also have been a result of the rapid conversion of the molecule since it can cause toxicity in plants when present in excess (Krapp, 2015).
Ammonium is assimilated via the GS/GOGAT cycle (Krapp, 2015). Thus, the observed increases in GS activity appears to have occurred as a positive-feedback response to increased ammonium levels (Ishiyama et al., 2004). Thus, the activity of the enzyme was higher in roots and leaves of plants grown in full light than in plants under shading and matching with the observed increases in ammonium levels. In contrast, plants that were treated with 75 kg of urea ha -1 had lower ammonium content and higher GS activity, compared with the other nitrogen treatments. This may be a result of the transient depletion of ammonium since enzyme activity was high at the time of sample harvesting.
The performance of GS together with the GOGAT complex functions to incorporate the ammonium into glutamate, which can be metabolized in other amino acids as needed. The total amino acid content in roots was approximately 5.0-fold higher than in the leaves. Taken together, these, along with the high levels of GS activity in the leaves observed, suggest that nitrogen metabolism varies between the two organs. Thus, the high content of amino acids in the roots can be attributed to the transfer of the assimilated nitrogen from leaves to roots via the phloem. Similar results were observed in Coffea arabica (Gonçalves, Gallo, & Favarim, 2007). In this work, shading had no influence on the concentration of total amino acids in leaves or roots of reproductive-stage yarrow. Furthermore, the addition of 75 and 150 kg of urea ha -1 decreased amino acid accumulation in roots. The ammonium ion should stimulate amino acid formation, by increasing levels of their precursors. However, the increased availability of nitrogen may have stimulated the production of other nitrogenous compounds, such as proteins and nucleic acids necessary for the growth, flowering and fruiting of the plant, thus reducing the free amino acid content.
At the vegetative stage, increased amino acid content occurred in parallel with nitrogen supply increases. The amino acid content also increased when shaded plants were provided with urea, which may be a characteristic of an adaptive shade. In other words, increased accumulation of amino acids may be associated with decreased utilization, since the low-light condition limits growth. Indeed, yarrow produced decreased quantities of dry matter when they were grown in the shade (Lima et al., 2011). Chen et al. (2017) showed that most enzymes involved in the biosynthesis of amino acids were down-regulated by dark treatment. In addition, the authors suggested that the proteolysis of chloroplast proteins contributed to amino acid accumulation in dark-treated leaves of Camellia sinensis. The differences in amino acid content found in plants at vegetative and reproductive stages are probably due to the allocation of nitrogen to reproductive organs in reproductive-stage plants since this phase demands a high level of energy and nutritive investment.
The yields of essential oils from shaded leaves were higher than those yields of unshaded plants. Similar results were observed in Tetradenia riparia grown with 50% shading (Araujo, Melo, Paula, Alves, & Portes, 2018). Essential oil production is usually associated with higher irradiance levels, as demonstrated in studies with peppermint (Mentha piperita; Costa et al., 2014) and basil (Ocimum basilicum; Paulus, Valmorbida, Ferreira, Zorzzi, & Nava, 2016). However, the excess in light can cause photoinhibition in some species of plants, and consequently, decrease essential oil yield (Araujo et al., 2018). This suggests that the effects of shading may be species-specific. On the other hand, the increased essential oil yield observed in flowers of plants fertilized with urea (75 kg ha -1 ) was attributed to enhanced oil biosynthesis, since there was no observed increase in the biomass of treated flowers (Lima et al., 2011). In basil treated with three concentrations of ammonium nitrate, the highest yield of essential oil was found in plants treated with 300 kg of N ha -1 , demonstrating that nitrogen enhances essential oil yield in other medicinal species (Sifola & Barbieri, 2006). In our study, however, the highest concentration of urea (150 kg ha -1 ) did not enhance oil yield, which indicates that oil yield is not a concentration-dependent parameter.
A MIC concentration of 12.5 mg mL -1 essential oil from either leaves or flowers of yarrow produced strong inhibition activity; moderate activity was associated with 25 mg mL-1 essential oil. The MIC values of 50, 100 and > 100 mg mL -1 were considered weak, very weak and without inhibition activity, respectively (Aligiannis, Kalpoutzakis, Mitaku, & Chinou, 2001;Sartoratto et al., 2004). In addition, for strains of S. aureus ATCC 25923, B. cereus, S. aureus, and S. epidermidis the inhibitory activity of essential oils from yarrow leaves was considered strong to moderate. Similarly, the inhibitory activity of yarrow oil was considered strong against C. albicans ATCC 18804 and C. albicans. On the other hand, the inhibitory activity of leaf oil was considered weak or very weak E.coli ATCC 8739, Salmonella sp. and E. coli. The essential oil of flowers strongly inhibited the bacterial strain B. cereus, as well C. albicans ATCC 18804 and C. albicans fungal strains. Inhibition was considered moderate for S. aureus, E. faecalis ATCC 19433, S. aureus and S. epidermidis, and weak to very weak for E. coli ATCC 8739, Salmonella sp., E. aerogenes, and E. coli strains.
The E. faecalis ATCC 19433 and E. aerogenes strains were inhibited by essential oils from flowers, but not leaves; a finding that indicated that only some compounds present in the flowers are effective against these species of microorganisms. The differences in the antimicrobial activity of the essential oils extracted from leaves and flowers of A. millefolium can be explained by variability in the chemical compositions of the oils, which contain monoterpenes, sesquiterpenes and phenolic compounds such as sabinene, β-phenyl alcohol, kaempferol, farnesol, caryophyllene, linalool and eugenol (Alvarenga et al., 2015;Mohammadhosseini et al., 2017).
Gram-negative bacterial species are generally most resistant to essential oils than gram-positive organisms, due to the composition of their peptidoglycan lipid bilayer (Semeniuc, Pop, & Rotar, 2017). This barrier is responsible for restricting diffusion of hydrophobic compounds, prevents their direct exposure to the oil and its bioactive components. Many substances, which are qualitatively and quantitatively different in each oil, have specific capacities to break or penetrate bacterial membranes. In order to determine the mode of action of the oils, it is necessary to evaluate the effects of each active ingredient, both in combination and separately. Only after these analyses are performed will researchers be able to determine whether they have bactericidal or bacteriostatic action individually or in combination with other compounds (Mahmoud, El-Baky, Ahmed, & Gad, 2016).

Conclusion
Full light exposure enhanced nitrogen metabolism in A. millefolium. However, the species adapts well to shading being able to produce increased yields of essential oils compared to plants grown under full light. Regarding nitrogen supply, intermediate concentrations of urea (75 kg ha -1 ) resulted in optimal nitrogen metabolism and essential oil yields. In addition, the antimicrobial activity demonstrated that the essential oil of leaves and flowers of A. millefolium is of great interest to the pharmaceutical industry, since knowledge regarding the components of this oil may enhance research facilitating the production of bioactive products that act predominantly against gram-positive bacteria.