Influence of nutrition and water stress in Hyptis suaveolens

Industrial Crops & Products 125 (2018) 511–519 Contents lists available at ScienceDirect Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop Influence of nutrition and water stress in Hyptis suaveolens a a Maria Valdiglezia de Mesquita Arruda , Francisco Fábio Mesquita Oliveira , Maria Vanilse Sampaioa, Mônica Danielle Sales da Silva Fernandesa, Nildo da Silva Diasb, ⁎ Cynthia Cavalcanti de Albuquerquea, Cleyton dos Santos Fernandesb, a b T Faculty of Exact and Natural Sciences, State University of Rio Grande do Norte, Mossoró, Rio Grande do Norte, 59600-000, Brazil Center of Agrarian Sciences, Federal Rural University of the Semiarid, Mossoró, Rio Grande do Norte, 59625-900, Brazil A R T I C LE I N FO A B S T R A C T Keywords: Bamburral Bioaccumulation Field capacity Physiological response Poultry manure Soil fertility Hyptis suaveolens (L.) Poit. is a medicinal plant that produces essential oil with high therapeutic potential, but its essential oil production is still low and it may vary according to growing conditions. The objective was to evaluate the effect of water stress and fertilization on the production of biomass and essential oil of H. suaveolens (L.) Poit. In factorial experiment (3 × 3), with eight replicates, H. suaveolens were grown in three substrates: natural soil (NS) without fertilization, NS + NPK, and NS + Poultry manure (PM), at three levels of pot capacity (PC) (field capacity for soil in pots): 100, 50, and 25% PC. At 30 days after planting, the variables were evaluated: shoots and root dry matter, root/shoots ratio, plant height, stem diameter, relative water content in the plant, chlorophylls, protein, proline, carbohydrates, content of nutrients: N, P, K, and Na+ in plant tissue, oil yield and translocation factor. Cultivation in NS + PM improved plant growth at the water availability levels of 100 and 50% PC, except root dry matter, whose best result was obtained in NS + NPK. The contents of all biochemical variables increased due to organic fertilization, proline and carbohydrates, when plants were grown in soil under water regime of 25% PC. The substrates NS + NPK and NS + PM increased the contents of N, P, and K. The Na+ contents were high and accumulated in the roots of plants grown with poultry manure, regardless of the studied water conditions. The best oil yield was promoted by PM fertilization. Hyptis suaveolens developed under good conditions of water and fertilization. Under water and salt stress conditions there was reduction of growth, osmotic adjustment and Na+ storage in the root. Oil production was favored by fertilization, but it is still low compared with the literature. 1. Introduction Medicinal plants have long been used by humans as a valuable natural resource. The use of phytotherapy in the treatment of diseases has aroused the interest of the scientific community in conducting studies that particularly aim to increase the yield of medicinal plants, without compromising their active principles. However, there is a concern to promote rational use of medicinal plants, taking into consideration the traditional medical practices and scientific rigor in health care (Silva, 2010). Studies focusing on agronomic conservation and management of many species become necessary because of the scarce information on the cultivation of medicinal plants in the literature. Nevertheless, it has been known that biomass production and synthesis of active principles in medicinal plants depend on various factors, such as genetic and environmental, including biotic and abiotic stresses (Sales et al., 2009). ⁎ Water and nutrients have direct influence on the quality and quantity of bioactive constituents produced by medicinal plants (Mapeli et al., 2005), so that adequate water conditions ensure normal vegetative development (Meira et al., 2013), guaranteeing the production at satisfactory levels of the main active principles contained in plant structures. In this context, studies seeking species that guarantee a good development with low cost of water, without compromising production and yield in medicinal plants, are of extreme importance (Maia et al., 2008; Meira et al., 2013). Nutritional support has also been one of the main factors responsible for the increase of yield in plants (Maia et al., 2008) and organic fertilization, besides providing nutrients, promotes benefits in the physical and chemical structure of the soil (Rosal et al., 2011). The Lamiaceae family includes approximately 258 genera and 7193 species, 40% of which have aromatic properties (Malendo et al., 2003). Among the genera, Hyptis stands out for being rich in species with great Corresponding author. E-mail address: cleyton1959@hotmail.com (C. dos Santos Fernandes). https://doi.org/10.1016/j.indcrop.2018.09.040 Received 18 October 2017; Received in revised form 18 September 2018; Accepted 19 September 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved. Industrial Crops & Products 125 (2018) 511–519 M.V. de Mesquita Arruda et al. Table 1 Physical-chemical characteristics of the samples of the growing substrates at the beginning and after the experiment. Mossoró, Brazil, 2015. Properties Initial substrate −1 Silty (g kg ) Clay (g kg−1) Sand (g kg−1) AD (g cm3) TDS (ppt) pH (H2O) OM (g kg−1) N (g kg−1) P (mg/dm3) K+ (mg/dm3) Na+ (mg/dm3) Ca2+ (cmolc/dm³) Mg2+ (cmolc/dm³) Al3+ (cmolc/dm³) H + Al (cmolc/dm³) SB (cmolc/dm³) t (cmolc/dm³) CEC V (%) m (%) ESP (%) Final substrate Natural soil Natural soil + NPK Natural soil + Poultry manure Natural Soil Natural soil + NPK Natural soil + Poultry manure 138.5 37.3 824 1.53 74.8 5.30 15.98 0.63 37.4 156.9 91.9 3.70 2.10 0.05 1.16 6.60 6.65 7.76 85 1 5 107.5 29.7 861.5 1.56 88.0 5.50 16.20 0.77 11.5 130.6 60.5 3.70 2.10 0.05 1.32 6.40 6.45 7.72 83 1 3 78 18 903 1.48 232 8.20 18.61 0.63 378.5 3642.6 1192.4 2.50 3.20 0.00 0.00 20.20 20.20 20.20 100 0 26 – – – – – 5.70 – 1.05 2.4 296.5 30.3 3.00 1.10 0.05 2.15 4.99 5.04 7.14 7.0 1 2 – – – – – 6.00 – 1.26 23.7 680.0 434.4 2.80 1.20 0.00 2.15 7.63 7.63 9.77 78 0 19 – – – – – 7.60 – 1.68 370.5 103.3 208.0 6.50 1.90 0.00 0.00 9.57 9.57 9.57 100 0 9 AD = Apparently density; TDS = Total dissolved solids; pH (H2O) = Potential of hydrogen in water; OM = Organic matter; N = Nitrogenium; P = Phosphorus; K+ = Potassium; Na+ = Sodium; Ca2+ = Calcium; Mg2+ = Magnesium; Al3+ = Aluminium; H + Al = Potential acidity; SB = Sum of bases; t = Effective CEC; CEC = Cation exchange capacity; V = Base Saturation; m = Saturation by aluminum; ESP = Exchangeable sodium percentage. agronomic studies become necessary, aiming to increment biomass and, consequently, the production of essential oil, whose active constituents are important from the pharmacological point of view (Maia et al., 2008; Vijay et al., 2011). Considering the adaptation capacity of this species to semi-arid conditions, low essential oil yield and lack of information on its nutritional aspects, it becomes relevant to investigate the influence of these factors on biomass production and essential oil content. Thus, this study aimed to evaluate the effect of water stress and fertilization on the production of biomass and essential oil of H. suaveolens (L.) Poit. economic and ethnopharmacological importance (Falcão and Menezes, 2003). Hyptis suaveolens (L.) Poit. is an annual species that produces essential oil rich in mono and sesquiterpenes (Martins et al., 2006). It is commonly known as ‘bamburral’, ‘erva-canudo’, ‘sambacoité’, ‘alfazema-brava’, ‘alfazema-de-caboclo’, ‘salva-limão’, and ‘alfavacão’ (Lorenzi and Mattos, 2002). This species has been widely studied due to its essential oil, which has high antifungal, antibacterial, anticarcinogenic and antiseptic activity (Malele et al., 2003; Moreira et al., 2010), besides exhibiting nematicidal and larvicidal activity, due to the presence of D-limonene and menthol (Falcão and Menezes, 2003). In popular medicine, it has been used as antitussive, diaphoretic, antispasmodic and is useful in the treatment of gout (Corrêa and Penna, 1984). It has also been a promising alternative in the combat against larvae and mosquitoes of Aedes albopictus and Aedes aegypti (Noegroho and Srimulyani, 1997; Conti et al., 2012). Studies by Benelli et al. (2012) confirmed the activity of essential oil of H. suaveolens in adults of the barn weevil Sitophilus granarius, due to the presence of the constituents sabinene, β-caryophyllene, 4-terpineol and trans-αbergamotol. This same study, together with works by Conti et al. (2011, 2012) registered average variations in the major constituents of the oil, even though they maintained the same cultivation conditions, such changes were justified by the climatic variation. It is well acknowledged that the H. suaveolens EO chemical composition and biological activity change as a function of the origin and collecting period of the plants (Tchoumbougang et al., 2005). This is a common feature among secondary metabolites and from essential oils of Lamiaceae plants in particular. Several authors reported a large variability in the chemical compounds of this family due to genetic, geographical and seasonal factors (Baydar et al., 2004; Tonzibo et al., 2009; Kodakandla et al., 2012; Bachheti et al., 2015). In general, there is little information available, from the agronomic perspective that demonstrates the behavior of medicinal, aromatic and condiment plants when subjected to agricultural production techniques (Pravuschi et al., 2010). For H. suaveolens, it becomes important to study fertilization as a function of water conditions, because this species is highly susceptible to the influences of the surrounding environment, and the chemical constituents of its essential oil can increase, decrease or even change the composition according to the environmental conditions. Therefore, 2. Material and methods The study was carried out at the Universidade do Estado do Rio Grande do Norte – UERN, from February to March 2015, in a greenhouse, protected with white shade cloth that attenuates 30% of solar radiation. During the experimental period, the average temperature in the greenhouse was 35.5 °C and relative air humidity was 69.4%. The soil used in the experiment was collected in the same site of occurrence of the studied species, close to the UERN (05°12′ 10S and 37°18′ 57W) in the municipality of Mossoró, Brazil. The soil was sampled in the superficial layer, at depth of 0.02 m, and classified as Quartzipsamments. The experiment was set in completely randomized design, in a 3 × 3 factorial scheme, with three types of substrate and three levels of pot capacity (PC), totaling 9 treatments with eight replicates each and one plant per plot. The treatments consisted of three growing substrates: Natural soil (NS); NS + mineral fertilization (NPK) and NS + poultry manure (PM), and three levels of water availability: 100, 50, and 25% of PC. Poultry manure came from an egg-laying chicken farm, located in the municipality of Mossoró, and was aged for 90 days, dried, sieved and mixed to the soil. After prepared, the substrates were characterized regarding physical and chemical aspects, according to Embrapa (Silva, 2009). (Table 1). Plants in the experiment came from seeds cultivated in polyethylene containers with capacity for 250 mL, containing NS as substrate. At 20 days after planting, H. suaveolens seedlings were transplanted to pots with capacity for 8 L containing the respective substrates. After 512 Industrial Crops & Products 125 (2018) 511–519 M.V. de Mesquita Arruda et al. OY % = EOW (g )/ LFW (g )* 100 Table 2 Summary of the analysis of variance for shoots dry matter (SDM), root dry matter (RDM) and root/shoots ratio (R/S) in Hyptis suaveolens plants subjected to fertilization and different water regimes. Mossoró, Brazil, 2015. Source of variation Degrees of freedom Fertilizing (F1) Pot Capacity (F2) F1 x F2 Treatments CV (%) 2 2 4 8 Where OY% is the oil content in percentage; EOW is the essential oil weight (g) and LFW is leaf fresh weight (g). The obtained data were subjected to analysis of variance (ANOVA) and the means were compared by Tukey test at 0.05 significance level using the statistical software Assistat® version 7.7. Values of F SDM (g) RDM (g) ** 147.8646 4.8309* 7.9724** 42.1601** 10.46 ** 6.7967 5.7450* 0.8092 ns 3.5400* 25.00 R/S 21.0540** 3.3525 ns 0.1083* 6.1558** 25.43 3. Results 3.1. Effect of water and nutritional stress on H. Suaveolens growth Hyptis suaveolens responded to the stimuli imposed by water stress and to the supplied sources of nutrients, easily adapting to the growing conditions of each treatment. This response was clearly observed in both development and physiological conditions. Growth analyses demonstrate the variation in its biomass production in response to the nutritional conditions of the growing substrate and field capacity of each treatment. According to the ANOVA, there was significant interaction between the studied factors for the variables SDM, PH, and SD (p < 0.01) and for the R/S ratio (p < 0.05). For RDM, there was no interaction between factors, but the results were significant for both factors individually (Table 2). According to the obtained results, the highest mean values of shoots dry matter (SDM) (76.6 g), plant height (PH) (49.4 cm) and stem diameter (SD) (10.8 cm) (Fig. 1a, c, and d) were caused by the interaction between the factors poultry manure fertilization and 100% PC. These statistical data correspond to what was observed during the study, i.e., plants were taller with greater number of leaves, more branched and with thicker stems. Plants grown in substrate containing NPK also showed greater development compared with those in the control treatment, with mean values of 26 to 32 g for SDM, 43 to 49 cm for PH and 8.7 cm for SD, regardless of the studied level of field capacity. This indicates that H. suaveolens responded well to organic and inorganic fertilization; however, the lower yield in the treatments with NPK, in comparison to poultry manure, may have resulted from an insufficient dose to meet the nutritional requirements of the species. Regarding R/S ratio, interaction between factors was significant (p < 0.05). Control plants and those cultivated with NPK exhibited greater balance in water absorption and distribution and in the translocation of photoassimilates, for both shoots and roots (Fig. 1b). Plants grown with poultry manure showed greater crown development and lower root development, which was possibly due to a greater allocation of nutrients to the shoots. Root dry matter (RDM) was influenced only by the individual factors, fertilization (p < 0.01) and PC (p < 0.05). Plants grown with NS + NPK exhibited the highest mean values of growth (Fig. 2a) at 100% PC (3.5 g), with reduction of growth as the PC decreased (Fig. 2b). For the relative water content (RWC), only the factor fertilization caused significant difference (Table 3). Plants grown with NPK and poultry manure showed values lower than 40%, which can be related to the Na content at the end of the experiment in the substrates with poultry manure and NPK (Table 1). The Na content in the abovementioned substrates probably induced osmotic stress on the plants. The natural substrate (control) led to higher RWC, but it did not exceed 50% (Fig. 2c). ** 1% level of significance (p < 0.01). * 5% level of significance (p < 0.05); ns, not significant (p > = 0.05); CV, Coefficient of variation. Transformed values to x=√x. establishment, seedlings were subjected to daily irrigation regime, according to the pot capacity (100, 50 or 25%) of each treatment. Pot capacity was previously established through the capillarity method, in which the pots containing dry substrates were weighed and placed in a container with a known volume water, which rose by capillarity to saturate the substrate. After reaching full saturation, the substrate was weighed to determine pot capacity using Eq. (1). PC = (WSW + PW )−(DSW + PW ) × 100 DSW (3) (1) Where, PC = pot capacity (%); WSW = Wet soil weight (g); PW = Pot weight at the moment of weighing (g); DSW = Dry soil weight (g). In order to maintain PC, the pots with the plants were daily weighed on digital scale to replace the water and maintain the maximum water retention capacity of each treatment. In the treatments with chemical fertilization, the soil received 1.54 g dm−3 of the 10-10-10 commercial NPK formulation per week, accumulating at the end of the experiment a total NPK content of 6.16 g dm−3, according to the N dose described by Maia et al. (2008). The treatment with poultry manure consisted in natural soil + aged poultry manure, at proportion of 3:1, respectively. At 30 days after PC establishment, plant growth evaluations started and all analyses were performed in triplicate. The following variables were evaluated: root dry matter (RDM) and shoots dry matter (SDM), obtained by drying the plant material in forced-air oven at 70 °C; plant height (PH), measured with a tape measure; stem diameter (SD), measured with a digital caliper; root/shoots ratio (R/S), calculated according to Benincasa (1988); relative water content (RWC), according to Slavik (1979); proline contents, according to Bates et al. (1973); contents of chlorophylls a, b, and total, through the method of Welburn (1994); total proteins, according to Khan and Robinson (1994), quantified by the methodology of Bradford (1976), and total carbohydrates, according to Dubois et al. (1956). In addition, contents of nutrients (N, P, K, and Na+) were also evaluated. P, Na+, and K were quantified in leaves and roots through the wet digestion method, according to Plank (1992). P was determined using the vanadate/molybdate method (yellow method) (Carmo et al., 2000), whereas Na+ and K were quantified through atomic absorption spectrophotometry. Total N was determined through the semi-micro Kjeldahl method (Association of Official Analytical Chemists - AOAC, 1990) and quantified by spectrophotometry, as described by Baethgen and Alley (1989). Na+ translocation factor in the plant were quantified using Eq. (2): (2) 3.2. Physiological responses of H. Suaveolens to water and nutritional stress Where TF = Translocation factor and NaC = Sodium content, in mg kg−1H. suaveolens essential oil yield (OY) was also evaluated and it was extracted through the hydrodistillation process in modified Clevenger apparatus using all the biomass per plant, in 2 L of distilled water heated during 2 h. Oil yield was calculated using Eq. (3). In the biochemical analyses, there was significant interaction between factors for the variables free proline and total carbohydrates (p < 0.01). For the contents of total proteins, only the factor fertilization was significant at 0.05, and there was no negative interference of water stress on protein synthesis (Table 4). TF = (NaC shoots )/(NaC roots ) 513 Industrial Crops & Products 125 (2018) 511–519 M.V. de Mesquita Arruda et al. Fig. 1. Mean of the interaction for shoots dry matter (a), root/shoots ratio (b), plant height (c), and stem diameter (d) in Hyptis suaveolens plants, as a function of fertilization and water availability. Means followed by the same letters, lowercase for fertilization and uppercase for field capacity, do not differ by Tukey test (**p ≤ 0.01). Bars in the columns indicate the standard error of the mean. According to the data, the accumulation of carbohydrate and proline in plants grown with poultry manure increased as soil water availability decreased from 50 to 25% (Fig. 3a and b). The contents varied from 2.5 to 3.9 μmol.g−1 of fresh matter for free proline and from 26.2 to 43.4 mg mL−1 for total carbohydrates in the plants at lower PC. The increase in the synthesis of these solutes is a response to the low water content in leaf tissues (Fig. 2c) caused by the low water availability in the soil, which combined with the high Na+ content in the poultry manure (Table 1) led to a more severe water limitation to the plant. Protein contents were not affected by water stress, since the results did not show differences (Table 4). However, there was an increment in the protein content of plants fertilized with poultry manure (Fig. 2d). At all tested levels of water availability, N content was higher in plants fertilized with poultry manure and such difference was significant in plants at 25% PC (Fig. 4a). This means that, in plants fertilized with compost rich in N, such as poultry manure, water deficit was not a negative factor for protein production. The production of photosynthetic pigments was not affected either, Table 3 Summary of the analysis of variance for plant height (PH), stem diameter (SD) and relative water content (RWC) in Hyptis suaveolens plants as a function of fertilization and different water regimes. Mossoró, Brazil, 2015. Source of variation Degrees of freedom Values of F PH (cm) Fertilizing (F1) Pot Capacity (F2) F1 x F2 Treatments CV (%) 2 2 4 8 ** 50.9798 17.3250** 6.2915** 20.2219** 11.38 SD (cm) RWC ** 198.7592 9.0968** 6.8969** 55.4125** 8.10 6.3116** 1.71448 ns 0.7148 ns 2.3640 ns 11.8 ns, not significant (p > = 0.05); CV, Coefficient of variation. ** 1% level of significance (p < 0.01). even in plants fertilized with poultry manure, which showed lower RWC and greater osmotic adjustment. The highest values of chlorophyll a, b and total were observed in plants treated with organic fertilizer, regardless of the studied water conditions (Table 5). These results Fig. 2. Mean of the isolated factors for root dry matter - RDM, factor fertilization** (a), root dry matter - RDM, factor water availability* (b), relative water content** - RWC (c), and total proteins* (d) in Hyptis suaveolens plants, as a function of fertilization. Equal letters in the columns do not differ by Tukey test at ** (p < 0.01) and * (p < 0.05). Bars in the columns indicate the standard error of the mean. 514 Industrial Crops & Products 125 (2018) 511–519 M.V. de Mesquita Arruda et al. Because of the influence of Na on the absorption of K and since it was found in high contents in the soil fertilized with poultry manure, the Na+ content was evaluated and higher contents were found in the root tissues of plants fertilized with this type of organic compost (Fig. 6a). High Na+ content in the roots of plants grown with poultry manure was probably due to the phytoextraction process, through which plants fertilized with this type of compost accumulated greater amount of Na in the root system. This fact was evidenced by the results of the translocation factor (TF), which demonstrate that, although all substrates showed TF below 1, this value was significantly lower in plants fertilized with poultry manure, in comparison to the other treatments (Fig. 6b). In the shoots, there were no significant differences between treatments regarding Na content. For H. suaveolens essential oil yield (OY), only the factor fertilization was significant and poultry manure led to higher oil percentage in the plants (0.17%) compared with NPK treatment (0.13%) and control treatment (0.12%) (Fig. 5c). Table 4 Summary of the analysis of variance for the contents of free proline, total soluble carbohydrates and total proteins in Hyptis suaveolens plants under the effect of the interaction between fertilization and water availability. Mossoró, Brazil, 2015. Source of variation Fertilizing (F1) Pot Capacity (F2) F1 x F2 Treatments CV (%) Degrees of freedom Values of F Free proline Total soluble carbohydrates Proteins 2 2 64.5328** 36.7923** 24.7349** 33.6845** 4.7049* 2.8461 ns 4 8 15.7570** 33.2098** 22.4 11.3040** 20.2569** 22.4 1.7011 ns 2.7383* 12.7 ** 1% level of significance (p < 0.01). * 5% level of significance (p < 0.05); ns, not significant (p > = 0.05); CV, Coefficient of variation. corroborate the observations made during the experiment, in which plants fertilized with poultry manure exhibited a much more accentuated green color compared with the other treatments, which justifies a high level of photosynthetic pigments. 4. Discussion The beneficial effect of poultry manure on H. suaveolens, increasing SDM production, PH and SD, had already been reported by Maia et al. (2008), who claimed that the increment of biomass was attributed to the increasing availability of nutrients to the crop. According to the authors, poultry manure has been indicated as a rich source of N, P and other nutrients, besides promoting better chemical and physical conditions in the soil. Probably, the reduction of growth in plants under severe water stress results from morphological and physiological alterations caused by the low water availability in the soil, which lead to reduction in leaf area production, stomata closure, acceleration of senescence and leaf abscission in the plants (Taiz and Zeiger, 2013). Plants grown in substrate containing NPK also showed higher mean values of SDM, PH, and SD, compared with the control, confirming that H. suaveolens responded well to organic and inorganic fertilization, but the yield in the chemical treatment was lower and it may have resulted from an insufficient dose of nutrients to meet the nutritional requirements of the crop. In a study on the effects of the interaction between water availability, organic fertilization (poultry manure), and inorganic fertilization (NPK) on okra growth, Willie et al. (2016) demonstrated that plants showed higher values of height when subjected to 100% water availability and fertilized with poultry manure or NPK. In contrast, Lourenço et al. (2013) obtained greater shoots and root dry matter production in common bean under mineral fertilization (NPK), due to the greater availability of N and P in the soil. As evidenced in Fig. 1b, R/S ratio remained balanced between the control treatment and those containing NS + NPK, regardless of the water availability level. Although the nutritional conditions of the natural soil (control) and that fertilized with NPK were not much favorable initially, they allowed a balanced development of the plants, with resources well distributed between shoots and roots. Plants grown with NS + PM showed a different result, because most of the produced energetic resources were allocated to shoots growth, allowing a crown with more leaves and a lower root development. Different results were 3.3. Nutritional aspects of H. Suaveolens under fertilization and water stress Regarding the accumulation of nutrients, there was interaction between the factors fertilization and PC (p < 0.05) for N and P contents in the leaves (Fig. 4A and B). Poultry manure incorporation to the soil promoted higher leaf N contents (0.9 mg mL−1) in H. suaveolens plants maintained at lower water availability (25% PC) (Fig. 4a). For the other treatments, the levels of this nutrient were similar, without difference, when plants were under good water conditions. The physical-chemical analysis of the substrates (Table 1) demonstrates that N contents were initially similar between treatments without fertilization and with organic fertilization, with a small difference in treatments with NPK. However, even under limited water conditions, poultry manure promoted the highest accumulations of this nutrient in H. suaveolens plants. NPK was the fertilizer that caused greater increment in P content in the shoots, but it decreased in the plant tissue as soil water potential decreased, although there was no statistical difference between treatments (Fig. 4b). For P contents in the roots, only plants fertilized with poultry manure showed greater root increment, and this treatment was statistically different from the others (Fig. 5d). For K contents, no interaction was observed between fertilization and water availability. In the shoots, the highest K accumulation occurred in plants fertilized with poultry manure and with NPK (Fig. 5a). Regarding K contents in the roots, water limitation led to greater K accumulation in the roots of plants subjected to lower levels of water availability (Fig. 5b). Although there was no significant interaction between the studied factors for leaf K contents, both treatments with fertilization maintained the highest values of this nutrient in substrates containing poultry manure (24.2 g kg−1 FM) and substrates containing NPK fertilization (22.3 g kg−1 FM). Fig. 3. Interaction between water stress and fertilization for the contents of free proline Profree (a) and total soluble carbohydrates – TSC (b) in Hyptis suaveolens plants. Columns followed by equal letters, lowercase for fertilization and uppercase for water availability, do not differ by Tukey test (**p ≤ 0.01). Bars in the columns indicate the standard error of the mean. 515 Industrial Crops & Products 125 (2018) 511–519 M.V. de Mesquita Arruda et al. Fig. 4. Interaction between water availability and growing substrate for the contents of total nitrogen - NTotal (a) and total phosphorus PTotal (b) in the shoots of Hyptis suaveolens plants. Means followed by the same letter, lowercase for fertilization and uppercase for water availability, do not differ by Tukey test (p ≤ 0.05). Bars in the columns indicate the standard error of the mean. RDM was influenced by fertilization and water regime, but individually. The highest means of root growth were obtained by plants grown in NS + NPK (Fig. 2a), maintained at 100% PC, with a reduction of growth as the PC decreased (Fig. 2b). In addition, the root system in this treatment exhibited a healthier aspect, with lighter-colored, longer and well-branched roots, differing from those in the NS + PM treatment, which showed darkened color and were less developed. The low values of RDM in the treatments NS (control) and NS + PM may be respectively related to the low natural fertility of the soil and to the large amount of nutrients supplied by the poultry manure, since both the lack and excess are harmful to root development (Mapeli et al., 2005). Despite being well aged, poultry manure supplies twice as much nutrients as the manure of other animals and large amounts of nutrients may cause disorders in the root system and reduce absorption (Kiehl, 2010). In contrast with the results obtained in this research, Muhumed et al. (2014) observed that root dry matter in Zea mays convar. Saccharata var. rugosa (sweet corn) plants was significantly higher compared with the control, when subjected to organic fertilization, and with water restriction. On the other hand, Scalon et al. (2011) reported greater root growth under favorable water conditions, thus indicating that the response to fertilization under limiting water conditions may vary according to the substrate and plant species used. The highest RWC was maintained in plants grown only with NS (control) (Fig. 2c), and there was a reduction of this variable in the other treatments, regardless of the PC level. Lenhard et al. (2010) also observed that different water regimes did not influence RWC in Caesalpinia leiostachya (‘Pau ferro’) seedlings in the initial growth stage. Plants can be modulated by various environmental factors and, under stress conditions, respond in different ways, such as with synthesis of osmoregulators (Taiz and Zeiger, 2013). Fertilization with NS + PM promoted excellent shoots development in H. suaveolens. Table 5 Summary of the analysis of variance, means and standard errors of the contents of chlorophyll a, b, and total in Hyptis suaveolens plants subjected to fertilization and different levels of pot capacity. Mossoró, Brazil, 2015. Source of variation Fertilizing (F1) Pot Capacity (F2) F1 x F2 Treatments Fertilizing Degrees of freedom Chlorophyll a Chlorophyll b Total chlorophyll 2 7.5073** 4.1014* 9.9238** 2 3.8652 ns 0.5225 ns 1.8624 ns 4 8 1.2151 ns 3.4507* Means ± Standart 24.8 b ± 69.4 Natural soil (NS) NS + NPK NS + Poultry Manure CV (%) Values of F (Photosynthetic pigments - μg g−1 MF) 0.8532 ns 1.5826 ns erros 13.1 b ± 35.8 29.3 ab ± 92.2 17.5 ab ± 46.0 36.0 a ± 202.8 19.6 a ± 76.9 20.56 29.23 0.9372 ns 3.4152 ns 28.2 b ± 97.2 34.5 ab ± 102.5 41.5 a ± 218.1 18.29 Means followed by the same letter in the column do not differ by theTukey test at 5% probability (p < 0.05). ** 1% level of significance (p < 0.01). * 5% level of significance (p < 0.05); ns, not significant (p > = 0.05); CV, Coefficient of variation. Transformed values to x=√x. reported by Corrêa et al. (2010), who claimed that the R/S ratio of Origanum vulgare (oregano) was not influenced by poultry manure fertilization and that the distribution of photoassimilates to shoots and roots was equivalent. Fig. 5. Potassium contents in the shoots [K+]leaf* (a) and roots - [K+]roots* (b), essential oil yield** (c), and total phosphorus content in the roots (P[Total]roots)** (d) in Hyptis suaveolens plants as a function of fertilization and water availability. Equal letters in the columns do not differ by Tukey test at ** (p < 0.01) and * (p < 0.05). Bars in the columns indicate the standard error of the mean. 516 Industrial Crops & Products 125 (2018) 511–519 M.V. de Mesquita Arruda et al. Fig. 6. Na+ contents (a) and translocation factor (TF) in the roots (b) of Hyptis suaveolens plants as a function of fertilization and water availability. Equal letters in the columns do not differ by Tukey test at ** (p < 0.01) and * (p < 0.05). Bars in the columns indicate the standard error of the mean. The increment of poultry manure in the soil provided H. suaveolens plants with an excellent nutritional source of N, P, and K to the leaves. Nitrogen stood out in the conditions of 25% PC (Fig. 4a), but such reduction of water potential allowed a slight decrease in leaf P contents and, although not significant, it affected plant growth in this treatment. For Dominghetti et al. (2014), it occurs due to the lower P diffusion from the soil solution to the roots and can affect biomass production. The use of N in the synthesis of N compounds and sugars, such as proline (Fig. 3a) and carbohydrates (Fig. 3b), combined with high leaf K contents (Fig. 5a), favored the osmotic adjustment in plants fertilized with poultry manure and at 25% PC, in response to the water stress and osmotic effect. This fact may have been caused by the excess of Na+ in the soil, thus causing an indirect effect, such as unfavorable physical conditions for plant growth, especially root growth (Dias and Blanco, 2010). Corroborating with the results of the present study, Cunha et al. (2012) also observed increase of N content in the shoots of lemon grass plants fertilized bovine manure at low water availability conditions. Fertilization (organic and mineral) favored the increase of K contents in leaves and roots (Fig. 5a and b). The presence of high Na+ contents in the substrate prepared with poultry manure did not affect either absorption or translocation of K to the shoots, even with higher water restrictions. According to Rosolem et al. (2003), provided that it is not limiting to the plant, water stress does not interfere with the mechanisms of K transport to the root tissue. This nutrient has high mobility in the plant at any level of concentration, whether within the cell, in the plant tissue, or in the xylem and phloem (Cunha et al., 2012). Increment of K contents in the leaf tissue may be related to the need for it in cell osmoregulation processes, since K actively participates in cell expansion and stomatal conductance. In addition, under water stress conditions, K requirement in the plant is increased, to maintain photosynthesis and protect chloroplasts from oxidative damages (Cakmak, 2005). H. suaveolens essential oil yield (OY) increased as a function of the organic fertilization, which promoted increment in shoots biomass. OY was superior to that reported by Silva et al. (2003a, 2003b), who found oil yield of 0.037% in the same species. Oil yield similar to that of the present study was reported by Zoghbi et al. (2008), who also studied H. suaveolens and obtained values ranging from 0.5 to 0.1%. Many reports with other species also claim that organic fertilization favors essential oil production, since it is associated with higher biomass production (Silva et al., 2003a, 2003b; Castro et al., 2007; Costa et al., 2008). Nevertheless, Pinto et al. (2014) observed that the stress in different irrigation intervals on Cymbopogon citratus (lemon grass) increased its essential oil production, despite not causing difference in dry matter yield and plant height. Increment of essential oil production in citronella grass plants under chemical fertilization has also been reported (Seixas et al., 2013). Although the water stress conditions in the present study did not favor the increase in oil yield, compared with the literature, H. suaveolens is known to be highly susceptible to water variations in the soil. Therefore, new studies under slightly more severe conditions are However, as previously mentioned, plants in this treatment associated with a low PC showed high energy expenditure in the synthesis of free proline and total soluble carbohydrates. The increase in the synthesis of these solutes is a response of the plant to the low water content in the leaf tissues (Fig. 2c), caused by both low water availability in the soil and high Na+ content in the poultry manure (Table 1). The association between both factors led to even more severe water limitation to the plant, which was evidenced by the low RWC (Fig. 2c). Additionally, the contents of total proteins were higher in this treatment, compared with the others, reinforcing the idea that these proteins are directly related to the synthesis of these osmoregulators (Fig. 2d). However, there was no influence of water availability on this variable. These results can be justified by the benefits promoted by the addition of poultry manure to the soil, such as improvement in chemical, physical, biological, and nutritional conditions (Table 1). According to Hoffmann et al. (2001) and Silva et al. (2004), besides improving soil physical properties, organic fertilization provides nutrients to plants, improves soil microbiota and increases the organic matter content, water retention, and cation exchange capacity of the soil. The production of photosynthetic pigments, under water stress conditions, is particularly affected because the loss of chlorophyll causes a progressive reduction in the capacity of the plants to absorb light energy for the synthesis of photoassimilates (Silva et al., 2014), but different results were found in the present study. The production of chlorophylls a, b and total was higher in treatments that received fertilization, at all PC levels (Table 5), demonstrating that the availability of nutrients attenuated the deleterious effect of water stress on the production of chlorophylls. These results are consistent with the observations made during the experiment, which evidenced that plants showed an intense green color, much more accentuated than that in the control treatment, especially those fertilized with poultry manure. Organic fertilization (poultry and bovine) promoted significant increase in the contents of chlorophyll a and b in Origanum vulgare (oregano) (Corrêa et al., 2010) and Ocimum selloi (green pepper basil) (Costa et al., 2008). According to these authors, such increment is due to the greater availability of nutrients such as N and Mg in the soil solution, which participate in the chlorophyll molecule structure. In the present study, both poultry manure and NPK were favorable to the development of H. suaveolens. However, the addition of organic fertilization stood out, for promoting greater availability of nutrients to plants. It is important to reinforce that plants fertilized with manure and under water stress, despite showing higher chlorophyll contents compared with the other treatments, under the same water conditions, showed lower SDM and RDM. This result can be attributed to the fact that plants fertilized with poultry manure had to osmotically adjust because of the large amount of Na+ retained in this compost, and such adjustment requires energy, which in general is diverted from biomass production. Increments in chlorophyll contents in tree species after being subjected to water stress were observed by Liu et al. (2011) and this result, according to the authors, may be related to the osmotic adjustment, which, combined with other physiological processes, guarantees plant survival under adverse water conditions. 517 Industrial Crops & Products 125 (2018) 511–519 M.V. de Mesquita Arruda et al. necessary. Regarding Na+ in the root, the contents of this salt were extremely high (1174.91 mg kg−¹ DM) in plants grown with poultry manure (Fig. 6a), which may justify the differences found in the root system in this treatment, especially at 25% PC. However, even under severe conditions, H. suaveolens plants did not show any sign of toxicity in the shoots along the experiment, which indicates that even under saline conditions H. suaveolens was able to survive by adapting to the environment, using the ability to maintain high K levels and low Na+ levels in the leaf tissue, and the highest content was stored in the root tissue. According to Dias and Blanco (2010), this ability is one of the key mechanisms that contribute to exhibiting higher tolerance to salinity. Based on H. suaveolens behavior under the studied conditions, high values of the factor of Na translocation to roots (Fig. 6b) indicate that, apparently, this species can be an alternative to be used as bioaccumulator in salinized areas. However, more-detailed studies on its behavior in salinized regions are necessary. Cakmak, I., 2005. The role of potassium in alleviating detrimental effects of abiotic stresses in plants. J. Plant Nutr. Soil Sci. 168 (4), 521–530. https://doi.org/10.1002/ jpln.200420485. Carmo, C.A.F.S., Araújo, W.S., Bernardi, A.C.C., Saldanha, M.F.C., 2000. Métodos de análise de tecidos vegetais utilizados na Embrapa Solos. Embrapa Solos, Rio de Janeiro. Castro, H.G., Barbosa, L.C.A., Leal, T.C.A.B., Souza, C.M., Nazareno, A.C., 2007. Crescimento, teor e composição do óleo essencial de Cymbopogon nardus (L.). Rev. Bras. 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Moreira, A.C.P., Lima, E.O., Wanderley, P.A., Carmo, E.S., Souza, E.L., 2010. Chemical 5. Conclusions Hyptis suaveolens showed favorable development, under good conditions of water and fertilization. Poultry manure addition to the soil provided better growth conditions to the plants, expressed with higher values of shoots dry biomass, under 100 and 50% PC water conditions. Under water stress conditions of 25% PC, plants in this treatment showed a remarkable capacity of adaptation, using means such as: reduction of growth, osmotic adjustment and Na+ storage in the root, thus maintaining the high levels of production of total proteins and chlorophylls a, b and total. Because of the greater biomass production, promoted by poultry manure, oil production was favored by this type of fertilization. However, compared with the literature, the value obtained in the present study is still considered as low. 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