Effects of Water Stress, Nitrogen, Magnesium and their Interactions on Some Growth Characteristics and Essential Oil content of Rosemary ( Rosmarinus officinalis L.)

: A factorial experiment was applied with four replicates on rosemary plants ( Rosmarinus officinalis L.) grown in pots inside the glasshouse of the Department of Biology, College of Science, Salahaddin University, Erbil, Iraq, during April, 2019 to July, 2020, to determine the effects of soil moisture content ( SM1: 100% and SM2: 60% field capacity), nitrogen fertilizer (N1: 100, N2: 200 and N3: 300kg/hectare), and magnesium fertilizer (Mg1: 0.0, Mg2: 30 and Mg3: 60kg/hectare) and their interactions on some growth characteristics and essential oil content of rosemary plants. Two cuttings were taken from rosemary shoots (on March, 2020 and July, 2020) after 12 and 15 months of planting respectively. Results showed that cutting 1: SM2 decreased plant height, number of branches, Shoot fresh weight (FW), shoot dry weight (DW), leaves DW & stem DW. N3 increased plant height, number of branches, Shoot FW, shoot DW, leaves DW, stems FW & stem DW. Interaction treatment SM1N3Mg3 increased plant height, Shoot FW, shoot DW, leaves DW, stems FW and stem DW, while in cutting 2: SM2 decreased shoot DW, stems FW and stem DW. N3 increased Shoot FW and stem DW. Mg3 increased Root FW, but decreased stems FW. SM1N3Mg1 increased stem DW. In cutting 1+ cutting 2, SM2 decreased accumulative stem DW and biological yield FW. N3 increased accumulative Shoot FW; Accumulative leaves DW, accumulative stem FW, accumulative yield FW and biological yield DW . Mg3 increased biological yield FW, while SM1N3Mg3 treatment increased accumulative shoot DW, accumulative leaves DW, biological yield FW, and biological yield DW. In the second harvest, SM2 and Mg2 decreased rosemary essential oil %, N3 and SM1N3Mg2 increased EO% and concrete content, while SM1Mg2 increased EO.


Introduction:
Rosemary (Rosmarinus officinalis L.) is an aromatic medicinal plant, grown under a wide range of climates, endogenous to Europe, Asia and Africa, mainly in areas surrounding the Mediterranean Sea 1 .The plant is one of the Mediterranean aromatic shrubs containing phytochemicals with flavoring and pharmaceutical uses.The plant is impressive and handsome at all times, especially when covered with its lovely flowers that attract butterflies and bees (Fig. 1).Rosemary is a member of Lamiaceae family and well known for its biologically active essential oils 2 .The plant is cultivated mainly for essential oil production, food industry, medicine and in cosmetics 3 .The volatile oil of rosemary is used as perfume in soaps, shampoos and ointments.Morocco, Spain, USA, and Tunisia are considered as leading countries in production of rosemary essential oil 4 .Rosemary contains different chemical compounds such as phenolic acids, flavonoids, essential oils, triterpenic acids and triterpenic alcohols 5 .These chemical components, such as essential oils, are present in small concentrations in the plant tissues and organs.Rosemary production depends upon several biotic and abiotic factors such as soil moisture content, nutrient elements availability, environmental conditions such as light intensity and duration and others 6,7 .Soil moisture content is the most important factor in plant producing; plants absorb about 500 g of water to produce every gram of organic matter.Water makes up most of the mass of plant cells and water is considered a medium that most of the biochemical reactions and activities occur in through the plant life 8 , therefore water stress affects negatively on plant growth, development and production.It was reported that water deficit altered the morphology of rosemary plants, reduced growth parameters, oil yield, and photosynthetic productivity 9,10 .

Figure 1. Rosemary plant (shoots and flowers).
Nitrogen fertilization is considered the main factor for increasing plant production 11 , it plays an important role in various physiological processes.Nitrogen imparts dark-green color in plants, promotes leaves, stem, roots and other plant tissues growth and development and considered an essential constituent of proteins, amino acids, and chlorophyll.Nitrogen affected positively on the plant growth and increased their content of effective compounds such as volatile oils 12,13 .Magnesium (Mg) is one of the essential elements in plant nutrition because of its effectiveness on many physiological processes in the plant, such as photosynthesis and enzyme activation 14 , in addition to the presence of magnesium ion in the center of the chlorophyll molecule 15 .Magnesium plays important roles in many physiological activities such as formation and development of sink organs like seeds and roots 16 .Mg deficiency mostly occurs in the regions where highly acidic weathered soils are spread with its more removal from soils takes place 17 .Magnesium deficiency affects negatively on photosynthesis and carbohydrate partitioning in plants 18 .

Material and Methods:
A factorial pot experiment (2x3x3) was applied according to Completely Randomized Design (CRD) with four replications.The first factor was soil moisture (SM) (100% and 60% field capacity (FC)), the second factor was Nitrogen fertilizer (100, 200 and 300kg/ha), and the third factor was Magnesium (0.0, 30 and 60kg/ha).A sample of the soil used in the experiment was prepared after air drying, then grind and passed through a sieve with holes diameter of 2 mm.The soil sample was analyzed in the central laboratory of the College of Agriculture/ University of Mosul-Iraq (Table 1).Some soil minerals were estimated using X-ray Fluorescence (XRF) apparatus 19,20 .Nitrogen was estimated by Kjeldahl method, Potassium estimated by Spectrophotometer method, Mg and total phosphorus determined as mentioned by 21 .The field capacity (FC) of the soil was estimated using the method described 22 which was 35%.Soil texture was evaluated using the pipette method 23 ..using a sharp knife to take 7.5cm tall cuttings from young shoots just below the leaf joint at the stem.Leaves were stripped from the lower 4 cm, pulling them off by fingers; the cuttings were immersed two minutes in a solution (1000 ppm) of Indole-3-Butiric acid 27 .Pots were irrigated as needed according to field capacities.After 14 days of planting, the thinning and replanting processes were done to all pots in the study.Nitrogen fertilizer doses (100, 200 and 300 kg/hectare) were prepared from urea (46% N) and Magnesium doses (0.0, 30 and 60 kg/hectare) were prepared from Magnesium oxide MgO (56% Mg).Fertilizers were foliar applied on rosemary plant shoots once, except the nitrogen fertilizer which was applied in two equal half doses, the first half was applied in the same time of spraying other fertilizers and the second half was applied two months before harvesting, using hand sprayer.Tween 40 was used at 1:1000 (V: V) to the spray solution as a surfactant.Irrigation was carried out as needed using tap water according to the field capacities 28,29 .Plant shoots were harvested by cutting the shoots 15 cm above the ground on April, after twelve months of planting (1st cutting) and July (2nd cutting) respectively.Shoot fresh weights were registered and then dried in perforated paper bags (with holes) for ten days at room temperature with continuous stirring on both sides 26 .

Plant growth characteristics:
The following growth parameters were registered for cutting 1 and cutting Ten g of dry leaves (cut2) were extracted with 100 ml of n-hexane as a solvent in a Soxhlet apparatus for two hours, n-hexane was evaporated by rotary evaporator and rosemary concrete (a waxy, greenish semi-solid material) was obtained.The concrete was weighted for each sample, then dissolved in 15ml of Ethanol 95 % , alcoholic solution was cooled and filtered through filter paper at -18°C in freezer, ethanol with volatile oil (essential oils) passed throw the filter paper, while the fixed oil and wax particles were stay on the filter paper.The weight of concrete was recorded, also RMEO was determined using spectrophotometer at 300nm, using the calibration curve (Fig. 2), constructed by dissolving pure RMEO in ethanol at 1:4 (V: V), then a series of standard solutions were prepared, which were measured spectrophotometrically, ethanol 95% was used as blank 30,31 .Table 2 shows that water deficit (soil moisture 60% FC) decreased plant height significantly (37.6cm) compared to SM1 (100% FC) which registered 38.2 cm.This result is in agreement with results obtained by 32 .Water deficit also affected negatively on stomatal conductance and cellular turgor pressure, therefore affects negatively on plant growth, cell expansion and plant height.Nitrogen applied at 300kg/hectare N3 caused a significant increase in PH 38.9 cm, this result is in agreement with results of 33,34 , but it didn't agree with results of 35 .The interaction treatment SM2N1 decreased the PH significantly to 34.9 cm; N1Mg1 also decreased the rosemary PH significantly to 35 cm, which was in term with results of 36  Results are in agreement with those obtained by 39,40 .

Effects of Soil Moisture, Nitrogen and Magnesium application and their interactions on leaves dry weight (LDW g/ plant):
Table 2 shows that SM2 decreased rosemary leaves dry weight significantly 7.613g compared to SM1 which registered 8.162 g, but N3 increased the plant LDW significantly to 9.489g and the result does not agree with the results of 41 .Interaction between SM and N doses affected significantly on LDW, SM1N3 was superior and registered the highest LDW (10.168g).Interaction treatments N3Mg1, N3Mg2 and N3mg3 increased the LDW significantly.
Triple interaction treatment SM1N3Mg3 also increased the LDW significantly to 11.193g.

Effects of Soil Moisture, Nitrogen and Magnesium application and their interactions on stems dry weight (St. DW g/ plant):
Nitrogen fertilizer at 300kg/ha increased rosemary stem dry weight significantly (1.574g).SM1N3 also increased LDW significantly (1.792g).Interaction treatments N3Mg1, N3Mg2 and N3Mg3 also increased the LDW significantly.Triple interaction treatments SM1N3Mg1 and SM1N3Mg3 increased the LDW significantly to 1.903g and 1.883g respectively.

Effects of Soil Moisture, Nitrogen and Magnesium application and their interactions on basal stem diameter (St. D mm):
Significant drop in stem diameter was registered in SM2 3.49mm compared to 3.74mm in SM1, which was in term with the results obtained by 10 , who reported that deficit irrigation significantly reduced growth parameters.Interaction treatment SM2N1 also registered a significant decrease in St. D 3.24 mm.But triple interaction treatment SM1N3Mg3 caused a significant increase in St. D of rosemary plants 3.995mm, which was the highest value, while the lowest value was recorded in triple interaction treatment SM2N1Mg1 3.02mm, and results did not match with results of 36 .The means in each column followed by the same letters are not significantly different at P≤ 0.05 according to Duncan's Multiple Range Test.

Second Cutting (Cut.2)
Effects of Soil Moisture, Nitrogen and Magnesium application and their interactions on shoots fresh weight (Sh.FW g/ plant): Table 3 shows that all the factors and their interactions have no significant effects on shoot fresh weights of rosemary except the interaction treatment SM2N1 which reduced the Sh.FW significantly to 49.88 g compared to 57.82g for SM1N1.These results are in agreement with that obtained by 38  The means in each column followed by the same letters are not significantly different at P≤ 0.05 according to Duncan's Multiple Range Test.

Effects of Soil Moisture, Nitrogen and Magnesium application and their interactions on concrete % in leaves dry weight cut2:
Soil moisture deficiency (SM2) caused significant decrease in concrete% in leaves dry weight cut2 and registered 13.7%.Interaction treatment SM1N3 increased concrete% significantly to 16%.SM1Mg2 & SM2Mg1increased concrete % significantly by 15.8 & 14.9% respectively.Triple interaction treatment SM1N3Mg2 caused significant increase in concrete % by 17.9% compared to the treatment SM2N1Mg2 which registered 11.4% (Table 7).Effects of Soil Moisture, Nitrogen and Magnesium application and their interactions on RMEO g/plant in leaves dry weight cut2: Water deficiency (SM2) decreased the amount of RMEO g/plant significantly by 0.17g compared with 0.2g in SM1.N3 increased RMEO significantly (0.22g) compared to N1 and N2.SM1N2 and SM2Mg2 reduced RMEO significantly (0.12g) and the results were agreement with results of Said-Al Ahl 50 in Origanum vulgare L, but N3Mg1 and SM1N3Mg2 registered significant increase by 0.24g and 0.32g respectively.And the triple interaction SM1N3Mg2 increased the RMEO significantly to 0.32g.

Effects of Soil Moisture, Nitrogen and Magnesium application and their interactions on concrete g/plant in leaves dry weight cut2:
Water deficiency decreased the amount of concrete significantly to1.4g.N3 caused significant increase of concrete in leaves dry weight cut2 to 1.7g.Interaction treatments SM1N2 and SM2Mg2 reduced the concrete in rosemary plants significantly to1.3g and 1.1g respectively, but N3Mg2 registered significant increase (1.9g).The triple interaction treatment SM1N3Mg2 increased the concrete in leaves dry weight cut2 significantly to 2.4g.The means in each column followed by the same letters are not significantly different at P≤ 0.05 according to Duncan's Multiple Range Test.

Conclusions:
From the results of the present study, we can draw the following conclusions: Water adequacy (SM1) affects positively and records the highest increases in all rosemary plant growth characteristics, except the stem diameter.Nitrogen application at 300 kg/hectare increases all growth characteristics.Water deficiency (SM2) affects negatively and decreases growth characteristics.
Magnesium application at 60kg/hectare increases the roots fresh weight, roots dry weight and biological yield.The interaction treatment SM1N3 increases most of the growth characteristics of rosemary plants.Triple interaction treatment SM1N3Mg3 increases growth characteristics and plant biomass in cut1 and in cut1+2, except the number of branches/plant.Rosemary plants require 100% field capacity of water supply in order to gain the highest plant growth and biomass.Application of nitrogen and magnesium (at 300 and 60 kg/hectare respectively to rosemary plants) is necessary for high plant growth and biomass and to enable plants avoiding the adverse effects of water deficiency.Water deficiency affects negatively on rosemary essential oils%, but nitrogen supply mitigates the negative effects of water deficiency on essential oils.Interactions between adequate nutrient elements affects positively on rosemary essential oils.Further studies are necessary to determine the water stress line under which rosemary plants can't naturally grow and develop to avoid adverse effects on plant growth and development.More studies are necessary about the combined nutrient elements doses and their interactions to determine the proper fertilizer combinations and getting the highest plant growth, biomasses, essential oils and good quality.

Authors' declaration:
-Conflicts of Interest: None.-We hereby confirm that all the Figures and Tables in the manuscript are mine ours.Besides, the Figures and images, which are not mine ours, have been given the permission for re-publication attached with the manuscript.
-Ethical Clearance: The project was approved by the local ethical committee in Erbil Polytechnic University.

Figure 2 .
Figure 2. Calibration curve for evaluation rosemary essential oil concentration.
and Magnesium application and their interactions on RMEO % in leaves dry weight cut2, and RMEO g/plant in leaves dry weight cut2, concrete % in leaves dry weight cut2, concrete g/plant in leaves dry weight cut2: Effects of Soil Moisture, Nitrogen and Magnesium application and their interactions on RMEO % in leaves dry weight cut2: Table

Table 1 . Some chemical and physical properties of the soil used in the study.
Cuttings for implanting occurred in mid-April by selecting a healthy looking rosemary plants (more than one year old) with lots of young new growing branches on it

Effects of Soil Moisture, Nitrogen and Magnesium application and their interactions on shoot fresh weight (Sh. FW g/plant):
Table 2, shows that SM2 decreased rosemary shoot fresh weight significantly 23.613g.Researchers demonstrated that the shortage in soil moisture content decreases plant growth characteristics such as plant weight and plant height 32.N3 increased the plant Sh.FW significantly to 30.23g compared to N1&N2 21.128 &23.059g respectively.
38searchers showed that nitrogen application caused significant increases in the accumulated dry mass of the aerial plant parts38.Interaction between soil moisture and nitrogen doses affected significantly on Sh.FW, so SM1N3 was superior and registered the highest Sh.FW 33.014g.Interaction treatments N3Mg1, N3Mg2 and N3Mg3 also increased the Sh.FW significantly.Triple interaction treatment SM1N3Mg3 also increased the Sh.FW significantly to 35.655g and was superior compared to other triple interaction treatments.Effects of Soil Moisture, Nitrogen and Magnesium application and their interactions on shoot dry weight (Sh.DW g/ plant):Table2shows that SM2 decreased rosemary Sh.DW significantly (8.805g) compared to SM1 which registered 9.479g.N3 increased the plant Sh.DW significantly to 11.063g.Interaction between soil moisture and nitrogen doses effected significantly on Sh.DW and SM1N3 was superior and registered the highest Sh.DW 11.9 g.Interaction

of Soil Moisture, Nitrogen and Magnesium application and their interactions on basal stem diameter (St. D mm):
43,44 Mentha citrate plants.Table3shows that N3 caused a significant decrease in stem diameter 9.8 mm and results disagreed with results obtained by43,44about Jatropha curcas and Broccoli.Interaction treatment SM2N3 significantly decreased St. D of rosemary plants 9.399mm.All interaction treatments SM Mg, N Mg and SM N MG affected positively on St. D of rosemary plants.