Abstract: Background and Objective: Oregano herb has various pharmaceutical properties. Soil Moisture (SM) deficit has harmful effects on aromatic plants. So, the aim of this study was to decrease the harmful effect of SM deficit on oregano plants by adapting them through the use of glutamic acid (GLU). Materials and Methods: Oregano plants were divided into two main groups. The first group was subjected to different levels of SM: 100, 75, 50 and 25% corresponding to the Field Water Capacity (FWC), while second group was subjected to the same SM levels but GLU was added at 0.3 g L–1 as foliar spray. Fresh Mass (FM), Dry Mass (DM), Volatile Oil (VO) percentage and milliliter 100 plant–1, constituents of VO and proline (PRO) were identified. Results: The SM with GLU treatments increased the FM and DM of herb, VO and constituents of VO and PRO contents compared with the treatments of SM without GLU. Greatest FM, DM and VO were obtained with the treatment of 75% SM×GLU. The values were 59.2, 73.9, 23.8 and 29.8 g plant–1 and 7 and 9 mL 100 plant–1 of both seasons. The maximum values of PRO were resulted under the treatment of 25% SM×GLU with the values of 2.3 and 2.7 μmol g–1. The highest amounts of major components (carvacrol, p-cymene and γ-terpinene) were recorded with the treatments of 25% SM×GLU with the values of 42.9, 24.1 and 17.6%. About 25 and 50% of SM×GLU treatments resulted in the highest values of monoterpene hydrocarbons, MCH (47.5%) and oxygenated monoterpenes, MCHO (47.1%) while 75% of MS without GLU treatment produced the highest amounts of sesquiterpene hydrocarbons, SCH (6.2%) and oxygenated sesquiterpenes, SCHO (5.4%). Conclusion: It can use GLU to decrease the harmful effect of SM deficits.
INTRODUCTION
Origanum vulgare L., family Lamiaceae is commonly named oregano. It is cultivated and distributed in many places of the world of temperate climates of North Africa, Europe, Asia and America1-2. It is used to treat cough, sore throats and relieve digestive complaints3. The VO of oregano has different antimicrobial4 and antioxidant activities5.
The SM deficit limits the production of the agricultural lands in the world6. Yield and metabolites of agricultural crops can be affected by SM factor7. In the aspect of aromatic plants, drought causes significant changes in growth, yield and some metabolite products such as PRO and VO compositions8.
The chemical constituents of cumin herb were significantly affected by SM deficit conditions9. Morphological measurements (FM and DM) were reduced as SM decreased but the contents of PRO were increased of some aromatic plants such as basil species, calendula, lemon balm, apple geranium and black cumin herbs10-14.
The SM was effective in changing the yield (FM and DM) and VO composition of oregano plants15. In some pervious studies, Fatima et al.16 found that citronella grass VOs were increased under SM deficit factor. The VOs and its main constituents of basil species, calendula, lemon balm, apple geranium and black cumin were promoted under SM deficit conditions10-14. By contrast, rosemary and anise VOs were decreased17-18. On the other hand the Achillea VO yield was increased under limited SM but the main constituent was decreased19.
Accumulations of amino acids were detected in plants under a biotic stress factors such as SM deficit which has different roles in plants such as acting as osmolyte, regulation of ion transport, modulating stomatal opening, detoxification of heavy metals, synthesis and activity of some enzymes, gene expression and redox-homeostasis20. On the other hand, FM and DM of croton plants increased with GLU treatments21. The FM and DM of datura, lemon grass and basil were significantly increased under some amino acids treatments22-25. Amino acids promoted FM, DM, VO and main constituents of VO (farnesene, bisabolol oxide B, α-bisabolol, chamazulene and bisabolol oxide) extracted from chamomile flowers26. Amino acids had significant effects on the FM and DM, VO and its constituents (2,2-dimethyl butanoic acid, isobutyl isobutyrate, α-isophorone, thymol, fenchyl acetate and linalool) of khella plants27.
Some studies are available where accumulation of GLU in cotton and rice under SM deficit factor28-29. Thus, this study aimed to reduce the hazards effects of SM deficit on oregano plants by adapting them through the use of GLU.
MATERIALS AND METHODS
Experimental: Experiments were carried out in a greenhouse at National Research Centre, Egypt, during 2015 and 2016. Oregano seedlings were obtained from the Institute of Medicinal and Aromatic Plants, Egypt. Uniform seedlings were transplanted into plastic pots (30 cm diameter and 50 cm height). In the 1st week of June during both seasons, the pots were transferred to a greenhouse adjusted to natural conditions. Each pot was filled with 10 kg of air-dried soil. Three weeks after transplanting, the seedlings were thinned to three plants per pot. Pots were divided into two main groups. The first group was subjected to different levels of SM: 100, 75, 50 and 25% corresponding to the Field Water Capacity (FWC) determined in the field (by weight). The second group was subjected to the same treatments but GLU was added at 0.3 g L–1 as foliar spray. All agricultural practices were conducted according to the main recommendations by the Ministry of Agriculture, Egypt. Physical and chemical properties of the soil used in this study were determined according to Jackson30 and Cottenie et al.31 are presented in Table 1.
Harvesting: At full bloom, the plants were harvested twice (first and second harvests) during the growing seasons by cutting the plants 5 cm above the soil surface. Total FM and DM (g plant–1) were recorded.
Volatile oil isolation: The FM (aerial part) was collected from each treatment during the 1st and 2nd harvests in both seasons; air dried and weighed to extract the Volatile Oil (VO), then 100 g from each replicate of all treatments was subjected to hydro-distillation (HD) for 3 h using a Clevenger-type apparatus32. The VO content was calculated as a relative percentage (v/w). In addition, total VOs (mL 100 plant–1) were calculated by using the DM. The VOs extracted from oregano were collected during the 1st and 2nd harvests in both seasons from each treatment and dried over anhydrous sodium sulfate to identify the chemical constituents.
| Table 1: | Physical and chemical properties of the soil used |
| OM: Organic matter, EC: Electronic conductivity | |
GC-MS analysis: The GC-MS analysis was carried out with an agilent 5975 GC-MSD system. The DB-5 column (60 m×0.25 mm, 0.25 mm film thickness) was used with helium as carrier gas (0.8 mL min–1). The GC oven temperature was kept at 60°C for 10 min and programmed to 220°C at a rate of 4°C min–1 that was kept constant at 220°C for 10 min and followed by elevating the temperature to 240°C at a rate of 1°C min–1. Split ratio was adjusted at 40:1. The injector temperature was set at 250°C. Mass spectra were recorded at 70 eV. Mass range was m/z 35-450.
Gas chromatography analysis: Gas Chromatography (GC) analysis was carried out using an agilent 6890N GC system using Flame Ionization Detector (FID) temperature of 300°C. To obtain the same elution order with GC-MS, simultaneous auto injection was done on a duplicate of the same column at the same operational conditions. Relative percentage amounts of the separated compounds were calculated from FID chromatograms.
Identification of components: Identification of the VOs components were carried out by comparison of their relative retention times with those of authentic samples or by comparison of their Retention Index (RI) to series of n-alkanes. Computer matching against commercial (Wiley GC/MS Library, Mass Finder 3 Library)33-34 and in-house "Baser Library of Volatile Oil Constituents" built up by genuine compounds and components of known oils. Additionally, the previous study of Joulain and Koenig35 were also used for the identification.
PRO determination: The PRO was determined at both seasons in fresh leaves according to Bates et al.36 as follows: Samples: Fully expanded (sun) leaves from pot-grown oregano plants were sampled, purified PRO was used to standardize the procedure for quantifying sample values. Reagents: Acid-ninhydrin was prepared by warming 1.25 g ninhydrin in 30 mL glacial acetic and 20 mL, 6 M phosphoric acid with agitation until dissolved. Kept cool (stored at 4°C) the reagent remains stable 24 h; there are following procedure: (1) Approximately 0.5 g of plant material was homogenized in 10 mL of 3% aqueous sulfosalicylic acid and the homogenate was filtered through whatman No. 2 filter paper, (2) Two milliliter of filtrate was reacted with 2 mL acid ninhydrin and 2 mL of glacial acetic acid in test tube for 1 h at 100°C and the reaction terminated in an ice bath, (3) The reaction mixture was extracted with 4 mL toluene, mixed vigorously with test tube stirrer 15-20 sec and (4) The chromophore containing toluene was separated from aqueous phase, warmed to room temperature and the absorbance read at 520 nm using to standard curve and calculated on a fresh weight basis as follow:
(μg PRO/mL×mL toluene/15.5 μg/μmol) {(g sample)/5} = μmoles PRO/g of fresh material
Statistical analysis: In this experiment, 2 factors were considered: SM: 100, 75, 50 and 25% and GLU (with and without). For each treatment there were 5 replicates, each of which had 10 pots; in each pot 3 individual plants were planted. The experimental design followed a complete random block design. According to Snedecor and Cochran 37 the averages of data were statistically analyzed by using 2 ways analysis of variance (ANOVA). Significant values determined according to p-values (p<0.05 = significant, p<0.01 = moderate significant and p<0.001 = highly significant). The applications of that technique were according to the STAT-ITCF program38.
RESULTS
Effect of SM, GLU and their interactions on the FM and DM: The SM with or without GLU affected yield of herb (FM and DM) during both seasons (Table 2). In general, FM and DM decreased under the various SM levels, especially at 50 and 25% FWC. The SM with GLU treatments caused an increase in FM and DM compared with SM without GLU treatments. The heaviest FM and DM were recorded at the treatment of 75% FWC×GLU with the values of 59.2, 73.9 and 23.8, 29.8 g plant–1 during 1st and 2nd seasons, respectively. The changes in FM and DM were highly significant for SM with or without GLU except the DM at 2nd season were insignificant for the interactions between SM and GLU.
Effect of SM, GLU and their interactions on VO composition: The deficit of SM (less than 100%) caused an increase in VO (%) during both seasons (Table 3). The VO (%) was increased under SM levels with GLU compared with SM without GLU treatments. The highest VOs (%) were detected at the lowest SM level (25%)×GLU with the value of 0.5% at both seasons. The VO yields (mL 100 plant–1) affected by the amount of SM with or without GLU. The SM×GLU caused an increase in VO yield comparison with SM without GLU. About 75% of SM×GLU treatment recorded the highest yield of VO with the values of 7 and 9 mL 100 plant–1 during both seasons. The changes in VO (% or yield) were significant or highly significant for SM or GLU treatments while it was insignificant for SM×GLU treatments (Table 3).
Quantity and quality of constituents present with SM and GLU levels in oregano VO were investigated. Twenty two components were detected, ranged from 99-99.9% of total VO and classified into four chemical classes i.e., monoterpene hydrocarbons (MCH), oxygenated monoterpenes (MCHO), sesquiterpene hydrocarbons (SCH) and oxygenated sesquiterpenes (SCHO) (Table 4). Monoterpenes (MCH+MCHO) was the major class (more than 85%).
| Table 2: | Effect of SM, GLU and their interactions on the yield (FM and DM) |
| GLU: Glutamic acid, M: Mean, SM: Soil moisture, SD: Standard deviation, FM: Fresh mass, DM: Dry mass | |
| Table 3: | Effect of SM, GLU and their interactions on VO (percentage and mL 100 plant–1 ) and PRO contents |
| GLU: Glutamic acid, M: mean, SM: Soil moisture, SD: Standard deviation, VO: Volatile oil | |
| Table 4: | Effect of SM, GLU on the VO constituent |
GLU: Glutamic acid, M: Mean, SM: Soil moisture, SD : Standard deviation, MCH: Monoterpene hydrocarbons, MCHO: Oxygenated monoterpenes, SCH: Sesquiterpene hydrocarbons, SCHO: Oxygenated sesquiterpenes, RI: Retention index; α: alpha, β: beta, γ: gamma, p: Para |
|
Carvacrol, p-cymene and γ-terpinene were detected as major components which gave the highest values with all SM or GLU×SM treatments. All major components increased under SM or the interaction between SM and GLU treatments compared with control (100% of FWC). The highest amounts of major components were recorded with the treatments of 25% SM×GLU with the values of 42.9, 24.1 and 17.6%, respectively. The highest amounts of MCH (47.5%) and MCHO (47.1%) were obtained from the treatments of 25 and 50% of SM×GLU. The greatest values of SCH (6.2%) and SCHO (5.4%) were obtained from the treatment of 75% of MS without GLU. The changes in all constituents were insignificant for investigated treatments except the components of borneol and β-caryophyllene were significant. The changes in all chemical classes were significant for different treatments.
Effect of SM, GLU and their interactions on the PRO content: Treating oregano plants by various levels of SM, GLU and their interactions promoted the accumulation of PRO. The highest amount (2.3 and 2.7 μmol g–1) of PRO content produced from the treatment of 25% SM content×GLU during both seasons.
DISCUSSION
The decrease of herb yield (FM and DM) under low SM levels (50 and 25% of FWC) during both seasons may be due to exposure to injurious SM causing reduction of turgor which would result in reducing plant growth and development of cells, especially in the herb (stems and leaves)39. Low SM reduces plant cell development, so the leaves and plant size will be smaller40. When the size of leaf is smaller, the capacity to trap light reduces too and the capacity of total photosynthesis reduces, i.e., photosynthesis is restricted in low SM cases, with a subsequent inhibition in plant growth and performance40. Low SM resulted in significant reduces in fresh and dry mater of Japanese mint, basil species, calendula, lemon balm, apple geranium and black cumin41,10-14. On the other hand, under SM deficit the available water does not move into the root cells. Water loses in transpiration and not be completely replaced, resulting in turgor loss. In the guard cells which surrounding the stomatal pore, the turgor decreases, the cells fill the pore and the stomatal pore reduces, so the transpiration reduces. The uptake of CO2 and the carbon assimilation rate of the plant are reduced when the stomata are closed. The duration of water deficit affected in reducing the crop production and causing injury to chloroplasts. There may also be an interaction with other stresses, such as heat stress, when transpiration is reduced that will also contribute to the strain on the plant42. The effect of SM on VO contents, its constituents and chemical classes may be due to the influence on enzyme role and metabolism activities of VO productivity43. The VO contents of Parthenium argentatum, peppermint, hyssopus and anise were enhanced and there were significant quantitative variations among the VO in terms of chemical constituents44-47. Bunium persicum VO and its constituents were affected by soil SM treatments48. Regarding to the oregano VO composition, similar constituents were found by Said-Al Ahl15 and Teixeira et al.49, they said that the major constituents of VO extracted from oregano herb were carvacrol, p-cymene and γ-terpinene as well as the components belong to different classes (MCH, MCHO, SCH and SCHO). The accumulations of PRO were promoted by decrease of SM levels during the first and second seasons. These results are in accordance with those obtained by Slama et al.50 as well as Blum and Ebercon51, they reported that PRO is regarded as a source of energy, carbon and nitrogen for recovering tissues under soil moisture deficits.
The positive effects of GLU on FM, DM, VO and PRO contents under SM deficit confirmed by some previous studies, i.e., GLU promoting the auxin synthesis in plants52, auxins play an important role in plant development such as growth of root system, vascular tissue differentiation, auxiliary bud formation, apical dominance and flower initiation under stress factors53. Azimi et al.54 revealed that amino acids have a positive effect on plant, root development, yield and significantly mitigates injuries caused by environmental stresses. Amino acids are crucial to sustain cellular functions under the soil moisture deficits55. Amino acids can improve the yield and PRO content under SM deficits56-57. The VOs (percentage, yield and constituents) of chamomile and khella were significantly affected by amino acids treatments26-27. Omer et al.58 indicated that amino acids increase the VO content and major constituents of chamomile. Saburi et al.59 reported that basil VO was improved with the treatments of amino acids.
CONCLUSION
It can be concluded that SM resulted in a highly significant reduction of FM and DM of oregano herb while PRO, VO and main constituents of VO were increased. The GLU×SM recorded higher values of all measurements than SM treatments. Adapting oregano plants to SM conditions through the use of GLU is very important especially in arid and semi arid regions for increasing the yield and active constituents such as VO of oregano plants.
SIGNIFICANT STATEMENT
Previous studies indicated that reducing SM limits the quantity and quality of oregano herb. In this study the effect of GLU on oregano herb were carried out under SM stress factor. Results showed that GLU promotes yield and active principal of oregano herb under SM deficit. It means, using GLU to decrease the harmful effect of SM deficits.