Effect of irrigation system and soil conditioners on growth and essential oil of Rosmarinus officinalis L. cultivated in Egypt

A relevant improvement of the cultivar conditions of Rosmarinus officinalis L. in desert areas was achieved by a specific combination between irrigation system and soil conditioner. A drastic reduction of water employment was obtained without affect the quality of the plants, determined by monitoring growth parameters and essential oil characteristics. In particular, the effect of surface and subsurface drip irrigation systems and different soil conditioners on growth parameters, yield, and essential oil constituents of rosemary plant was assessed. Field experiments at the Agricultural Research Station (Al-Adlya farm), SEKEM group Company, El-Sharkiya Governorate, Egypt, conducted over the two seasons revealed the effectiveness of the subsurface irrigation system in obtaining better performances, especially in terms of water saving. The combination of subsurface irrigation and the conditioner Hundz soil with bentonite showed the maximum mean values of growth characters compared with other soil amendments during both seasons. The possibility to employ a water-saving irrigation system as the subsurface one without any drawback in the resulting plants was also explored in terms of molecular composition. GC-MS analysis of the essential oil extracted from plants growth under different irrigation conditions revealed a comparable composition in both cases. The goodness of the most performing system was also confirmed by the comparable yield of the essential oil.


Introduction
Water is a very important and economical resource and it represents a major limiting factor for sustainable agriculture in arid and semi-arid regions [1]. In Egypt, all cultivated lands are characterized by an arid or semi-arid climate and the water required for agricultural and horticultural crops is obtained mainly through irrigation systems which consume about 83% of the country's available fresh water [2]. The demanding need of water is in part determined by the low field application efficiency [3], which in most traditional irrigation methods is less than 50% or lower, often under 30% [4]. Thus, the interest toward developing and adopting new and efficient water irrigation systems, particularly in arid and semi-arid regions, is very high [5]. In areas like Egypt, this problem is particularly evident, as the growing competition for optimizing the scarce water resources has led to the development of new techniques for maximizing the water use efficiency and improving crop yields and quality [6].
The first step toward the optimization of water demand should consist in reducing the current excessive application of water in agricultural lands. This, generally entails losses due to surface run-The environmental characteristics, as well as the climatic and physical data of the soil employed for the present research are reported in table 1. Year average RH = relative humidity; WS = wind speed; PSSH = potential sunshine hours.

Plant height (cm) and branches/plant number
At first, growth factors as plant height and number of branches have been monitored for plants growth with surface or subsurface irrigation and with conditioners. The aim of this first analysis was to check how different irrigation systems and by conditioners affect the overall growth of the plants. The results of the monitoring conducted over two seasons are reported in Table 2. Looking at the data reported in the Table 2, is possible to notice that no significant variation in terms of plant height and number of branches occurred by changing the irrigation system or growing the plants with different sources of soil conditioners. This suggests that the more water-saving subsurface irrigation system can be employed for rosemary cultivation without any loss in terms of growth parameters. The same analysis was conducted in terms of different soil conditioners: HUNDZ soil combined with bentonite reached showed the maximum mean values in terms of plant height and branches number under sub surface irrigation system. The correlation between growth parameters and irrigation systems, herein presented for the first time, represents an important novelty for the rosemary growth, as allows the optimization of the cultivation conditions in arid lands. The data reported in Table 2 suggest the possibility to growth Rosmarinus officinalis in a high quality employing less water as usual. Similar results were reported by Phene [38], who observed significant differences between subsurface drip and surface irrigation systems for tomatoes crops. The finding of significative differences between the two irrigation systems was not obvious. In fact, Bidondo reported no significant variations between the two irrigation systems as regards to the phenological response [39].

Herb Fresh and Dry Weights (g/plant and Kg/Fad.)
In order to assess the effect of the different irrigation systems and soil conditioners on the growth of the Rosmarinus officinalis L., the yields in terms of g per plant obtained (fresh and dry weight, table 3 and 4), and int terms of cultivated area (tables 5 and 6) have been determined.    Data presented in tables (3, 4, 5 and 6) indicate that fresh and dry herb (g / plant and Kg/Fad.) was insignificantly affected by drip irrigation systems in the 1 st cut during both seasons, while these treatments had a pronounced effect during 2 nd cut as well as sum of two cuts during both seasons. Generally, subsurface system gave the highest values of fresh and dry herb weights. Different sources of soil amendments treatments had no significant effect on fresh herb and dry weights of herb (g/plant and Kg/acre) during 1 st cuts of both seasons. On the other hand, these treatments had a pronounced effect during the 2 nd cut and sum of the 2 cuts of both seasons. During the 1 st cut of both seasons, HUNDZ soil + bentonite gave the highest values of these characters. Moreover, during the 2 nd cut herb fresh and dry weights (g/plant and Kg/acre) reached their maximum values as a result of bentonite treatment for both seasons. Regarding the effect of soil amendments on total fresh and dry weights (sum of three cuts), the data reported in Tables 3, 4, 5 and 6 show that HUNDZ soil + bentonite and bentonite are advised for 1 st and 2 nd seasons, respectively. Concerning the combined drip irrigation systems with different sources of soil amendments had no significant effect on herb fresh and dry weights during both seasons except for the herb dry weight for the 2 nd cut of the 2 nd season.

EO content (%) and yield (ml/plant)
During the first part of the study, was possible to implement a water-saving irrigation system without affect the growth of the plants in terms of plant/branches number and yield. In order to verify if the reduced amount of water and the specific soil conditioner employed affect the less evident chemical composition of the plants, the corresponding Essential Oil (EO) was extracted and analyzed.
The procedures of extraction and analysis are described in the experimental at the end of the document. The results obtained were expressed in terms of percentages (EO%), EO ml, and amount of EO per cultivated on a determined cultivated area has been assessed as shown in tables 7-9.   The data obtained revealed that the EO content (%) and yield (ml/plant and Kg/Fad.) are affected by the drip irrigation systems, the different sources of soil amendment and by the combined effect of both factors (Tables, 7, 8 and 9). Mean comparison between both drip irrigation systems showed that the highest EO content (%) and yield (ml/plant or L/acre) were obtained from plants grown under sub surface irrigation system. This observation is in agreement with the previous reported data relative to growth parameters and confirms the possibility to grow a good quality Rosmarinus officinalis with a minimum amount of water.
Concerning the effect of different sources of soil amendment, data presented in the same tables indicate that HUNDZ soil and HUNDZ soil + bentonite gave the highest mean value of EO content (%) for the 1 st cut of 1 st season while bentonite alone gave the maximum value of EO percentage for the 2 nd cut and total (sum of both cuts) of both seasons. The combination between drip irrigation systems and soil amendments had no significant effect on EO's percentage except during the 2 nd cut of 2 nd season. Generally, plants grown on bentonite under sub surface irrigation system gave the maximum mean value of EO percentage during both seasons except during the 1 st cut of 1 st season.
Concerning the effect of drip irrigation systems, different sources of soil amendments on EO yield (ml/plant and L/Fad.), data tabulated in Tables 8 and 9 indicate that the effect of these treatments gave a trend similar to the one observed in the case of EO percentage.

Composition of EO
The EO extracted from plants subjected to different treatments revealed the presence of twentyfour compounds, which account for more than 95% of the overall chemical composition detected by GC-MS (Table 10). The unidentified compounds ranged from 0.9% to 4.4% from the separated compounds. The major constituents of EO samples were camphor (38.6%-44.8%), α-pinene (14.5%-21.1%) and then eucalyptol (13.1%-15.5%).
Recently Melito et al. [26] studied the chemical composition of Rosmarinus officinalis EOs extracted from Sardinian plants. The authors evidenced a great variability in composition according to meteorological and environmental condition and several chemo types were identified. The chemical analysis highlighted the presence of seven major compounds among which α-pinene ranged between 26 and 28%, champhene from 5-8%, 1,8-cineole 15 and 25%, borneol from 5 and 11%, camphor from 3-12%, verbenone 6 and 15% and bornyl acetate from 4 and 7%. According to these results the different chemical composition between Sardinian and Egypt EOs could be related to the different geographical area of cultivation as well as the different environmental contest.
Regarding the comparison between growth different conditions, no considerable differences between the relative distribution of these major constituents and the specific treatment were observed. This specific result is of particular importance in the context of water harvesting in arid fields. In fact, the data obtained demonstrate for the first time that is possible to cultivate Rosmarinus officinalis L. employing less water as usual without altering its chemical composition.
Subsurface drip irrigation had a pronouncing effect on growth characters with respect to surface irrigation. In this regard, subsurface drip is a low-pressure, highly efficient irrigation method which uses buried drip tubes or drip tape to meet crop water needs. Subsurface irrigation saves water and improves yields by eliminating surface water evaporation and reducing the incidence of disease and weeds. A subsurface drip system may require higher initial investment and cost will vary due to water source, quality, and filtration need, choice of material, soil characteristics and degree of automation desired. This technology has been a part of irrigated agriculture since 1960, and advanced rapidly in the last two decades. A subsurface drip irrigation system is flexible and can provide frequent light irrigations. This is especially suitable for arid, semi-arid, hot, and windy areas with limited water supply. Farm operations also become free of impediments that normally exist above ground with any other pressurized irrigation system. The effect of the soil amendment is related to the providing a better environment for roots and plant growth: this includes the improvement of the soil structure and water holding capacity, the availability of nutrients, and the living conditions for soil organisms, which are important for the plants to grow. Furthermore, a better soil texture and better root growth avoids soil degradation during heavy rains or in windy regions. It also supports the nutrient cycle when organic amendments are used (e.g. manure). Beside soil amendment, there are several methods to provide soil moisture conservation such as soil cover and reforestation (living plants), mulching or several tillage techniques. It is also very important the adaptability of a certain planted crop to the specific climate. Basically, any organic or inorganic material that is added to the soil and improves its quality can be considered as soil amendment. The type of amendment chosen depends entirely on how the soil needs to be changed. By using soil amendment, almost every type of soil can be made fertile. Bentonite, historically employed for clarification procedures [40], is a volcanic ash rock consisting predominantly of montmorillonite, a clay mineral of Mg and Al, which determine the peculiar structural properties of the material [41]. It has also been recognized as a very good material for the improvement of coarse textured soils in different parts of the world [42].  Table 1. Also, some meteorological data and evapotranspiration during the growing season are presented in Table 1.

Experimental design
The field experiment was carried out as a split plot design. The experiment included eight treatments which represented the interaction between two irrigation systems combined with three soil amendments treatments and control with three replicates. The irrigation systems (surface and subsurface irrigation) were represented in the main plot while the soil conditioners treatments (control (0), HUNDZ soil (3%), bentonite (3%) and HUNDZ soil + bentonite) were placed in the sub-plot. The HUNDZ soil and bentonite conditioners were added to the soil preparation in a concentration of 3%. Plants row spacing was 0.75 m and the distance between each plant was 0.25 m in plots with area of 15 m 2 (3X5 m).

Irrigation setup
The drip irrigation lines were twin-wall drip tapes (GR), with outlets spaced every 0.5 m. Standard drippers of 4 L/h discharge at 1.5 bar working pressure were used. Drip irrigation lines were laid above and under ridges of plant rows, and the installation depth of the subsurface drip lines was 0.20 m.

Plant materials
Rosmarinus officinalis L., variety Spanish rosemary cuttings (at age 2 years) were imported from Bionorica Company, Germany.

Cultivation
The cuttings were cultivated in the nursery in foam trays filled with a mixture of sand:compost:petmos (1:1:1 volume) in the first week of October. Cuttings were covered with 100micron white plastic mulch after cultivation and then irrigated every 3-5 days by dripping irrigation. After 45 days, the plastic mulch cover was removed, and after the cuttings showed white roots (2-5 cm) which were ready for transferring in the permanent soil in the open field. Irrigation was performed as needed until the plants generated the first two true leaves stage, where the irrigation treatments were applied. The experimental soils were supplied with 20 m 3 /Fad. (Fad. = 4200 m 2 ) of mature compost. Routine agricultural practices were carried out as usually practiced in rosemary cultivation. Data for growth characters, yield, EO and its chemical constituents for all treatments were obtained during two harvests in June and in August in the two seasons. The data measurements included plant height (cm), number of branches/plants, fresh and dry weights of herb (g/plant).

EO production
EO percentage of each replicate at the two harvests was determined in the air-dried herb according to Guenther (1995) and expressed as ml/100g, while EO yield was expressed as ml /plant and L / m 2 .
The extraction procedure for the essential oil as carried out according to a previous literature data [43], A sample weighing 300 g of plant aerial part was subjected to hydro-distillation using a Clevenger type apparatus for 2h. The extraction was repeated twice, the obtained EO was collected separately, dried over anhydrous sodium sulfate (Na2SO4) and then stored at 4º C in amber glass vials until analysis.

Qualitative and quantitative analyses of EOs
Rosemary EO samples were analyzed through GC-MS with a Hewlett Packard 5890 GC equipped with a Hewlett Packard 5971 MS system operating in the EI mode at 70 eV. EO separation was performed on an HP-5 capillary column (30 m × 0.25 mm, film thickness 0.17 μm). The following temperature program was used: 60ºC hold for 3 min, then increased 4ºC/min till reach 210ºC, then held at 210ºC for 15 min, then increased 10ºC/min to 300ºC, and finally held at 300ºC for 15 min. Helium was used as the carrier gas at a constant flow of 1 mL/min for both columns. The data was analyzed using Agilent Chemstation software and the identification of the individual components performed by comparison with the co-injected pure compounds and by matching the MS fragmentation patterns and retention indices with the data reported in libraries or literature data (NIST/EPA/NIH 2008; HP1607 purchased from Agilent Technologies and Adams, 2011). The relative proportion percentages of the EO constituents were obtained by peak area normalization [44].

Statistical analysis
Data of each season were statistically analyzed separately according to Cochran and Cox. The differences between the means of the treatments were considered significant when they were more than Least Significant Differences (LSD) at 5%. The data were subjected to ANOVA test (MS DOS/ Costat Exe Program).

Conclusions
Aromatic plants cultivation in arid and semiarid regions can be achieved in a sustainable and effective way, with a special care on water consumption and quality of growth parameters. In the specific case of Rosmarinus officinalis L. was possible to determine improved cultivar conditions by selecting a combination of subsurface irrigation system and HUNDZ-bentonite soil conditioner. In particular, by switching from surface to subsurface irrigation system and by employing as soil conditioner a combination of HUNDZ conditioner and bentonite, was possible to reach the same plant quality as usual but saving a consistent amount of weather. This notable result was assessed both in terms of plant growth parameters, as well as in terms of chemical composition, determined by extracting the essential oil and subjecting it to GC-MS analysis. The approach herein presented, based on the study of the correlation between growth parameters and chemical composition, and growth procedure, can be further extended to other cultivations.