EFFECT OF CHEMICAL FERTILIZATION, PLANT SPACING AND DRYING METHODS ON THE HERB AND ESSENTIAL OIL PRODUCTION OF SUMMER SAVORY PLANT

Transcription

1 EFFECT OF CHEMICAL FERTILIZATION, PLANT SPACING AND DRYING METHODS ON THE HERB AND ESSENTIAL OIL PRODUCTION OF SUMMER SAVORY PLANT By AHMED ABD EL GHAFOUR AWAD EL SAYED B.Sc. Agric. Sci. (Ornamental Horticulture), Fac. Agric., Cairo Univ., 2009 M.Sc. Agric. Sci. (Ornamental Horticulture), Fac. Agric., Cairo Univ., 2013 THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY In Agricultural Sciences (Ornamental Horticulture) Department of Ornamental Horticulture Faculty of Agriculture Cairo University EGYPT 2017

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3 SUPERVISION SHEET EFFECT OF CHEMICAL FERTILIZATION, PLANT SPACING AND DRY METHODS ON THE HERB AND ESSENTIAL OIL PRODUCTION OF SUMMER SAVORY PLANT Ph.D. Thesis In Agri. Sci. (Ornamental Horticulture) By AHMED ABD EL GHAFOUR AWAD EL SAYED B.Sc. Agric. Sci. (Ornamental Horticulture), Fac. Agric., Cairo Univ., 2009 M.Sc. Agric. Sci. (Ornamental Horticulture), Fac. Agric., Cairo Univ., 2013 SUPERVISION COMMITTEE Dr. AHMED SALAMA EL-LEITHY Professor of Ornamental Horticulture, Fac. Agric., Cairo University Dr. SAFIA HAMDY MAHMOUD Professor of Ornamental Horticulture, Fac. Agric., Cairo University Dr. MOHAMED EL-DESOKI KHATTAB Researcher Professor of Medicinal and Aromatic Plants, NRC

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5 DEDICATION I dedicate this work to my father (Dr. Abdel Ghafour Awad) and my Mother (Dr. Iman Haiba) for all the support they lovely offered during my post graduate studies. Also, I dedicate this work to my sister (Dr. Ayah) and her husband (Dr. Islam) and my Nieces Hoor and Ahella Special dedication to my brother (Dr. Mohamed).

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7 ACKNOWLEDGEMENT First of all, full praise and gratitude is due to Allah I wish to express my hearty appreciation and sincere gratitude to DR. AHMED SALAMA EL-LEITHY, Professor of Medicinal and Aromatic Plants, Faculty of Agriculture, Cairo University, for his supervision, indispensable advice, valuable comments and constructive criticism during the performance of this investigation. I wish to express my indebtedness and profound gratitude to DR. SAFIA HAMDY MAHMOUD, Professor of Ornamental Horticulture, Fac. Agric., Cairo University, for suggesting the subject of this work, kind supervision, long lasting beneficial instructions and continuous guidance during the course of this work. I wish to express my indebtedness and profound gratitude to DR. MOHAMED EL-DESOKI KHATTAB,Researcher Professor of Medicinal and Aromatic Plants, National Research Centre, for his supervision, long lasting beneficial instructions and continuous guidance during the course of this work. I wish to express my indebtedness and profound gratitude to DR. SALAH SAYED AHMED, Researcher Professor of Medicinal and Aromatic Plants, National Research Centre, for long lasting beneficial instructions and continuous guidance during the course of this work. I wish to express my indebtedness and profound gratitude to DR. DALIA MOHAMED NASSAR, Professor of Agricultural Botany, Fac. Agric., Cairo University, for her help me in anatomical study using light microscope of this work.

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9 Name of Candidate: Ahmed Abd El Ghafour Awad El Sayed. Degree: Ph.D. Title of Thesis: Effect of Chemical Fertilization, Plant Spacing and Drying Methods on The Herb and Essential oil Production of Summer Savory Plant Supervisors: Dr. Ahmed Salama El-Leithy Dr. Safia Hamdy Mahmoud Dr. Mohamed El-Desoki Khattab Department: Ornamental Horticulture Approval: 19/ 11/2017 ABSTRACT This study was conducted at the experimental farm of the Ornamental Horticulture Department, Faculty of Agriculture, Cairo University and Medicinal and Aromatic Plants Research Department, National Research Center, Dokki, Giza, Egypt during the two successive seasons of 2014 and The aim of this study was to investigate the effect of nitrogen rates of 0.0, 40, 80 and 120 kg N/feddan, and plant spacing at 15 50, and cm on the growth, yield and chemical composition of summer savory, Satureja hortensis L. plant. Also, the effect of different drying methods sun, shade, oven at 45ºC and freeze at -45ºC on the dry weight %, essential oil % and constituents and pigments content were investigated. The results pointed out that the fertilized plants by N especially at high levels 80 and 120 kg N/fed. increased plant height, fresh and dry herbs yield, glandular hairs, essential oil yield / plant and per feddan, chlorophyll a, b and carotenoids and N, P and K contents. Stem and leaf anatomy was investigated. The narrowest spacing (15 50 cm) increased the yield of herb and essential oil per feddan, while the widest (45 50 cm) increased it per plant. The plant spacing had different effect on the pigments and nutrients contents at both cuts in the two seasons. The plants fertilized with the highest level of N (120 kg N/fed.) and cultivated at narrowest spacing significantly increased the herb and oil yields per feddan than the other combination treatments. Generally, seven components were identified in the essential oil of summer savory plant. Carvacrol and γ-terpinene % were the two major constituents, while the other components; ρ-cymene, terpinene, α- terpinene, α- pinene, α- thujene % were found in small contents indesending order. All components had no clear trend as affected by nitrogen fertilization and plant spacing at both cuts in both seasons. Sun drying method increased the dry weight of herb, while shade drying method increased the essential oil %. Moreover, the freeze drying method increased the carvacrol % in the oil and pigments contents of chlorophyll a, b and carotenoids compared to the other drying methods. Currently, the freeze drying method was the best for the high quality of the dry herb production by increaseing the green color in the dried herb. Key words: Summer savory, nitrogen fertilization, plant spacing and drying methods

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11 CONTENTS Page INTRODUCTION REVIEW OF LITERATURE Effects of chemical fertilization on growth and chemical constituents of medicinal and aromatic plants.. 4 a. Vegetative growth and yield... 4 b. Oil production and chemical constituents Effect of plant spacing growth and chemical constituents of medicinal and aromatic plants a. Vegetative growth and yield. 21 b. Oil production and chemical constituents Effect of drying methods on the essential oil content and composition MATERIALS AND METHODS 45 a. The First Experiment 45 b. The second experiment 57 RESULTS AND DISCUSSION.. 60 a. The First Experiment Vegetative growth and herb yield.. 60 a. Plant height (cm). 60 b. Number of branches / plant 64 c. Herb fresh weight (g / plant) and (ton / feddan) d. Herb dry weight (g / plant) and (ton / feddan) Essential oil production.. 82 a. Essential oil % in fresh herb b. Essential oil yield (ml/plant) and (L/feddan) 85 c. Essential oil components by GC Anatomical studies 125 a. Shoot anatomy using light microscope. 125 b. Number of glandular hairs using SEM Chemical composition a. Pigment contents Chlorophyll a content (mg/g F.W) Chlorophyll b content (mg/g F.W) Carotenoids content (mg/g F.W) 153 b. Nutrients content I

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13 1. Nitrogen (% D.W) Phosphorus (% D.W.) Potassium (% D.W.) Protein (% D.W.) Total flavonoid content (mg / g D.W.) 171 b. The second experiment Dry weight percentage Essential oil percentage Essential oil constituents Pigments contents 192 SUMMARY REFERENCES ARABIC SUMMARY... II

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15 LIST OF TABLES No. Title Page 1. Mechanical and chemical analysis of the experimental soil Monthly average of metrological data of the experimental farm of Giza, Egypt, during the 2014 and 2015 seasons Effect of nitrogen fertilization, plant spacing and their interaction on plant height (cm) of Satureja hortensis L. plant in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on plant height (cm) of Satureja hortensis L. plant in the second season, Effect of nitrogen fertilization, plant spacing and their interaction on number of branches of Satureja hortensis L. plant in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on number of branches of Satureja hortensis L. plant in the second season, Effect of nitrogen fertilization, plant spacing and their interaction on fresh weight (g/plant) of Satureja hortensis L. plant in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on fresh weight (g/plant) of Satureja hortensis L. plant in the second season, Effect of nitrogen fertilization, plant spacing and their interaction on fresh weight (ton/feddan) of Satureja hortensis L. plant in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on fresh weight (ton/feddan) of Satureja hortensis L. plant in the second season, Effect of nitrogen fertilization, plant spacing and their interaction on dry weight (g/plant) of Satureja hortensis L. plant in the first season, III

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17 12. Effect of nitrogen fertilization, plant spacing and their interaction on dry weight (g/plant) of Satureja hortensis L. plant in the second season, Effect of nitrogen fertilization, plant spacing and their interaction on dry weight (ton/feddan) of Satureja hortensis L. plant in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on dry weight (ton/feddan) of Satureja hortensis L. plant in the second season, Effect of nitrogen fertilization, plant spacing and their interaction on oil % of Satureja hortensis L. plant in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on oil % of Satureja hortensis L. plant in the second season, Effect of nitrogen fertilization, plant spacing and their interaction on oil yield (ml/plant) of Satureja hortensis L. plant in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on oil yield (ml/plant) of Satureja hortensis L. plant in the second season, Effect of nitrogen fertilization, plant spacing and their interaction on oil yield (L/feddan) of Satureja hortensis L. plant in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on oil yield (L/feddan) of Satureja hortensis L. plant in the second season, Effect of nitrogen fertilization and plant spacing and their interactions on the essential oil components (%) of Satureja hortensis L. plant at the first cut in the first season, Effect of nitrogen fertilization and plant spacing and their interactions on the essential oil components (%) of Satureja hortensis L. plant at the second cut in the first season, IV

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19 23. Effect of nitrogen fertilization and plant spacing and their interactions on the essential oil components (%) of Satureja hortensis L. plant at the first cut in the second season, Effect of nitrogen fertilization and plant spacing and their interactions on the essential oil components (%) of Satureja hortensis L. plant at the second cut in the second season, Measurements in micro-meter (µm) of certain histological characters transverse sections through the median portion of the main stem of summer savory plant aged 14 weeks from sowing date, just prior the first cutting, as affected by different levels of nitrogen fertilizer (Means of three sections from three specimens) Measurements in micro-meter (µm) of certain histological characters transverse sections through the blade of the leaf developed on the median portion of the main stem of summer savory plant aged 14 weeks from sowing date, just prior the first cutting, as affected by different levels of nitrogen fertilizer (Means of three sections from three specimens) Effect of nitrogen fertilization levels on the number of glands per unit surface area (0.04 cm 2 ) and per leaf of Satureja hortensis L. plants at the first cut in the second season, (2015) Effect of nitrogen fertilization, plant spacing and their interaction on chlorophyll a (mg/g f.w.) of Satureja hortensis L. leaves in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on chlorophyll a (mg/g f.w.) of Satureja hortensis L. leaves in the second season, Effect of nitrogen fertilization, plant spacing and their interaction on chlorophyll b (mg/g f.w.) of Satureja hortensis L. leaves in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on chlorophyll b (mg/g f.w.) of Satureja hortensis L. leaves in the second season, V

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21 32. Effect of nitrogen fertilization, plant spacing and their interaction on carotenoids (mg/g f.w.) of Satureja hortensis L. leaves in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on carotenoids (mg/g f.w.) of Satureja hortensis L. leaves in the second season, Effect of nitrogen fertilization, plant spacing and their interaction on nitrogen percentage of Satureja hortensis L. leaves in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on nitrogen percentage of Satureja hortensis L. leaves in the second season, Effect of nitrogen fertilization, plant spacing and their interaction on phosphorus percentage of Satureja hortensis L. leaves in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on phosphorus percentage of Satureja hortensis L. leaves in the second season, Effect of nitrogen fertilization, plant spacing and their interaction on potassium percentage of Satureja hortensis L. leaves in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on potassium percentage of Satureja hortensis L. leaves in the second season, Effect of nitrogen fertilization, plant spacing and their interaction on protein percentage of Satureja hortensis L. leaves in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on protein percentage of Satureja hortensis L. leaves in the second season, Effect of nitrogen fertilization, plant spacing and their interaction on total flavonoids (mg/g D.W.) of Satureja hortensis L. leaves in the first season, Effect of nitrogen fertilization, plant spacing and their interaction on total flavonoids (mg/g D.W.) of Satureja hortensis L. leaves in the second season, VI

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23 44. Effect of drying methods on dry weight (%) of dry herb of Satureja hortensis L. in both seasons Effect of drying methods on essential oil percentage of dry herb of Satureja hortensis L. in both seasons Effect of drying methods on the essential oil constituents of dry herb of Satureja hortensis L. plants in both seasons Effect of drying methods on chlorophyll b content (mg/g D.W.) of dry leaves of Satureja hortensis L. in both seasons Effect of drying methods on carotenoids content (mg/g D.W.) of dry leaves of Satureja hortensis L. in both seasons Effect of drying methods on carotenoids content (mg/g D.W.) of dry leaves of Satureja hortensis L. in both seasons VII

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25 LIST OF FIGURES No. Title Page 1. GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N0S1 treatment at the first cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N0S2 treatment at the first cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N0S3 treatment at the first cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N1S1 treatment at the first cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N1S2 treatment at the first cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N1S3 treatment at the first cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N2S1 treatment at the first cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N2S2 treatment at the first cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N2S3 treatment at the first cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N3S1 treatment at the first cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N3S2 treatment at the first cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N3S3 treatment at the first cut in the first season, VIII

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27 13. GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N0S1 treatment at the second cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N0S2 treatment at the second cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N0S3 treatment at the second cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N1S1 treatment at the second cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N1S2 treatment at the second cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N1S3 treatment at the second cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N2S1 treatment at the second cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N2S2 treatment at the second cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N2S3 treatment at the second cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N3S1 treatment at the second cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N3S2 treatment at the second cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N3S3 treatment at the second cut in the first season, Transverse sections through the median portion of the main stem of summer savory plant, at the age of 14 weeks from sowing date, as affected by different levels of nitrogen fertilizer 127 IX

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29 26. Transverse sections through leaf blade developed on the median portion of the main stem of summer savory plant, at the age of 14 weeks from sowing date, as affected by different levels of nitrogen fertilizer Scanning electron microscope (SEM) micrographs of the glandular hairs on the upper epidermis of mature leaf of Satureja hortensis L. plant treated with N0S2 treatment at the fist cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair Scanning electron microscope (SEM) micrographs of the glandular hairs on the lower epidermis of mature leaf of Satureja hortensis L. plant treated with N0S2 treatment at the first cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair Scanning electron microscope (SEM) micrographs of the glandular hairs on the upper epidermis of mature leaf of Satureja hortensis L. plant treated with N1S2 treatment at the first cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair Scanning electron microscope (SEM) micrographs of the glandular hairs on the lower epidermis of mature leaf of Satureja hortensis L. plant treated with N1S2 treatment at the first cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair Scanning electron microscope (SEM) micrographs of the glandular hairs on the upper epidermis of mature leaf of Satureja hortensis L. plant treated with N2S2 treatment at the first cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair Scanning electron microscope (SEM) micrographs of the glandular hairs on the lower epidermis of mature leaf of Satureja hortensis L. plant treated with N2S2 treatment at the first cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair Scanning electron microscope (SEM) micrographs of the glandular hairs on the upper epidermis of mature leaf of Satureja hortensis L. plant treated with N3S2 treatment at the first cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair 143 X

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31 34. Scanning electron microscope (SEM) micrographs of the glandular hairs on the lower epidermis of mature leaf of Satureja hortensis L. plant treated with N3S2 treatment at the first cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair GC chromatogram of Satureja hortensis L. essential oil distilled from plants dried by sun drying method at the first cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants dried by shade drying method at the first cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants dried by oven drying method at 45ºC at the first cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants dried by freeze drying method at -45ºC at the first cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants dried by sun drying method at the second cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants dried by shade drying method at the second cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants dried by oven drying method at 45ºC at the second cut in the first season, GC chromatogram of Satureja hortensis L. essential oil distilled from plants dried by freeze drying method at -45ºC at the second cut in the first season, XI

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33 INTRODUCTION Summer savory (Satureja hortensis L.) plant which belongs to Lamiaceae family is an annual herb up to 45 cm height with slender, erect, slightly hairy stems, linear leaves and small, pale lilac flowers. It is native to Europe, naturalized in North America. It is extensively cultivated in Spain, France, Yugoslavia and the USA and recently in Egypt for the production of essential oil. It is popular culinary herb, with a peppery flavour. It has been used therapeutically mainly as a tea for various ailments including digestive complaints (cramp, nausea, indigestion, intestinal parasites), menstrual disorders and respiratory conditions (asthma, catarrh, sore throat). Applied externally, the fresh leaves bring instant relief from insect bites, bees and wasp stings (Lawless, 1992). Plant nutrition is one of the most important factors that increase plant production. Nitrogen has the most recognized role in plant nutrition as it is in corporate in the structure of the protein molecule. Nitrogen is found in such important molecules as purines, pyrimidines, porphyrines and coenzymes. Purines and pyrimidines are found in the nucleic acids RNA and DNA essential for protein synthesis. Accordingly, nitrogen plays an important role in synthesis of the plant constituents through the action of different enzymes (Robert and Francis, 1986), Hassanzadeh et al. (2015) and Jalili (2015) and Balbalar et al. (2016) on summer savory, Satureja hortensis L. plants. 1

34 Plant spacing is an important factor in determining the microenvironment for the medicinal and aromatic plants. The optimization of this factor can lead to a higher growth, essential oil yield and a better chemical composition, by many investigators such as Akbarinia (2013) on summer savory, Satureja sahendica Bormn.; Gopichand et al. (2013) on Mentha piperita L.; Raina et al. (2013) on Ocimum sanctum and Yousefzadeh and Sabaghnia (2016) on dragonhead, Dracocephalum moldavica L. Essential oil of summer savory (Satureja hortensis L.) was extracted by hydro distillation from the whole fresh and dried herbs. An oleoresin is also produced by solvent extraction. The principal constituents are carvacrol, γ-terpinene, pinene, ρ-cymene, camphene, limonene, phellandrene and borneol. This essential oil and oleoresin are used in perfumery work and in most major food categories especially meat products and canned food, Lawless (1992); Zahedifar and Najafian (2015) and EL-Gohary et al. (2015) on Satureja hortensis L. plants. Total flavonoids are the largest group of phytonutrients, with more than types. Some of the best known flavonoids are quercetin and kaempferol. Flavonoids are powerful antioxidant with anti-inflammatory and immune system benefits. Diets rich in flavonoid containing foods are sometimes associated with prevention of cancer, neurodegenerative and cardiovascular disease. Many investigation determined the flavonoids in different medicinal and aromatic plants such as Eghami and Sadeghi (2010) on Achillea millefolium; Milan 2

35 (2011) on Marrubium peregrinum and Ovais Ullah Shirazi et al. (2014) on common herbs and spices. Drying is one of the common processes in medicinal and aromatic plants where controlled heating used to conduct removing of water by evaporation and sublimation resulting in reduction of water activity. Preventing microbial and enzymatic activities, reducing of harmful chemical interaction such as non-enzymatic browning and auto oxidation, volume and weight reduction to facilitate packing and transportation. Drying is one of the most important post-harvest processes of herbal plants in which regarding the type of effective substances such as alkaloids, essential oils and flavonoids.etc. (Blose, 2001). The objective of this study was to investigate the effect of nitrogen fertilizer, plant spacing and their interaction on the growth, herb, essential oil yield and constituents as well as chemical composition and total flavonoids of Satureja hortensis L. plant in sandy clay soil conditions. Also, the effect of different drying methods on the essential oil contents and constituents were investigated in this study. 3

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37 REVIEW OF LITERATURE 1. Effects of chemical fertilization on growth and chemical constituents of medicinal and aromatic plants Plant nutrition is one of the most important factors that increase plant production. Nitrogen has the most recognized role in the plant for its presence in the structure of the protein molecule. Nitrogen is found in such important molecules as purines, pyrimidines, porphyrines and coenzymes. Purines and Pyrimidines are found in the nucleic acids RNA and DNA essential for protein synthesis. The porphyrin structure is found in metabolically important compounds such as the chlorophyll pigments and the cytochromes essential in photosynthesis and respiration. Coenzymes are essential to the function of many enzymes, Robert and Francis (1986). Accordingly, nitrogen plays an important role in growth and synthesis of the plant constituents through the action of different enzymes. a. Vegetative growth and yield Afify et al. (1993) found that plant height, number of branches per plant, fresh and dry weights of Salvia officinalis plants were stimulated by increasing nitrogen rate up to 150 kg urea/fed. Jacoub (1995) fertilized Ocimum basilicum with a nitrogenous fertilizer at 0, 5, 10, 15 and 20 g/plant (N 1, N 2, N 3 and N 4 ) using ammonium sulphate (20.5% N). Constant levels of phosphorus and potassium fertilizers [10 and 5 g/plant, respectively (P 1 and K 1 )] were applied using triple super-phosphate (46% P 2 O 5 ) 4

38 and potassium sulphate (48% K 2 O), respectively. Nitrogen fertilization at all levels significantly increased plant height and number of branches/plant. N 2 P 1 K 1 and N 3 P 1 K 1 treatments resulted in the maximum fresh and dry weights/plant, whereas all nitrogen levels had no effect on the leaves: stems ratio. Zheljazkov et al. (1996) studied the effect of nitrogen (0, 151, and Kg N /ha), phosphorus (0 and 182 Kg P 2 O 5 /ha) and potassium (0, 110 and 240 Kg K 2 O /ha) on leaf yield of 3 Bulgarian cultivars (tundiga, zefir and clon 1) of Mentha piperita. Data showed that plant height and weight of all three cultivars were increased with increasing rates of fertilization. Yield of fresh material from the first cut was increased by 18 to 79 % as compared with non-fertilized control. El-Ghadban (1998) fertilized Mentha viridis and Origanum majorana plants with a mixture of N (as ammonium sulphate), P (as calcium superphosphate) and K (as potassium sulphate) at the ratio of 5: 2: 2 and rates of 400, 800 and 1200 kg/fed. In M. viridis, using NPK fertilization increased plant height, fresh and dry weights per plant, leaf area and leaves: stems fresh weight ratio. In O. majorana, NPK fertilization increased plant height, number of branches and fresh and dry weights per plant. It had no significant effect on the leaves: stems fresh weight ratio. El-Ghawwas et al. (2001) studied the effect of adding different levels of chemical fertilizers on Ambrosia maritima plant. The results showed a significant increase in plant height, fresh and 5

39 dry weights /plant by adding 150 Kg/fed ammonium sulphate, 150 Kg/fed calcium superphosphate and 75 Kg/fed potassium sulphate. Sakr (2001) on peppermint, found that NPK at 900 Kg/fed gave the highest fresh and air dry herb yields, compared to adding NPK at 300 or 600 Kg/fed. Abdou and El-Sayed (2002) supplied caraway plants with 200 Kg / fed of ammonium nitrate (33.5 % N) Kg / fed calcium superphosphate (15.5 % P 2 O 5 ) Kg / fed potassium sulphate (48 % K 2 O). This treatment improved plant height, number of branches / plant and stem diameter. Csizinszky (2002) on Italian parsley (Petroselinum crispum), summer savory (Satureja hortensis), sweet marjoram Origanum majorana, and french thyme (Thymus vulgaris), were treated by two rates of nitrogen and potassium (0.68 N and 0.74 K kg ha -1 day -1 and 1.8 N and K kg ha -1 day -1 in the growing season and 1.1 N and 0.9 K kg ha -1 day -1 and 2.2 N and 1.8 K kg ha -1 day -1 in the growing season) as fertilizer treatments. The yields of the herbs were higher at both fertilizer rates except for summer savory and Italian parsley, yields of the herbs at the two rates of nitrogen and potassium rates were inconsistent in the two seasons. Mohsen (2002) on Ocimum basilicum L., found that mineral NPK fertilization treatment (600, 800, 1000, 1200 kg/feddan) with a ratio of 25:10:10 increased leaves: stems fresh weight ratio. 6

40 Abd El-Azim (2003) on Salvia officinalis, found that fertilizing the plants with 150 Kg ammonium sulphate, 150 Kg calcium superphosphate and 100 Kg potassium sulphate /fed caused a significant increase in plant height, number of branches /plant, leaf area, fresh and dry weights /plant, and leaves: branches ratio. Supplying the plant with 150 kg ammonium sulphate, 100kg calcium superphosphate and 150 kg potassium sulphate / fed gave the highest fresh and dry herb yields / fed/ year. Munnu (2004) on rosemary plant, found that the fertilizer rates (0, 10 t vermicompost, 5 t vermicompost + 150:25:25 kg NPK/ha, 300:50:50 kg NPK/ha and modified urea material (prilled urea and 20% (DCD) coated urea), application of 300:50:50 kg NPK/ha, DCD coated urea produced the highest herbage yield. Sakr (2005) found that fertilizing senna plants with half dose of N fertilizer (200 Kg ammonium sulphate / fed) in association with biofertilization (Azospirillum brasilense, Bacillus polymyxa, Azotobacter chroococcum, Klebsiella pneumonia and Pseudomonas putida) increased total herb fresh weight per plant, full dose of N fertilizer gave the highest leaf: stem fresh weight ratio. PK were used as a basal dressing for all treatments at 300 Kg calcium superphosphate / fed and 100Kg potassium sulphate / fed. Abd El-Naeem (2008) recorded that NPK fertilization treatment ( 200 : 300 : 100 Kg / fed) increased considerably different vegetative growth characteristics of caraway plants such as 7

41 plant height, stem diameter, main and secondary branches number and herb fresh and dry weights. Tanious (2008) on fennel plants, added 200 Kg / fed of ammonium nitrate (33.5 % N) Kg / fed calcium superphosphate (15.5 % P 2 O 5 ) +100 Kg / fed of potassium sulphate (48 % K 2 O) and found that mineral NPK fertilization treatment increased plant height, stem diameter, main branches number and herb fresh and dry weights. Ali (2009) invstigated the response of Foeniculum vulgare to NPK at 100:100:50 and 200:200:100 Kg/fed on vegetative growth and seed yield. NPK treatements significantly increased plant height, number of main branches, fresh and dry weights of leaves, stems and roots per plant, root length, number of umbels/plant, weight of 100 seeds, fruit yield per plant and per feddan. Alizadeh et al. (2010) studied the effects of different amounts of complete fertilizer on the yield, fresh and dry weights of Satureja hortensis L. Different amounts of complete fertilizer (0, 500, 1000 and 1500 mg / plant) were applied. The results showed that the use of fertilizer increases fresh and dry weighs of S. hortensis. Babalar et al. (2010) studied the effects of nitrogen and calcium carbonate on growth and yield of Satureja hortensis L. Nitrogen fertilization significantly increased plant height, fresh and dry weights of herb. While the fresh and dry weights of herb were increased by applying 5 t ha -1 CaCO 3. 8

42 Biesiada and Kus (2010) studied the effect of nitrogen fertilization at the doses of 50, 150 and 250 kg N ha -1 on the herb yield of sweet basil, Ocimum basilicum L. The highest yields were recorded at the dose of kg N/ha. Chen and Lay (2010) on mint (Mentha spp.), investigated that the effects of three major nutrient elements, i.e., nitrogen (N), phosphorous (P) and potassium (K), on the growth of three different mint species, namely, Spearmint, Swiss mint and Japanese mint. Results indicated that growth, biomass, and the accumulation of dry weight of mints were mostly influenced by N fertilizer. The yield of Mentha spp plants with N fertilizer had a significant increase in the form of 20 to 40 percent higher than unfertilized plants. Mumivand et al. (2011) on Satureja hortensis L. (summer savory) studied the effects of nitrogen (0, 50, 100 and 150 Kg.ha -1 ) and calcium carbonate (0, 5 and 10 t.ha -1 ) application rates on the growth of S. hortensis. Nitrogen fertilization increased plant height, fresh and dry weights of herb, stem diameter, mean of leaf area (LA). Plant fresh and dry weights and mean of LA were increased by CaCO 3 up to 5 t. ha -1. Saad EL-Deen (2012) on Majorana hortensis L., found that the plants received 1/4 NPK dose with the biofertilizer gave higher values of vegetative characteristics than those received 1/2 NPK with the same biofertilizer (Azospirillum brasilense, Bacillus megatherium and Frateuria aurantia) during the three cuts of both seasons. 9

43 Sabry et al. (2012) on Marrubium vulgare L., three nitrogen doses N1 (80 kg N ha -1 ), N2 (120 kg N ha -1 ) and N3 (160 kg N ha - 1 )). Nitrogen fertilization had a significant effect on most of the agronomic parameters studied. N2 exhibited the best growth attributes, although the differences between N2 and N3 were not significant in most harvests. Akbarinia (2013) studied the effects of nitrogen fertilizer at levels of 0 (control), 40 and 80 kg/ha, on height, number of branches per plant and herb yield of Satureja sahendica Bormn. There were no significant difference in terms of height, number of branches per plant and the yield of flowering shoots using 40 and 80 kg/ha nitrogen treatments. However, higher values were recorded for both treatments compared to control. Raina et al. (2013) fertilized Ocimum sanctum L. with six fertilizer treatments. Results revealed that the conjunctive use of N at 60 kg/ha and vermicompost at 3 t/ha recorded maximum plant height (66.80 cm), number of branches (16.97), fresh and dry weights of leaves (77.92 and g, respectively), fresh and dry weights of inflorescence (46.16 and g/plant, respectively) and fresh herb yield (8.92 t/ha). Hassani et al. (2015) on peppermint (Mentha piperita L.), studied the conventional chemical fertilizers and nano-fertilizer of iron, zinc and potassium on plant height, the number of branches, number of leaves branch and nodes, and fresh and dry weights of leaves, stems and whole plants. The recorded results revealed the 01

44 whole plant height, the number of branches, leaves of branch and nodes, and fresh weights of leaves, stems and whole plant were cm, 58.2, 916.3, 16.6, 219 g, 26.7, 66.42, 23.1, g and 40.34g, respectively. Hassanzadeh et al. (2015) studied the effect of deficit irrigation and macro and micro fertilizers on morpho-physiological characteristics of Satureja. In a field experiment irrigation treatments consisted of normal, moderate and severe water stress as main plots and nine fertilizer allocated to sub-plots included of triple superphosphate and ammonium nitrate each at low and high levels (100 and 200 kg.ha -1 ), foliar application of zinc sulfate and iron sulfate each at low and high levels (0 and 0.5 kg.ha -1 ), and a control (no fertilizer). The results showed that irrigation treatment and fertilizer application had a significant effect on plant height and number of secondary branches. Jalili (2015) studied the effect of different amounts of nitrogen, phosphorus and micronutrients on yield of Satureja hortensis L. This experiment included three levels of nitrogen from ammonium nitrate 0,150,300 kg/ha, two levels of phosphorus from triple supper phosphates 50,100 kg/ha and two levels of Fe, Zn and Mn from the iron sulfate, zinc sulfate and manganese sulfate at 0.0 and 50 kg/ha. The results showed there were significant effects on plant height, dry weight, number of secondary branches, length of inflorescence, the interaction effect of nitrogen and phosphorous fertilizers were significant in dry weight of herb. 00

45 B. Oil production and chemical constituents Svoboda et al. (1990) analysis the oil obtained from growing summer savory in the west of Scotland through 4 contrasting seasons ( ) showed that, without exception, the quality and quantity of oil produced were of a commercially acceptable standard although the cultivars used had not been especially selected for growing in northern Britain. Optimal harvesting time for the maximum yield of good quality oil was found to be during early to mid-flowering. Hot and dry conditions did not enhance oil content and neither did the application of fertilizer or herbicide. It is concluded that summer savory will regularly produce commercially acceptable oil under Scottish conditions. Kandeel (1991) reported that, NPK treatments increased the plant herb and contents of N and P of celary herbs (Petroselinum crispum). Munsi (1992) investigated the effects of N (at 0, 60, 80, or 100 kg /ha) and P (at 0, 20, 40 or 60 kg /ha) on essential oil content of Mentha arvensis. He found that essential oil yield was increased linearly with increasing amounts of N and P, the highest essential oil yield was obtained with N at 100 and P at 60 kg /ha (119.1kg oil/ha, the increment was % greater than the control). However, the content of essential oil was not affected by the same treatments. Shalaby and Razin (1992) fertilized Thymus vulgaris plants with N (at 22, 33 and 44 kg/fed), P (at 8.16 and 24 kg/fed.) and K 01

46 (at 24 kg/fed.) They showed that, increasing fertilizer dosage increased oil yield/ unit area. The oil percentage was not influenced by fertilizer treatments. Afify et al. (1993) found that the nitrogen, phosphorus and potassium percentages in dried herb of Salvia officinalis plants were increased with raising the dose up to 150 kg urea/fed. Court et al. (1993) investigated the effect of different rates of nitrogen fertilizer (0, 69, 120, 180, 240 and 300 kg /ha) on chemical composition of the essential oil of Mentha piperita cv. Black Mitcham. They reported that oil yield increased with increasing fertilizer rate up to a maximum of 51 kg N / ha in the first season, and 102 kg oil/ha in the second season (N at 180 kg / ha). Many of the chemical constituents of the oil (menthol, alpha-terpineol, 1,8- cineole, limonene, alpha-pinene, beta-pinene, and sabinene) were not influenced by the rate of N fertilization, but menthone and isomenthone levels were increased with increasing N fertilization. Pulegone, linalool, beta-caryophyllene, terpinene-4-o1, menthol, and methyl acetate levels were decreased with increasing N fertilization. These differences in essential oil composition could be related to the delay in maturity which was observed at highest N rates. Abd El-Salam (1994) on anise, found that N, P and K contents in herb and fruits progressively increased by increasing the level of N-fertilization. 01

47 Jacoub (1995) fertilized sweet basil plants with N at 5, 10, 15 and 20 g/plant, and P and K at 10 and 5 g/plant, respectively. She found that N fertilization levels at 5, 10, 15 and 20 g/plant gradually increased the N contents in both leaves and stems, while P and K contents were decreased. Hammam (1996) fertilized anise plants with nitrogen at the rates of 20, 40 and 80 kg/fed. He found that the contents of total carbohydrates, nitrogen, phosphorus and potassium in herb were increased steadily by raising the rate of nitrogen fertilization. Lata and Sadowska (1996) reported that, foliar sprays of N, P, K and Zn significantly increased the N, P and K content in leaves in comparison with the controls of Catharanthus roseus, L. plants. Shylaja et al. (1996) found that alkaloids yield in leaves of Catharanthus roseus was highest with the highest N and P applications at 150 kg/ha and 90 kg/ha, sequentially. Kassem (1997) showed that, NPK fertilization of Rosmarinus officinalis at 150 kg N + 50 kg P 2 O kg K 2 O /fed. increased N and P contents. Prasad et al. (1997) gained a significant increase in the contents of N, P and K in chamomile plants supplied with nitrogen fertilization compared to the control. Wander and Bouwmeester (1998) treated Anethum graveolens with 0, 30, 60, 90 and 120 kgn/ha. The nitrogen content of the seeds increased with increasing nitrogen rate. 04

48 El-Ghadban (1998) supplied Mentha viridis and Origanium majorana plants with NPK fertilization (5:2:2) at the rates of 400,800 and 1200 kg /fed. In M. viridis, NPK fertilization increased the essential oil percentage in the fresh herb, chlorophyll a and b contents as well as nitrogen, phosphorus and potassium contents in the dry herb were increased as a results of NPK fertilization compared to the control. Raising rate of NPK fertilization increased the limonene content in the essential oil. In O. majorana, NPK treatments increased the oil yield and gave significantly high oil contents of α-pinenes, α-terpineol, β-pinene, cineole and citronellol, while the linalool and α-terpineol acetate contents were decreased. NPK fertilization (especially at the rates of 400 or 800 kg /fed) gave the highest content of minor components in the essential oil. Youssef et al. (1998) reported that Ocimum basilicum plants received 4 g N/pot gave the highest contents of N, P and K in the leaves. Essa (1999) showed that, the higher percentages of nitrogen, phosphorus and potassium in chamomile plant tissues were obtained from using high soil fertilizer level. Jacoub (1999) on Ocimum basilicum, pointed out that NPK fertilizer (5N:2P 2 O 5 : 2K 2 O) at 1200 Kg/fed was the most effective treatment for increasing chlorophyll a, b and carotenoid contents in the leaves compared to using 400 or 800 Kg NPK/fed. NPK at 800 or 1200 Kg/fed increased the total carbohydrate content in both leaves and stems. NPK at all rates increased P content in leaves and 05

49 stems. NPK at 1200 Kg/fed increased K content in leaves and stems. On Thymus vulgaris, NPK (5N: 2 P 2 O 5 : 2K 2 O) at 400 or 1200 Kg/fed increased chlorophyll a, b and carotenoid contents. All NPK treatments increased the N, P and K contents in both leaves and stems. Ali (2000) studied the effect of ammonium sulphate and urea at the rates of 50, 100 and 200 Kg N / fed on Nigella sativa, L. plants and pointed out that, 100 Kg N / fed increased chlorophyll a, b, total chlorophylls and carotenoid contents in the leaves. Osman (2000) stated that, among different chemical fertilization of NPK, the medium level (80: 32: 24) increased N, P and K contents in both herb and fruits of Coriandrum sativum plant. Agina et al. (2001) on Cymbopogon spp., studied the effect of N fertilization at kg / fed, P and K at kg / fed either alone or in combination, and found that the combination of N P K at 60: 45: 45 kg / fed significantly increased oil percentage in the first cut which was always more higher than the second cut. Barbara (2002) on goldenrod (Solidago spp), found that an increase in flavonoids accumulation was observed with increasing nitrogen level. Borella et al. (2001) evalute the effect of varying NPK treatments on the total flavonoid content on Baccharis trimera plants. They reported that mineral fertilization (N-P-K) exhibited increase flavonoid content. 06

50 Abd El-Azim (2003) fertilized Salvia officinalis plant with 150 kg ammonium sulphate, 150 kg calcium superphosphate and 100 kg potassium sulphate /fed. cause a significant increase in N, P and K contents in the plant. Abd El-Kader and Ghaly (2003) fertilized coriander plants with NPK and recorded an increment in the volatile oil percentage and yield / plant and per feddan. Abdou et al. (2004) on coriander plant, observed that oil percentage and oil yield / plant were promoted with NPK treatments especially with increasing the rates of fertilizers. Niakan et al. (2004) demonstrated that fertilizing Mentha piperita with urea at the rate of 200 kg / ha, calcium superphosphate at 100 kg/ha and 200 kg / ha potassium sulphate gave the highest essential oil content. Sakr (2005) found that fertilizing senna plants with half dose of N fertilizer (200 Kg ammonium sulphate / fed) in association with biofertilization (Azospirillum brasilense, Bacillus polymyxa, Azotobacter chroococcum, Klebsiella pneumonia and Pseudomonas putida) increased N, P and K contents in leaves more than using nitrogenous or bio-fertilizers singly. P K were used as a basal dressing for all treatments at 300 Kg calcium superphosphate / fed and 100Kg potassium sulphate / fed. Al-Shareif (2006) on caraway, proved that mineral NP fertilization treatment at ( kg/fed) gave the highest contents of chlorophyll a, b and carotenoids. 07

51 Golcz et al. (2006) studied the influence of nitrogen fertilization (NH 4 NO 3 ) on Ocimum basilicum. They found that the highest nitrogen dose significantly increased the nitrogen content. Shaheen et al. (2007) on Cynara scolymus, showed that applying ammonium sulphate at 120 kg N/fed gained the best values of N content, while 80 kg N/fed. was the best treatment for increasing K content. On the other hand, nitrogen fertilization showed insignificant increase in P content. Abd El-Naeem (2008) on caraway pointed out that mineral NPK fertilization treatment enchanced chlorophyll a, b and caroteniod contents in the fresh leaves. Azzaz and Hassan (2008) recorded that, secondary metabolites of fennel plants, essential oil and crude oil were increased when applied with different mineral and organic fertilizers. Hemdan (2008) pointed out that treating anise plant with mineral NPK fertilization at recommended dose +Minia Azotein led to significant increase in chlorophyll a, b and carotenoids in the leaves comparing with mineral NPK fertilization without Minia Azotein. Abd El-Naeem (2008) on caraway plants, proved that NPK ( Kg/ fed) led to significant increase in essential oil percentage and oil yield/ plant and per feddan. Ali (2009) invstigated the response of Foeniculum vulgare to NPK at 100:100:50 and 200:200:100 Kg/fed on essential oil yield 08

52 and essential oil constituents. Chemical fertilization significantly increased volatile oil percentage and volatile oil yield/plant. Alizadeh et al. (2010) investigated that the effect of different amounts of complete fertilizer on essential oil composition, total phenolic contents and antioxidant activity of Satureja hortensis L. Different amounts of complete fertilizer (0, 500, 1000 and 1500 mg/plant) were applied. The results showed that the use of fertilizer at 1500 mg/plant increased the essential oil percent and yield. Nineteen components were identified in the essential oil of S. hortensis. that represented % of the oil. The major components were carvacrol ( %), gamma-terpinene ( %), alpha-terpinene ( %) and p-cymene ( %). The effect of different amounts of fertilizer on the essential oil components was vary slight and insignificant. But the percentages of carvacrol, γ-terpinene and α-terpinene were changed with using fertilizer. Mumivand et al. (2011) of Satureja hortensis L. (summer savory). Studied the effect of nitrogen at (0, 50, 100 and 150 Kg / ha -1 ) and calcium carbonate (0, 5 and 10 t / ha -1 ) application rates on the essential oil content and oil yield. The content and the yield of oil were increased by nitrogen and calcium carbonate application. The interaction effect of N and CaCO 3 application was significant for leaf N and essential oil contents. Akbarinia (2013) studied the effects of nitrogen fertilizer at levels of 0 (control), 40 and 80 kg/ha, on the essential oil yield of 09

53 aerial parts of Satureja sahendica Bormn. There was no significant difference in terms of oil content between using 40 and 80 kg/ha nitrogen treatments. However, higher values were recorded for both treatments compared to control. Raina et al. (2013) fertilized Ocimum sanctum L. with six fertilizer treatments. Results revealed that the conjunctive use of N at 60 kg/ha and vermicompost at 3 t/ha recorded maximum essential oil yield (16.02 litre/ha). Jalili (2015) studied the effect of different amounts of nitrogen, phosphorus and micronutrient on yield and essential oil of Satureja hortensis L. This experiment included three levels of nitrogen from ammonium nitrate 0,150,300 kg/ha, two levels of phosphorus from triple supper phosphates 50,100 kg/ha and two levels of Fe, Zn and Mn from the iron sulfate, zinc sulfate and manganese sulfate at 0.0 and 50 kg/ha. The results showed a significant effect on essential oil and content of chlorophyll a contents. The effect of phosphorus fertilizer on the length of florescent, essential oil, chlorophyll b and total chlorophyll contents. El-Gohary et al. (2015) on summer savory, Satureja hortensis L. found that the main constituents of the essential oil were γ-terpinene ( %), carvacrol ( %) and p- cymene ( %). EL-Ziat (2015) on Ocimum spp., O. basilicum and O. citriodorum, reported that the highest values of chemical constituents such as total chlorophylls, N, P and K contents were 11

54 obtained from plants treated with NPK in the third cut of Ocimum spp. plants. Babalar et al. (2016) on summer savory plant, found that treated plants by five levels of ammonium sulfate consist of: control (without fertilizer), 40, 60, 80 and 100 kg/h were applied as split application (three weeks after sowing). At full flowering stage, the plant samples of all treatments were harvested and measured for the desired attributes such as chlorophyll a, b and total carotenoids, essential oil percentage, yield and components (consist of 26 compounds by GC-MS apparatus). The results showed a significant difference between treatments. With increasing ammonium sulfate concentration, essential oil percentage and yield significantly increased. 2. Effect of plant spacing on growth and chemical constituents of medicinal and aromatic plants Plant spacing is an important factor in determining the microenvironment in the different aromatic plants. The optimization of this factor can lead to a higher vegetative growth, yield, essential oil yield, and a better chemical composition a. Vegetative growth and yield Shalaby et al. (1993) planted Melissa officinalis at distances of 40, 60 or 80 cm (in rows spaced 60 cm apart). The wider plant spacing increased individual plant parameters, closer spacing produced greater herb yield/unit area. A spacing of 40 cm was the best. 10

55 Leto et al. (1996) on Origanum heracleoticum L. planted in rows 100 cm apart at plant spacings of 25, 50, 75 or 100 cm. Fresh herbage yields not affected by plant densities. Singh (1996) showed a significant effect was obtained on the herb and oil yields of patchouli (Pogostemon patchouli) by plant spacings at 45 x 45, 60 x 45 or 60 x 60 cm. Closer plant spacing of 45 x 45 cm gave higher yields compared with those of wider spacings (60 45 and cm). Singh et al. (1996) on lemongrass [Cymbopogon flexuosus (Steud.) Wats. var. Cauvery], found that a plant spacing of 45 x 45 cm resulted in a higher herb yield than a spacing of 60 x 60 cm. Galambosi et al. (1998) planted a Hungarian peppermint clone at an inter-row spacing of 50 cm, and interplant spacings (within the rows) of 10, 20 and 30 cm. They found that a high planting density produced higher fresh and dry biomass yields compared with a low planting density. Ibrahem (2000) on Foeniculum vulgare Mill., showed that increasing spacing between plants to 50 cm significantly increased number of umbels seed and yield/plant in both seasons. Yadav et al. (2000) conducted a field experiment on fennel cv. PF-35 to determine the optimum row and plant spacing. There were three rows (20, 30, and 40 cm) and three plant spacings (15, 20 and 25 cm). They indicated that the maximum plant height (182.0 cm), number of primary branches / plant (6.55) and number of umbels / plant (30.5) were recorded at cm spacing. 11

56 However these growth parameters were recorded minimum at cm spacing. Maximum fruit yield (24.16g/ ha -1 ) was produced at cm spacing and minimum at cm spacing. Yadav and Khurana (2000) on seedlings of fennel cv. HF-33 transplanted on 22 and 24 Oct. in combinations of 2 row spacings (30 and 45 cm) and 4 plant populations ( , , and plants/ha). They found that plant height was highest with plants/ha. No significant effect was observed in 1996/97. The highest number of primary branches was recorded from plants/ha. Row spacing had no significant effects on plant height and number of primary branches per plant. The highest numbers of primary, secondary and tertiary umbels per plant, and umbellets in the primary umbel during both seasons were recorded from plants / ha; again, row spacing had no significant effects. The highest seed yield was recorded from plants/ha which was statistically at a par with plants/ha. Aiello et al. (2001) studied the effect of plant spacing on growth and yield of medicinal plants. This study in Italy. For Hypericum perforatum cv. Topaz, grown at densities of 5, 6.3, 8, 10 and 12.5 plants/m 2, the 6.3 plants/m 2 spacing gave a dried production of 2.8 t/ha (whole plants), similar to that obtained with denser plantings in the first year, and a higher yield (4.1 t/ha flowering tops) in the second year. For Hyssopus officinalis, grown at densities of 4.2, 5.6 and 8.3 plants/m 2, total yield of aerial parts was highest at the 8.3 plants/m 2 spacing. For Rosmarinus officinalis 11

57 grown at densities of 1, 1.3 and 2 plants/m 2, production of leafy twigs (6.8 t/ha) and dried leaves (1.3 t/ha) were highest at a spacing of 2 plants/m 2 in the first year, with average yields of 12.1 and 2.3 t/ha, respectively. For Solidago virgaurea, grown at densities of 5.6, 6.7, 8.3 and 11.1 plants/m 2, spacing had no significant effects on production. Baruah (2001) studied the effect of plant spacing (10, 20 or 30 cm) on growth and seed yield of fennel during and They found that plant spacing and its interaction with sowing dates had a significant influence on plant height only in El-Gendy et al. (2001) on Ocimum basilicum L. cv. Grande Verde, studied the effect of plant spacing (15, 25, 35 and 45 cm) on herbage yield. Both fresh herb and dry leaf yield/feddan were highest at the closest plant spacing (15 cm) compared to 45 cm spacing by 16 and 21%, and 21 and 17%, in 1997 and 1998, respectively. El-Gendy et al. (2001) transplanted Ocimum basilicum L., found that the transplanting at four plant spacing levels (15, 25, 35 and 45 cm). They found that widening plant spacing significantly increased the number of branches, herb fresh and dry weights per plant, while decreasing plant height. Kandeel et al. (2001) reported that decreasing plant spacing to 30 cm using one plant per hill led to an increase in plant height and seed yield/fed. of fennel plants. 14

58 Nofal et al. (2001) on Ammi visnaga L., recorded that decreasing sow spacing to 30 cm using one plant per hill led to increase plant height and yield / fed, while sowing at 50 cm using one plant / hill led to an increase in the number of umbels/hill and seed yield / hill. Singh et al. (2001) studied the effect of row spacings (30, 45, 60 or 75 cm) and plant spacings (15.0, 22.5 or 30.0 cm) on the performance of fennel cv. Azad Saunf-1. They found that a row spacing of 60 cm resulted in the highest average seed yield. Seed yield was also highest with a plant spacing of 22.5 cm. Badran and Hafez (2002) studied the effect of nine plant densities (10 44 plants / m 2 ) on Nigella sativa. They found that increasing plant density caused a gradual reduction in basal and lateral branch number / plant and fresh and dry weights/ plant. They added that plant height was gradually reduced by the gradual widening of the distance between plants. Yadav et al. (2002) planted fennel cv. PF-35 at three rows (20, 30 and 40 cm) and three plant spacing (15, 20 and 25 cm), to determine the optimum row and plant spacing. They observed that the fruit yield (24.16 q/ha) was highest at 20 cm row and 15 cm plant spacing. However, fruit yield per plant (17.22 g), umbels per plant (30.5), primary branches per plant (6.65) and plant height (182.0 cm) gave maximum record at cm row and plant spacing treatments. 15

59 Ramachandra et al. (2003) planted patchouli (Pogostemon patchouli) cultivars Johore and Java at 60x60, 60x45, 45x45 and 45x30 cm in a field experiment conducted in Karnataka, India during Java was more robust and recorded higher dry herbage yield compared to Johore. Plant spacing of 60x45 cm resulted in the highest cumulative dry herbage (4.21 t/ha) of Java, whereas spacing of 60x45 cm resulted in the highest cumulative dry herbage (3.15 t/ha) and of Johore. Badi et al. (2004) on thyme (Thymus vulgaris), cultivated in rows of 50 cm apart with inter-row spacing of 15, 30 or 45 cm. The maximum yield of dry and fresh herbage obtained in 15-cm spacing and beginning of blooming stage. However, 15-cm spacing and harvesting in the beginning of blooming was the best treatment in terms of yield of dry matter per unit area. Masood et al. (2004) investigated the effect of different row spacings (40, 50, 60 and 70 cm) on seed production of fennel (Foeniculum vulgare). They stated that 40 cm gave the greatest plant height (114.7 cm) and seed yield / ha (3697 kg), while the lowest plant height (78.1 cm) and seed yield/ha (1925 kg) were recorded with the 70 cm spacing. Munnu (2004) on rosemary plant, studied the influence of plant spacings at (45x30, 45x45 and 45x60 cm), showed that plant spacing of 45x30 cm produced the highest herbage yield. Sar (2005) studied the effect of row spaces (45 and 70 cm) and planting spaces (10, 20 and 30 cm) on the yield of Sideritis 16

60 perfoliata L. This study was conducted in Turkey in Planting density had no significant effects on the yield components of the crop except for green herbage yield. Crop yield was significantly higher during 2003 than during Mechanization was easier with 70 cm plant spacing. Hussein et al. (2006) on Dracocephalum moldavica L. plants, found that wider plant spacing showed the greatest effect on growth characteristics. Generally the maximum rate of compost (39.6 t/ha) combined with wider distance between plants (40 cm) had a favorable effect on most of growth characteristics. Al-Kiyyam et al. (2008) found that the highest density of Origanum syriacum L. plants (14 plants/m 2 ) gave the highest fresh yield, and resulted in a significant increases in fresh weight, branches, number of leaves and fresh weight of leaves produced per unit area. Verma et al. (2008) on Indian basil, Ocimum basilicum L., investigated the influence of different nitrogen levels (0, 80, 100 and 120 kg ha -1 ) and spacings (45x30, 45x45, 50x50, 60x30, 60x45 and 60x60 cm) on herb and yield. They found that nitrogen application significantly increased the herb and yield particularly in the narrow plant spacing (45x30 cm). Nitrogen at 100 kg ha -1 increased the herb yield by 400% over the control. Mishra et al. (2009) showed that growing Rosmarinus officinalis L. at the widest spacing (60 30 cm) was the most favourable for plant height, plant spread, number of branches, stem 17

61 diameter and herbage yield per plant in the first year, whereas a cm spacing was the best for these traits, except herbage yield per plant, in the second year. Elham et al. (2010) found that the highest grain yield and root yield of Valeriana officinalis L. were achieved with plants sown on 10 th August, at a planting density of 8 plants/m 2. Zawislak (2011) on hyssop, Hyssopus officinalis L. studied the effect of plant spacing (30 30, and cm) upon yielding and quantity of herb. The results revealed that the highest fresh herb yield (1.47 kg/m 2 ) was obtained from plants grown in the spacing of cm. Sabry et al. (2012) planted horehound, Marrubium vulgare L., at spacings of 25, 35 and 50 cm apart within the row, 50 cm between the rows. Showed that the maximum mean values of plant height were obtained with a plant spacing of 35 cm. The lowest number of branches and fresh and dry weights of plant were recorded with 25 cm plant spacing. Gopichand et al. (2013) on peppermint, Mentha piperita L. cultivated at plant spacings of 25 15, and cm. The crop yield was not affected by variation in plant spacing. However, planting of M. piperita at cm spacing and application of FYM at 45 t/ha was found to be favorable for biomass production. Raina et al. (2013) on Ocimum sanctum, reported that the cultivation at closer plant spacing recorded maximum fresh herb yield (8.09 t/ha.). 18

62 Youselfadeh and Sabaghnia (2016) on dragonhead Dracocephalum moldavica L. cultivated the plants at various plant sowing densities of 10, 15, 20 and 40 cm, found that the fourth level of sowing density (40 cm) was the best in number of flowering branches, number of secondary branches and stem diameter while the second level of sowing density (15 cm) gave the best yield. B. Oil production and chemical constituents Shalaby et al. (1993) on Melissa officinalis planted at distances of 40, 60 or 80 cm (in rows spaced and 60 cm apart). The wider plant spacing increased individual plant parameters, closer spacing produced greater oil yield/unit area. A spacing of 40 cm was the best. All increases in oil yields were as a result of increased herb yield rather than increased essential oil concentrations in the plant. Leto et al. (1996) on Origanum heracleoticum L. planted in rows 100 cm apart at a plant spacing of 25, 50, 75 or 100 cm. Plant density had no significant effect on yield or essential oil content. Essential oil content did not differ significantly between years. Singh (1996) found a significantly effect on the herb and oil yields on patchouli (Pogostemon patchouli) by plant spacings of 45 x 45, 60 x 45 or 60 x 60 cm Closer plant spacing of 45 x 45 cm gave higher oil content compared to those of wider spacings (60 45 and cm). Saha et al. (1999) on Mentha piperita studied the effect of different plant spacing between the rows (30, 40, 50 and 60 cm), 19

63 and within rows (0, 10 and 20 cm). Planting in February, resulted in the higher essential oil yield and menthol content. Total carbohydrates and ascorbic acid contents were highest with 40 x 20 cm spacing. Aiello et al. (2001) studied the effect of plant spacing on growth and yield of medicinal plants. For Hypericum perforatum cv. Topaz, grown at densities of 5, 6.3, 8, 10 and 12.5 plants/m 2, the 6.3 plants/m 2. The content and yield of hypericin were 0.024% and 625 g (first year) and 0.071% and 2572 g (second year), respectively. For Hyssopus officinalis, planting density did not influence the content and total yield of essential oil. For Rosmarinus officinalis in the second year, essential oil composition did not vary. For Solidago virgaurea, grown at densities of 5.6, 6.7, 8.3 and 11.1 plants/m 2, spacing had no significant effects on oil production, but did influence on total flavonoid yield and production. El-Gendy et al. (2001) on Ocimum basilicum L. cv Grande Verde, found that the effect of plant spacing (15, 25, 35 and 45 cm) on essential oil yield. Oil yield/feddan increased by 38% and 38% cultivated at 35 or 45 cm in 1997 and 1998, respectively. Similarly, at 15 cm spacing, oil yield/feddan increased by 15% and 19% in 1997 and 1998, respectively, compared to 45 cm spacing. Chemical analysis of essential oil revealed that linalool was the main component in sweet basil oil followed by eugenol. Other components such as alpha -pinene, beta -pinene, myrcene, 1,8- cineole [eucalyptol], ocimene, methyl chavicol, methyl eugenol and 11

64 farnesol represented only small percentages of oil composition. Plant spacing had no effect on either essential oil content or compopsition. Planting at 35 cm resulted in, an increase in eugenol in both seasons. Ramachandra et al. (2003) planted patchouli (Pogostemon patchouli) cultivars Johore and Java at 60x60, 60x45, 45x45 and 45x30 cm in a field experiment conducted in Karnataka, India during Plant spacing of 60x45 cm resulted in the highest oil yield (60.65 l/ha) of Java, whereas spacing of 60x45 cm resulted in the highest oil yield (86.52 t/ha) of Johore. Badi et al. (2004) cultivated thyme (Thymus vulgaris), in rows of 50 cm apart with inter-row spacing of 15, 30 or 45 cm. The maximum yield and content of oil and thymol yield were obtained in 15-cm spacing and beginning of blooming stage. Maximum thymol content was observed in the beginning of blooming and 45- cm spacing. However, 15-cm spacing and harvesting in the beginning of blooming was the best treatment in terms of yield of oil and thymol per unit area. Gopichand et al. (2006) found that oil yield of Curcuma aromatica was maximum at 50 x 50 cm spacing (213.5 kg/ha) as compared to closer spacing of 25 x 25 cm (191.6 kg/ ha). The pooled oil constituents of the first order and second order rhizomes showed an increment in the 1, 8 cineole content with the increase in plant spacing from 25 x 25 cm (14%) to 50 x 50 cm (17%). 10

65 Irrespective of the treatments, camphor was the major oil component, followed by 1, 8 cineole and isobornyl alcohol. Hussien et al. (2006) on Dracocephalum moldavica L. plants, reported that the wider plant spacing showed the greatest effect on chemical constituents. Generally, the maximum rate of compost (39.6 t/ha) combined with wider distance between plants (40 cm) treatment gave the highest mean value for essential oil yield during the first season, while the same compost rate combined with the medium distance (30 cm) gave the highest value during the second season. Menaria and Maliwal (2007) conducted a field experiment to determine the influence of plant density on fennel. The results revealed that planting at a density of plants/ha produced higher seed yield, chlorophyll content and total soluble sugars compared to the densities of and plants/ha. Mannu (2008) on patchouli, Pogostemon cablin (blanco) Benth plant, showed that spacing of cm was superior to cm. Total essential oil yield (10.9%) was greater than that of cm. The quality of the essential oil was not influenced by plant spacing. Verma et al. (2008) on Indian basil, Ocimum basilicum L. plant, found that the narrow plant spacing (45 30 cm) significantly increased the essential oil yield. The spacing of cm registered the essential oil yield 100% higher than wider spacing of cm. The quality profile of essential oil was not influenced either 11

66 nitrogen application at different levels or by spacing (population density). Jalal et al. (2009) carried out an experiment on Foeniculum vulgare, Mill. var. Soroksary to study the effect of five planting densities on yield and essential oil components. Five plants spaces were 10, 15, 20, 25 and 30 cm on the row. The distance between rows in all treatments was 40 cm. The higher essential oil percentage (3.53%) was obtained with the lowest densities of planting. The higher percentage of anethole (83.07%), estragol (3.47%), fenchone (8.04%), p-cymene (4.45%), α-terpinene (0.54%), sabinene (0.51%), and α-pinene (0.48%) were obtained with space between plants 25, 10, 20, 20, 15, 20 and 25 cm, respectively. Mishra et al. (2009), on Rosmarinus officinalis L., showed that a spacing of cm was the best for essential oil yield per hectare. Elham et al. (2010) found that the highest essential oil yield of Valeriana officinalis L. was achieved at a planting density of 8 plants/m 2, and 20 th September sowing date, while the highest essential oil percentage was achieved from plants sown at the same date using a planting density of 4 plants/m 2. Zawislak (2011) on hyssop, Hyssopus officinalis L. plant, reported that no significant effect of plant spacing recorded on the L-ascorbic acid, chlorophyll, carotenoids, essential oil tannins and flavonoids. However, demonstrated that the harvest term 11

67 significantly affects the contents of L-ascorbic acid, chlorophyll, carotenoids and essential oil in hyssop herb. Sabry et al. (2012) on Marrubium vulgare L. plant, found that, cultivating plants at 25, 35 and 50 cm apart within the row, 50 cm distance between rows and three nitrogen doses were N1 (80 kg/ha), N2 (120 kg/ha) and N3 (160 kg/ha). The results indicated that the oil content was not influenced by plant spacing in the three harvests. Gopichand et al. (2013) on peppermint, Mentha piperita L., found that the essential oil content and composition were not affected by variation in plant spacing. However, planting of M. piperita at cm spacing was associated with increased production of menthone. Raina et al. (2013) on Ocimum sanctum L. plant, indicated that the closer plant spacing recorded maximum essential oil yield (13.37 Litre/ha). Using the dose of N (60 kg/ha) and vermicompost at 3t/ha with a plant spacing of cm recoded maximum essential oil yield (19.88 litre/ha). Yousefzadah and Sabaghnia (2016) on dragonhead, Dracocephalum moldavic L., found that the medium distance (15 cm) can be used for achieving high yield and essential oil performance. 14

68 3. Effect of drying methods on the essential oil content and composition After harvesting the herb and leaves of aromatic plants become brown quickly therefore it should be dried immediately. Drying the different organs after harvesting is one of the most important processes. The process involves the removal of moisture which is a critical threshold for the product to be stored for a long time. Dry is also associated with stoppage of enzymatic activities as well as bacterial and yeast proliferation. (Soysal and Ostekin, 2001). The purpose of this study is to evaluate drying methods include sun drying, shade drying, oven drying and freeze drying. The freeze drying can be used to avoid damage caused by heat, producing a product with superior physical and chemical qualities for different aromatic plants. Several studies were done on effect of drying methods of the several aromatic plants on the essential oil content and composition. Venskutonis (1997) on thyme (Thymus vulgaris L.) and sage (Salvia officinalis L.), found that the volatile oil constituents of herbs (fresh, freeze-dried and oven dried at 30 C and WC) were isolated by distillation-extraction methods and analysed by GC and GC-MS. In total, 68 compounds were identified in thyme and 44 in sage, and more than 100 components were screened quantitatively. A significant reduction in the amount of extracted volatile oil was found only in the case of drying at WC, mainly as a result of the loss of non-oxygenated monoterpenes. 15

69 Maroto et al. (2002) the effect of different drying treatments on the volatile oil of parsley (Petroselinum crispum L.) was studied. Air drying at ambient temperature resulted in few losses in volatile oil compounds compared with the fresh herb, whereas oven drying at 45 C and freeze-drying caused a decrease in the concentrations of the majority of the volatile oil components. Fatemeh et al. (2005) stated that the aerial parts of Satureja hortensis, were collected at the full-flowering stage and dried by three different drying methods (sun-drying, shade-drying and ovendrying at 45º C). The essential oils were obtained by hydrodistillation of the aerial parts. In addition, the essential oil of shadedried sample was obtained by two other distillation methods (waterand steam-distillation). The oils were analyzed by capillary GC and GC MS. Statistical analysis showed no significant difference between oil yield (w/w) of the oven-dried sample (1.06%) compared to shade-dried (0.94%) and sun-dried (0.87%). The oil content of the shade-dried sample, obtained by hydro-distillation, was higher (0.94%) than that of the steam-distilled (0.27%). Twenty-three components were identified in the oil of S. hortensis in the different drying methods, including carvacrol ( %) and γ-terpinene ( %) as main components. Seventeen compounds were characterized in the oils of different distillation methods, including carvacrol ( %) and γ-terpinene ( %). Although the drying methods had no significant effect on oil composition of S. hortensis, the distillation changed the percentage of main 16

70 components sharply (significant at 1%). The steam-distillation method produced the lowest amount of carvacrol and highest amount of γ-terpinene. The results showed that extraction by hydrodistillation gave the best results for S. hortensis, based on oil yield and carvacrol percentage. Sefdkont et al. (2006) found that in an experiment with savory, Satureja hortensis L., among the different drying methods (shading, sunlight, and oven drying at 45º C), the highest and lowest essential oil contents were obtained with oven drying at 45º C and sun drying, respectively. Khalid et al. (2008) the herbs of Lemon balm, (Melissa officinalis L.) were dried by different drying methods of shadedrying, sun-drying and oven-drying at 40 C and it compared with the fresh herb through two harvesting times. The essential oil were obtained by hydrodistillation of the herbs, and were analysed by GC-MS. Fresh herbs had the highest essential oil content followed by shade drying, oven drying and sun drying respectively, during the first and the second harvesting. The essential oil content of Melissa officinalis L. was significantly decreased towards the second harvesting. Drying methods had no effect on the number of chemical components of the essential oil, as 43 components were identified in the essential oil of each drying method. The major components were citronellal, citronellol and geranyl acetate during the first and second harvesting. Drying of Melissa officinalis L. by sun-drying conditions was the most suitable for a high percentage of 17

71 monoterpene hydrocarbons (during the first harvesting) or ovendrying at 40 C (during the second harvesting). On the other hand, oven-dried herb at 40 C was the most suitable for a high-percentage of sesquiterpene hydrocarbons and oxygenated sesquiterpene components through the first and second harvesting. Khorshidi et al. (2009) on rosemary, Rosmarinus officinalis L. investigated the influence of different methods of drying, extraction time, and type of organ on the essential oil percentage. Three drying methods investigated were oven drying (45ºC), shade drying and sun drying. Four extraction times were: 1, 2, 3, 4 hours and three organ type were leaf, stem, mixed leaf and stem. Results showed that effect of drying methods, extraction time, and organ type on the essential oil percentage were significant. The maximum essential oil percentage (1.8%) was obtained to leaf sample, 3h of extraction, and shade drying. While the minimum essential oil percentage (0.12%) was obtained to stem sample, 1h of extraction, and oven drying. Massoud et al. (2010) investigated the effect of cuts and different drying methods (room, air and oven at 50 ºC) on volatile oil quality and quantity of sweet basil (Ocimum basilicum L.) plant. The results showed that the oven drying method was the best treatment to produce the highest volatile oil percentage, while the room drying method gave the highest percentage of volatile oil components. Also, the second and third cuts gave the highest volatile oil percentage and yield compared to the first cut. The GLC 18

72 of the volatile oil of the fresh leaves revealed a total of 7 compounds. The total identified compounds were 92.62, and % in the oil of the room, air and oven at 50 ºC drying methods, respectively, compared to the fresh plants (91.0 %). The percentages of the main component (Linalool) were 87.77, and 79.86% in the oil of the room, air and oven at 50 ºC drying method, respectively compared to the fresh plants (84.55 %) at the second cut in the second season. Stanisavljević et al. (2010) studied the effect of different methods of drying on the content and chemical composition of the essential oil from the herb of Mentha longifolia (L.) Hudson. The drying of plant material was carried out naturally in the shade of draughty place, in the laboratory oven at temperature of 45 C and absorptive low temperature condensation drying oven at 35 C (low temperature drying). Isolation of essential oil from dried samples in three different ways was conducted by hydrodistillation, while the chemical analysis was carried out by GC/FID and GC/MS methods. The highest yield of the essential oil was obtained from the herb which was dried at low temperature (1.1%) and the lowest from that dried in the laboratory oven (0.6%). The highest content of the dominant component of essential oils, piperitone, was recorded in the oil from low temperature dried herb (71.7%), while those isolated from naturally dried drug and from the laboratory oven contained piperitone in lower concentrations (50.8% and 43.1%, respectively). 19

73 Abdollah et al. (2013) studied the effect of different drying methods, i.e., sun-drying, shade-drying, oven-drying at 45º C and 65º C and freeze-drying for the aerial parts of Bakhtiari savory (Satureja bachtiarica, Bunge.) on oil production. The essential oils of fresh and dried samples were obtained by hydro-distillation, and analyzed using GC MS. The highest essential oil yields (v/w on dry weight basis) were obtained by oven-drying at 45º C (2.3%) followed by freeze-drying (2.1%), oven-drying at 65º C (2.0%), shade-drying (1.7%), sun-drying (1.6%) and fresh sample (1.2%). Twenty-seven components were determined in essential oils of S. bachtiarica, which were mostly oxygenated monoterpenes and hydrocarbons monoterpenes. The main components in essential oils of fresh and dried Bakhtiari savory aerial parts were carvacrol ( %), γ-terpinene ( %), thymol ( %) and p-cymene ( %). Drying of aerial parts in the oven at 45º C was the most suitable drying method considering short drying time and high-oil yield. Angel et al. (2013) evaluated the influence of drying method on aroma compounds of thyme (Thymus vulgaris L.). The drying methods tested were convective drying, vacuum microwave drying, and freeze drying, as well as a combination of convective predrying and VM finish drying. Volatile oil compounds of thyme samples were extracted by hydrodistillation and analyzed by GC. Thirtythree compounds were identified; thymol, terpinene, p-cymene, caryophyllene, and α-terpinene were the major components. The 41

74 total quantity of volatile oil of fresh thyme (1.167 mg / 100 g) was reduced by most of the drying treatments, with the exception of VM at 240 and 360 W. The combined method with 40º C and 240W was the best option for drying thyme; the time required was relatively short (301 min) and aroma quality was good according to instrumental data (total concentration of volatile oil (1.127 mg /100 g). Ghasemi et al. (2013) investigated the effect of different drying methods (shade-drying at 25 C with three levels; the shadow alone, the shadow with the fan air flow and the shadow with the fan air flow + rummage; 40 C oven-drying for 24, 48 and 72 hrs; microwave-drying with 500 and 1000 W of power at 20 minutes and freeze-drying with three levels of 7, 15 and 24 hrs) on the essential oil content and components of lemon balm (Melissa officinalis L.). Essential oil content and composition were determined by Clevenger type apparatus and GC/MS method. The maximum essential oil content (0.43%) obtained from 48 hrs oven-drying, while minimum content (0.03%) obtained from drying under microwave with the power of 500 W. Citral and Citronellal percentages in shade-drying with an air flow fan were more than other drying methods. Finally it could be concluded that ovendrying method had better results compared to the other methods. Slavica et al. (2013) reported that the essential oil glands and trichomes of savory are located on the surface of stems, leaves and calyces, accordingly drying and processing of savory have huge 40

75 influence on essential oil content in savory drugs. Drying kinetics and influence of drying temperatures (35, 40, 45, 50, 55 and 60 C) were investigated on selected parameters of savory collected in winter The percentage of dried leaves in fresh herb, essential oil content and proportion of dried leaves in fresh and dried herb were defined. The average of initial water content was %, essential oil content ml / 100g and the proportion of dried leaves was % in the dried herb. Higher drying temperatures expectedly reduced the essential oil content. The reduction of essential oil content at 45 C was 14.8 %, while further temperature increase to 50 C resulted in a 59.4 % lower content of essential oil in the drug. Vahid and Sharareh (2013) on common sage (Salvia officinalis L., studied the effects of three drying methods on the chemical composition of the essential oil. The aerial parts of Saliva officinalis were collected at full-flowering stage and dried by three different drying methods: sun-drying, shade-drying and oven-drying at 50 C. The essential oil was extracted and analyzed by GC-MS components were detected in the oil of Saliva officinalis under different drying methods, including, α-thujone ( %), 1,8-Cineole ( %), Viridiflorol ( %), Thujone ( %), Camphor ( %), Borneol ( %), α-pinene ( %), β-pinene ( %) and humulene ( %), as main components. The presence of comparatively high concentration of oxygenated compounds mainly thujones, 1,8-41

76 cineole and camphor in sage oils. The study revealed that sage can be dried by different methods in full flowering stage of the plants. Ayyobi et al. (2014) on dill, Anethum graveolens L. and peppermint, Mentha piperita L., investigated the effect of different drying methods on appropriate drying time, essential oil yield, phenol content and antioxidant capacity. Four drying methods were used in this experiment. Results indicated that different drying methods had a significant effect on all characteristics. Increasing oven temperature led to reduction of essential oils yield. Minimum and maximum essential oil were obtained when dill and peppermint were oven dried at 75º C (20.11 ml/m 2 ) and dried in shading (28.44 ml / m 2 ). However, drying at 60º C may be an appropriate temperature for drying peppermint and dill plant materials. A significant reduction of drying time with no adverse effects on the essential oil yields. Oven drying at 75º C reduced antioxidant of dill while shade drying increased antioxidant capacity of peppermint. Lotfi et al. (2014) on Satureja hortensis investigated the different drying conditions. The drying methods including (shadow, sun, oven (70º C and 100º C), and microwave). Results revealed that the most changing in color occurred after 5 % removing of water and no significant changes observed. The changes in Satureja color was a function of time and temperature drying. Sana and Seyede (2014) studied the effect of drying conditions on the color of savory (Satureia hortensis) leaves. Leaves were dried at four temperature levels of 40, 50, 60 and 70 C, 41

77 in a laboratory scale cabinet dryer. Results showed the effect of temperature on the color was significant. Drying at 40 C caused minimal changes in color of product. Regarding these results, the most suitable treatment in relation to conserving color of savory leaves was the temperature of 40 C. Marina et al. (2015) on the two peppermint (Mentha piperita L.) cultivars Krasnodarskaja and Peppermint, the leaves were dried using different methods. The highest content of essential oil (0.77%) and chlorophyll (1.69%) was found in the cv. Peppermint. Chlorophyll a to b ratio was different in the fresh peppermint leaves: in the cv. Peppermint it was 1.35 and in the cv. Krasnodarskaja it was Peppermint leaves were dried using active ventilation, convection, infrared, vacuum, microwave, and sublimation methods. The quality of dried herbs depended on the properties of plants and drying techniques. The highest content of essential oil ( % of dry mass) was found in the variously dried peppermint leaves of the cv. Krasnodarskaja. The lowest essential oil content (0.08% and 0.065% of dry mass) was determined in microwave dried herbs. The highest content of chlorophyll was found in the lyophilized peppermint leaves (715.0 mg 100 g-1 of dry mass) of the cv. Peppermint. 44

78

79 MATERIALS AND METHODS a. The First Experiment This study was carried out at the Department of Ornamental Horticulture, Faculty of Agriculture, Cairo University, and Medicinal and Aromatic Plants Research Department, National Research Center, Dokki, Giza, Egypt, during the two successive seasons of 2014 and The objective of this study was to investigate the effect of nitrogen fertilization, plant spacing and their interactions on growth, essential oil yield and constituents and biochemical compositions of Satureja hortensis L. plant grown in sandy clay soil conditions. Also, glandular hair and anatomy were investigated. 1. Layout of the first experiment This experiment was designed using a factorial split plot design, the main plots were assigned to different nitrogen levels (N0, N1, N2, and N3 (0.0N, 40 N, 80 N and 120 kg N/feddan, respectively) (feddan= 4000m 2 net area). While the subplots were assigned to plant spacings S1, S2 and S3 (15, 30 and 45 cm respectively) between the plants. 2. Experimental procedures a. Plant material The seeds of summer savory (Satureja hortensis L.) were imported from Jellitto Standensamen Gmbh, Schwarmstedt, Germany by Sekem Company, Egypt, through Dr. S. Hendawy, 54

80 National Research Center, Dokki, Giza, Egypt. The seeds were sown on 15 th February 2014 and 2015 (in the two seasons) at nursery beds inside greenhouse in peatmoss medium. After 45 days from sowing the seeds, i.e. on 1 st April 2014 and 2015, when the seedlings were cm. height, they were transplanted to prepared plots in the experimental field. The physical and chemical characteristics of the soil experiment field were determined according to Jackson (1973) and are shown in Table (1). Table 1. Mechanical and chemical analysis of the experimental soil. Mechanical analysis of the samples taken from the experimental soil. Year Sand Silt Clay Texture SCL SCL Chemical analysis of the samples taken from the experimental soil. Year ph E.C. Millie equivalent/liter (dsm -1 ) Cations Anions (2.5:1) (5:1) Ca ++ Mg ++ Na + K + -- CO 3 - HCO 3 Cl SO Nil Nil b. Soil preparation The soil was prepared on 15 th March 2014 for the first season and 20 th March 2015 for the second season: compost at 5m 3 /fed and superphosphate 15.50% P 2 O 5 at (200kg/fed) were added during soil preparation, while potassium sulphate 48% K 2 O (100 kg /fed) was added in two doses, the first dose added during the soil preparation, while the second dose was added after the first cut. 54

81 c. Cultivation procedures The experiment included 12 treatments, each experimental unit (plot) was m (3.2 m 2 ) and divided into 4 rows with 50 cm apart and 15, 30 and 45 cm between the plants, i.e. the plots contained 36, 24 and 16 plants, respectively. Then, the treatments replicated three times (36 plots). d. Nitrogen fertilization treatments Nitrogen fertilizer at different levels of N0, N1, N2 and N3; 0, 40, 80 and 120 kg N/ feddan were added as equivalents, 0.0, 200, 400 and 600 kg ammonium sluphate 20.5 % N in two doses, the first dose was added 21 days after transplanting, while the second dose was added after the first cut in the two seasons. The experiment involved 12 treatments as following: 1- N0S1= 0.0 kg N/fed. (15 50 cm) 2- N0S2= 0.0 kg N/fed. (30 50 cm) 3- N0S3= 0.0 kg N/fed. (45 50 cm) 4- N1S1= 40 kg N/fed. (15 50 cm) 5- N1S2= 40 kg N/fed. (30 50 cm) 6- N1S3= 40 kg N/fed. (45 50 cm) 7- N2S1= 80 kg N/fed. (15 50 cm) 8- N2S2= 80 kg N/fed. (30 50 cm) 9- N2S3= 80 kg N/fed. (45 50 cm) 10- N3S1= 120 kg N/fed. (15 50 cm) 11- N3S2= 120 kg N/fed. (30 50 cm) 12- N3S3= 120 kg N/fed. (45 50 cm) 54

82 3. Metrological data Maximum, minimum and average air temperature and relative humidity of the experimental farm area during the growing period are presented in Table (2). Table 2. Monthly average of metrological data of the experimental farm of Giza, Egypt, during the 2014 and 2015 seasons. Air temperature C Relative Humidity Month Max Min Average % First season, 2014 Jan Feb Mar Apr May June July Aug Sept Second season, 2015 Jan Feb Mar Apr May June July Aug Sept

83 4. Recorded data a. Vegetative growth. The plants were harvested two times at the early bloom stage (on 2 nd June and 5 th August 2014 for the first and the second cuts, respectively, in the first season and on 10 th June and 13 th August 2015 for the first and the second cuts, respectively, in the second season). The plants were harvested by cutting vegetative parts at 10 cm above the soil surface. Data were recorded in the two seasons as the following: 1. Plant height (cm). 2. Number of branches / plant. 3. Fresh weight of herb (g / plant): Fresh weight was measured immediately after harvest. 4. Fresh weight of herb (ton / fed). 5. Air dry weight of herb (g / plant). 6. Air dry weight of herb (ton / fed). Number of plants/feddan= S1 (53,333), S2 (26,666) and S3 (17,777) plants. b. Essential oil production 1. Essential oil percentage in the fresh herb. 2. Essential oil yield (ml / plant). 3. Essential oil yield (L / feddan). 4. Essential oil components by GC. 54

84 Essential oil percentage in the fresh herb The oil percentage was determined in fresh herb in both seasons using the hydro-distillation method by Clevenger apparatus according to Guenther (1961). A known weight of fresh herb (100 g) was placed in a flask of 1 L capacity for distillation, and an adequate amount of water was added. A proper essential oil trap and condenser were attached to the flask and enough water was added to fill the trap. The distillation continued for 3.0 hours until no further increase in the oil was observed. After finishing the distillation process the apparatus was left to be cooled, and the essential oil percentage was estimated as follows: Essential oil vol. (Measuring pipette reading) 100 = % oil Essential Weight of sample The oil was dried by sodium sulphate anhydrous. Essential oil yield/ plant (ml) Essential oil yield per plant = oil percentage fresh herb weight/plant. Essential oil yield/ feddan (L) Oil yield per feddan = oil yield/ plant (ml) number of plants/fed. * 1000 Essential oil components Samples taken from the essential oil obtained in the two seasons were analyzed using GC analysis, to determine their main constituents. The use of GC in the quantitative determinations was performed using the methods described by Tatjana et al. (2009). 45

85 The GC analysis of the essential oil samples were carried out in the two seasons using gas chromatography instrument stands at the Laboratory of Medicinal and Aromatic Plants, National Research Center with the following specifications. Instrument: capillary GC-2010 plus Gas Chromatographs (Shimadzu Corp., Japan), coupled with a Shimadzu FID 2010 Plus detector (Flame Ionization Detector). The GC system was equipped with a Stabilwax column (30 m x 0.25 mm i.d., 0.25 μm film thickness). Analysis were carried out using helium as carrier gas at a flow rate of 1.0 ml/min at a split ratio of 1:10 and the following temperature program: 40º C for 1 min; rising at 4.0º C/min to 150º C and held for 6 min; rising at 4º C/min to 210º C and held for 1min. The injector and detector were held at 210º C and 250º C, respectively. Diluted samples (1:10 hexane, v/v) of 0.2 μl of the mixtures were always injected. Most of the compounds were identified using GC standards. The obtained chromatogram and analysis report for each sample were analyzed to calculate the percentage of the main volatile oil components. The area of each peak was first calculated by an automatic integrator. The areas were then summed, and the total area of the peaks represented the whole sample. The percentage of each component was the ratio between its peak area to the total peak areas, multiplied by

86 c. Anatomical studies 1. Shoot anatomy using light microscope It was intended to carry out a comparative microscopical examination on plant material which showed extreme or prominent response of plant growth to investigated treatment with nitrogen fertilizer. Specimens of summer savory were taken from the median portion of the main stem as well as from the blade of the corresponding leaf. Plants used for examination were taken throughout the second season of 2015, at the age of four months from sowing date, just prior the first cutting. Specimens from the stems and leaves were killed and fixed for at least 48 hours in F.A.A. (10 ml formalin, 5 ml glacial acetic acid and 85 ml ethyl alcohol 70%). The selected materials were washed in 50% ethyl alcohol, dehydrated in a normal butyl alcohol series, embedded in paraffin wax of melting point 56ºC, sectioned to a thickness of 20 micro-meter(µm), double stained with safranin/light green, cleared in xylene and mounted in Canada balsam (Nasser and EL-Sahhar, 1998). Sections were read resulted from investigated treatments with nitrogen fertilizer and photomicrographed. 2. Anatomical characteristics (glandular hairs by SEM) Pieces of 3 to 10 mm size were cut out of the leaves in the following treatments: plants treated with N0, N1, N2 and N3 (0.0, 40, 80, and 120 kg N/fed., respectively, and cultivated at medium 45

87 spacing (30 50 cm) between the plants at the first cut of the second season. The cut faces were sealed with glue (conductive carbon glue or two-component epoxy glue). The pieces were mounted on scanning electron microscope (SEM) stubs with a conductive adhesive tab. The fresh samples were examined without metal coating (Mendez Vliess and Diaz, 2010). d. Chemical constituents 1. Pigment contents: Determination of chlorophyll a, b and total carotenoids (mg/g fresh leaves) Chlorophyll a, b and total carotenoid contents were determined in representative fresh leaves samples according to Moran (1982) as follows: Ten ml N,N-Dimethylformamide was added to 1 g of fresh leaves in dark tubes then they were placed overnight in the refrigerator. The obtained extracts from previous materials were measured by spectrophotometer at the wavelength of 663 nm for chlorophyll a, 647 nm for chlorophyll b and 470 nm for carotenoids, using N,N-Dimethylformamide as a blank. The contents of the different leaf pigments were calculated using the following formulas: Chl.A (mg/l) = (E 663) 2.79 (E 677). Ch.B (mg/l) = (E 647) 4.62 (E 663). 45

88 Total carotenoids (mg/l) = 1000 E chl.a 104 chl.b / 229 (mgl -1 ). 2. Determination of flavonoids and elements: The dried leaves of samples in the two seasons were grounded and kept in desiccators for chemical analysis, which included the following patterns: a. Total flavonoid content (mg/g D.W.) Extraction of plant materials One gram of each sample was extracted with 25 ml of methanol using a sonicator Bandelin Sonorex (Germany) at 35 khz and 200 W for 60 min at room temperature. The extracted samples were filtered through Whatman filter paper No. 4 and the filtrate was evaporated to dryness under vacuum and the yield of the extract from each sample was calculated. Each extract was dissolved in 10 ml methanol, 0.1 ml of sample solution, 1.5 ml of methanol, 2.8 ml of water, 0.1 ml of potassium acetate (1M) and 0.1 ml of aluminum chloride (10% in methanol) were added and mixed. After incubation at room temperature for 30 min, the decrease in absorbance was measured at 415 nm. The total flavonoids content was expressed as milligrams of Quercetin equivalent per 100 g of dried plant (mg Quercetin/100 g) (Sandra and Paula, 2011). b. Elements determination Dry herb samples were oven- dried at 70º C until a constant weight was obtained. The dried samples were then digested for extraction of nutrients, using the method described by Piper (1947). 45

89 Five ml concentrated sulphuric acid were added to 0.5 g dried samples and heated for 10 min., and then 0.5 ml perchloric acid was added and heating continued till a clear solution was developed. The digested solution was quantitatively transferred to a 100 ml volumetric flask using deionized water. The percentages of the three main nutrients (N, P and K) in the extract were determined as follows: Nitrogen determination Nitrogen determination was carried out using the modified micro-kjeldahl method, as described by Pregl (1945). 25 mg of dry plant tissues were weighed in a porcelain boat. The boat contents were transferred to a microdigestion tube and digested with 1 ml. of concentrated H 2 SO 4 and few mg of 3:1 CuSO 4 / K 2 SO 4 mixture. When the solution was partially clear, two or three drops of Merck ' s reagent Superoxol (30 percent hydrogen peroxide) were added and the digestion completed. The acid digest was diluted with 1 ml. of ammonia-free water and washed into the distillation apparatus. After adding 7 ml. of 30% NaOH containing 5% Na 2 S 2 O 3 the mixture was distilled with steam for 5 minutes. The ammonia was collected in 10 ml of 0.01 N H 2 SO 4. Distillations were boiled and titrated with 0.01 N NaOH to a methyl red end point. The indicator, introduced by means of glass thread, was prepared by adding an excess of methyl red 0.01 N NaOH. Acid and base were measured in 10 ml microburettes. The apparatus was steamed for 30 minutes 44

90 before each series of determinations. Blank determinations amounted to 0.05 to 0.06 ml of 0.01 N NaOH. Protein determination The protein content was determined according to the following formula: Protein % = nitrogen % in the herb dry weight Phosphorus determination The phosphorus content was estimated after wet shaking by using molybdic acid to form a phosphomolybdate complex. It was then reduced with amino-naphtholsulphonic acid to the complex molybdenum blue, which measured colourimetrically at (650 nm) using a standard curve of potassium dihydrogen phosphate, as recommended by King (1951). Potassium determination Potassium was determined by using a "Pye Unicam, Model SP-1900" atomic absorption spectrophotometer with a boiling airacetylene burner according to Isaac and Kerber (1971). Statistical analysis of data Data recorded on growth, oil content and oil yield were statistically analyzed, and separation of means were performed using the Least Significant Difference (L.S.D.) test at the 5% level, as described by Little and Hills (1978). 44

91 b. The second experiment Drying methods experiment This study was carried out at the Department of Ornamental Horticulture, Faculty of Agriculture, Cairo University and Medicinal and Aromatic plants Research Department, National Research Center, Dokki, Giza, Egypt, during the two successive seasons of 2014 and The objective of this study was to investigate the effect of different drying methods, sun drying, shade, oven at 45º C and freeze drying at -45º C on the dry weight percentage, essential oil percentage, essential oil constituents and chlorophyll a, b and carotenoid content of summer savory, Satureja hortensis L. plants. Plant material In this experiment, the fresh herb samples (100 g) of summer savory plants were obtained from plants grown at the first experiment which fertilized with N2 (80 kg N/fed.) and planted at medium spacing S2 (30 50 cm), N2S2 treatment at both cuts in the two growing seasons. Layout of the experiment This experiment was designed using complete randomized design (CRD), with four drying treatments, i.e. sun drying, shade drying, oven at 45ºC and freeze drying (-45ºC). Each treatment replicated three times (12 replicates). Procedures Fresh herb samples (100 g) were dried as follows: 44

92 Sun drying: the samples were placed on mesh plastic trays directly exposed to direct sunlight for 4 days until reaching a constant weight. Shade drying: the samples were placed on mesh plastic trays at room temperature 25ºC for 7 days until reaching a constant weight. Oven drying: the samples were placed in the laboratory oven at 45ºC for 48 hr. until reaching a constant weight. Freeze drying: the samples were placed in freeze dryer (Instrument: Christ Mode (beta 1-8 LD plus, Germany) at -45ºC for 72 hrs. until reaching a constant weight. All dried samples obtained from different drying methods were packed in paper bags and kept in desiccators, then the following data were recorded at both cuts in both seasons: 1- Dry weight percentage (g/100g F.W.) 2- Essential oil percentage in dried samples (25g) were used to determine the essential oil percentage according to the method described in the first experiment (Guenther, 1961). 3- Essential oil constituents identified by GC according to the method described in the first experiment (Tatjana et al. 2009). 4- Chlorophyll a, b and carotenoid contents (mg/g D.W) were determined according to the method described in the first experiment (Moran, 1982). 44

93 Statistical analysis of data Data recoded on the dry weight percentage, essential oil percentage and pigments contents were statistically analyzed and separation of means were performed using the Least Significant Difference (L.S.D) test at the 5% level as described by Little and Hills (1978). 44

94

95 RESULTS AND DISCUSSION a. The First Experiment The effect of nitrogen fertilization and plant spacing on 1. Vegetative growth and herb yield a. Plant height Data presented in Tables (3 and 4) show the effect of N fertilization, plant spacing and their interactions on plant height of summer savory plants in the two seasons. Table (3) shows that, the nitrogen fertilization treatments at N1, N2 and N3 (40, 80 and 120 kg/fed.), respectively insignificantly increased plant height at the first cut. The mean values were 43.52, and cm for the plants fertilized with N1, N2 and N3, respectively as compared with cm for the control plants. N2 and N3 treatments slightly decreased plant height compared with N1 which recorded the highest value for plant height at the first cut of the first season. All nitrogen fertilization levels (N1, N2 and N3) significantly increased plant height compared to the untreated plants (N0) at the second cut in the first season. The mean values were 33.81, and cm, respectively as compared to cm for the control plants. Data presented in Table (4) show that all nitrogen levels (N1, N2 and N3) significantly increased plant height compared to untreated plants (N0) at both cuts in the second season, the mean values were 42.70, and cm, respectively compared to 06

96 Table 3. Effect of nitrogen fertilization, plant spacing and their interaction on plant height (cm) of Satureja hortensis L. plant in the first season, 2014 Plant Spacing Nitrogen fertilization Mean N0 N1 N2 N3 First cut S S S Mean L.S.D at 0.05 for N N.S. S 0.74 N x S 3.48 Second cut S S S Mean L.S.D at 0.05 for N 2.54 S N.S. N x S 4.41 N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm Table 4. Effect of nitrogen fertilization, plant spacing and their interaction on plant height (cm) of Satureja hortensis L. plant in the second season, 2015 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N 1.32 S 1.88 N x S 2.29 Second cut S S S Mean L.S.D at 0.05 for N 1.50 S 1.26 N x S 2.51 N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm 06

97 40.63 cm for control plants at the first cut. Also the mean values were 33.74, and cm, respectively at the second cut while the control plants recorded cm. Generally, all nitrogen fertilization levels gradually increased the plant height with increasing nitrogen level at both cuts of the first and second seasons, compared to the control plants. The effect of nitrogen fertilization on increasing the plant height may be due to N supply as a precursor of protein syntheses and as vacuolar osmoticum. The osmotic compounds in the cell sap are important in order to allow cell elongation. N also has its effect on the biosyntheses of polyamines involved in cell division and production of some plant hormones like auxins, which promote laterality buds growth. These findings are in agreement with Alizadeh et al. (2010), Babalar et al. (2010), Mumivaud et al. (2011) and Jalili (2015) on summer savory, Satureja hortensis L. Also, Biesiada and Kus (2010) on sweet basil, Ocimum basilicum, Sabry et al. (2012) on Marrubium vulgare L., Raina et al. (2013) on Ocimum basilicum and Hassani et al. (2015) on peppermint, Mentha piperita L. who found that nitrogen fertilization significantly increased plant height. Data presented in Tables (3 and 4) show that, S1 significantly increased plant height as compared to S2 and S3 at the first cut in the first season, the values were (44.42, and cm, respectively), while all plant spacings gave insignificantly effect on the height of summer savory plant in the second cut. The narrowest plant spacing (S1) gave the highest value of plant height 06

98 (36.58 cm) compared to S2 and S3 (34.97 and cm) at the second cut in the first season, respectively. In the second season, the tallest plants were significantly produced from narrow spacing S1 at both cuts as compared with the medium (S2) and wide (S3) spacings with no significant differences between them. The mean values were 45.44, and cm at the first cut and 34.69, and cm at the second cut, respectively. The increase in the height of Satureja hortensis L. plants as a result of narrow spacing may be attributed to high density of the plants, causing excessive shading of the plants, i.e a reduction in light intensity to which the shoots are exposed. A similar conclusion was obtained by Masood et al. (2004) on Foeniculum vulgare L., Verma et al. (2008) on Ocimum basilicum L., Zawislak (2011) on Hyssopus officinalis L. and Raina et al. (2013) on Ocimum sanctum plants. Regarding the interaction between the effects of nitrogen fertilization and plant spacing on the plant height of Satureja hortensis L., data presented in Tables (3 and 4) showed that significant differences were recorded in both seasons. In the first season the tallest plants (46.00 and cm were obtained at the first and second cuts, respectively) for those at the narrowest spacing and supplied with the highest N rate (N3), whereas, the shortest plants (40.33 and cm) resulted from widest spacing with N2 level treatment at the first cut and widest spacing with unfertilized N (N0) treatment, respectively. In the second season, the tallest plants (47.67 and cm) at the first and second cuts, 06

99 respectively were those planted at narrowest spacing and supplied with N2 at the first cut and N3 at the second cut, respectively. b. Number of branches / plant Data presented in Tables (5 and 6) show the effect of N fertilization, plant spacing and their interactions on number of branches / plant of Satureja hortensis L. plants in the two growing seasons. The data presented in Table (5) indicated that the number of branches formed by summer savory plants was significantly affected by nitrogen fertilization treatments. In the first season, the plants fertilized by all nitrogen levels, N1, N2 and N3 (40, 80 and 120 kg N/fed.) significantly reduced the number of branches / plant compared to unfertilized plants (N0) at the first cut, the mean values were 21.19, and compared to branches / plant, respectively. On the other hand, at the second cut all nitrogen treatments N1, N2 and N3 significantly increased it as compared to the unfertilized plants (control), the mean values were 13.11, and compared to branches / plant, respectively. In the second season Table (6) at first cut, all nitrogen levels had no significant effect on the number of branches / plant compared to control plants (unfertilized plants), whereas in the second cut, all N fertilization levels insignificantly decreased number of branches / plant. 06

100 Table 5. Effect of nitrogen fertilization, plant spacing and their interaction on number of branches of Satureja hortensis L. plant in the first season, 2014 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N 0.83 S N.S. N x S 1.44 Second cut S S S Mean L.S.D at 0.05 for N 1.10 S 1.07 N x S 1.91 N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm Table 6. Effect of nitrogen fertilization, plant spacing and their interaction on number of branches of Satureja hortensis L. plant in the second season, 2015 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N N.S. S 1.41 N x S 1.99 Second cut S S S Mean L.S.D at 0.05 for N N.S. S 0.96 N x S 2.39 N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm 06

101 In general, the results recorded at the first cut in both seasons and at the second cut in the second season, gave less number of branches in comparison with unfertilized plants. Similar results recoded by Csiziszky (2002) on summer savory, Satureja hortensis L., and Akbarinia (2013) on Satureja sahendica Bormn., found that there was no significant difference in number of branches / plant by using 40 and 80 kg N/ha treatments. The results presented in Tables (5 and 6) show that, the plant spacing had a marked effect on the branching of summer savory plants. In the first season, at the first cut all plant spacing (narrow, medium and wide) had no significant effect on the number of branches / plant, the mean values were 21.83, and branches / plant, respectively. Whereas, opposite trend was noticed in the second cut, in the first season, the widest plant spacing significantly increased the number of branches / plant compared to medium and narrow spacing, the mean values were 15.86, and branches / plant for S3, S2 and S1, respectively. In the second season, at both cuts, the widest plant spacing (45 50 cm) significantly increased the number of branches / plant compared to medium and narrow plant spacing (30 50 and 15 50, respectively) the mean values were 16.28, and at the first cut and 17.94, and branches / plant at the second cut, respectively. Currently, the widest plant spacing gave the highest number of branches / plant at the second cut in the first season (2014) and at 00

102 both cuts in the second season (2015). The increase in the number of branches / plant formed on Satureja hortensis L. plants at widest spacing may be attributed to the low plant density, which lead to a higher supply of nutrient and light to the plants and consequently a promotion of lateral growth and an increase in branching. These findings are in close conformity with the results of Hussein et al. (2006) on Dracocephalum moldavica L., Mishra et al. (2009) on Rosmarinus officinalis and Youselzadeh and Sabaghnia (2016) on Dracocephalum moldavica L. plants, who found that the widest plant spacing significantly increase the number of branches / plant. Regarding the interaction between the effects of nitrogen fertilization and plant spacing on the number of branches / plant, data presented in Tables (5 and 6) show that, significant differences were recorded between these two factors at both cuts in the two growing seasons. In the first season, at the first cut the highest number of branches / plant (24.00 branches / plant) was obtained with unfertilized plants (N0) and planted at widest spacing, whereas, the lowest number of branches / plants (20.22 branches / plant) was recorded from plants fertilized at N2 (80 kg N/fed.) and cultivated at medium spacing (30 50 cm). The opposite trend was noticed at the second cut, the highest number of branches (18.55 branches / plant) was recorded on the plants fertilized with N2 (80 kg N/fed.) and planted at widest spacing (45 cm), whereas the minimum number of branches / plant (11.00 branches / plant was obtained by unfertilized plants (N0) and transplanted at narrowest spacing (15 cm). 06

103 In the second season, at both cuts, the highest number of branches / plant (17.00 and branches / plant) was recorded on plants fertilized with N1 (40 kg N/fed.) and cultivated at widest spacing (45 50 cm), meanwhile, the lowest number of branches / plant (13.44 and branches / plant) was obtained on the plants receiving N1 and planted at narrowest spacing (15 50 cm) at both cuts, respectively. c. Herb fresh weight (g / plant) and (ton / feddan) 1. Fresh weight (g / plant) Data presented in Tables (7 and 8) show the effect of N fertilization, plant spacing and their interactions on fresh weight (g/plant) of Satureja hortensis L. plants in the two growing seasons. Nitrogen fertilization at all levels N1, N2 and N3 (40, 80 and 120 kg N/fed.) significantly increased fresh weight of plant (g/plant) compared to unfertilized plants (N0) at both cuts in the two growing seasons. Nitrogen fertilization at the highest level (120 kg N/fed.) had a maximum increase on fresh weight as compared to the other treatments (40 and 80 kg N/fed.) at both cuts in the two seasons, the mean values were , and g / plant at the first cut and , and g/plant at the second cut in the first season for N3, N1 and N2, respectively. Moreover the mean values were , and g / plant at the first cut and , and g / plant at the second cut in the second season for N3, N1 and N2, respectively, whereas, the lowest fresh weight 06

104 Table 7. Effect of nitrogen fertilization, plant spacing and their interaction on fresh weight (g / plant) of Satureja hortensis L. plant in the first season, 2014 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N 8.57 S N x S Second cut S S S Mean L.S.D at 0.05 for N S 8.87 N x S N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm Table 8. Effect of nitrogen fertilization, plant spacing and their interaction on fresh weight (g / plant) of Satureja hortensis L. plant in the second season, 2015 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N S N x S Second cut S S S Mean L.S.D at 0.05 for N 7.24 S N x S N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm 06

105 values were resulted from unfertilized plants (N0) at both cuts in both seasons. These results agreed with Alizadeh et al. (2010), Mumivand et al. (2011) and Jalili (2015) on Satureja hortensis L. they found that N fertilization increase fresh weight. The plants grown at the widest spacing (45 50 cm) significantly increased fresh weight of plant compared to medium (30 50 cm) and narrow (15 50 cm) distances at both cuts in the two seasons. The highest mean fresh weight values were and g / plant in both cuts respectively in the first season and and g / plant in both cuts respectively in the second season in plants grown at cm, followed by plants grown at a medium spacing of cm (with mean values of and g / plant at both cuts in the first season and and g / plant at both cuts respectively in the second season. On the other hand, the lowest fresh weights ( and ; and g / plant at both cuts in the first and second seasons, respectively were obtained from plants grown at narrow spacing (15 50 cm). The favorable effect of wide spacing on fresh weight of summer savory may be attributed to the low density of the plants and the consequent greater availability of water and nutrients lead to more vegetative growth resulting in a higher fresh weight. Similar results were reported by EL-Gendy et al. (2001) on Ocimum basilicum L., Mishra et al. (2009) on Rosmarinus officinalis L., 66

106 found that the widening plant spacing significantly increased the herb fresh weight/plant. Regarding the interaction between the effects of nitrogen fertilization and plant spacing on the fresh weight / plant, data presented in Tables (7 and 8) show that, significant differences were recorded between these two factors at both cuts in the two seasons. In the first season (2014), the highest plant herb fresh weights and g / plant were obtained from plants fertilized with high level of N (120 kg N/fed.) and planted at widest spacing (45 50 cm) at both cuts, respectively. Whereas the lowest herb fresh weights and g / plant were recorded in unfertilized plants and planted at narrowest spacing at both cuts, respectively. Similar results were obtained at both cuts in the second season (2015). 2. Fresh weight (ton / feddan) The results presented in Tables (9 and 10) show the effect of N fertilization, plant spacing and their interaction on herb fresh weight (ton/feddan) of Satureja hortensis L. plants in the two seasons. Nitrogen fertilization at all levels (N1, N2 and N3 (40, 80 and 120 kg N/fed., respectively) significantly increased the fresh weight of herb compared to unfertilized plants (N0) at both cuts in the two growing seasons. Increasing the N fertilizer application rate caused gradual increases significantly in the herb fresh weight / fed. at both cuts in 66

107 Table 9. Effect of nitrogen fertilization, plant spacing and their interaction on fresh weight (ton / feddan) of Satureja hortensis L. plant in the first season, 2014 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N S N x S Second cut S S S Mean L.S.D at 0.05 for N S N x S N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm Table 10. Effect of nitrogen fertilization, plant spacing and their interaction on fresh weight (ton / feddan) of Satureja hortensis L. plant in the second season, 2015 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N S N x S Second cut S S S Mean L.S.D at 0.05 for N S N x S N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm 66

108 the two seasons. The maximum values of herb fresh yield / fed. (7.249 and ton/fed.) were recorded in plants fertilized by the highest level of N application (120 kg N/fed.) at both cuts in the first season, respectively compared to the other nitrogen rates (40 and 80 kg N/fed.) and control plants. In the second season, at both cuts, similar trend was recorded, i.e increasing nitrogen level resulted in a gradual steady increases in the herb fresh yield (ton / feddan). This effect may be attributed to the important role of nitrogen in increasing the vegetative growth (herb fresh weight) of the medicinal and aromatic plants especially summer savory plant. These results agree with the findings obtained by Mumivand et al. (2011) on Satureja hortensis L., Akbarima (2013) on Satureja sahendica, Bormn. and Hassani et al. (2015) on Mentha piperita L. plants. Growing summer savory plants at the narrowest spacing S1 (15 50 cm) gave significantly higher herb fresh weights (with mean values of and ton/fed. for the first and second cuts in the first season, respectively) in comparison with the medium spacing S2 (30 50 cm), the mean values were and ton/fed for both cuts, respectively. On the other hand, widest spacing S3 (45 50 cm) resulted in the lowest mean value for herb fresh weights (4.327 and ton/fed.) for both cuts, respectively. This effect on the herb fresh yield / feddan may be attributed to increase the number of plants per feddan at the narrow spacing it was 53,333 plants / fed. in comparison with 26,666 and 17,777 66

109 plants / fed. at medium and widest spacing, respectively. Similar trend was noticed in both cuts of the second season (2015). The increase in the herb fresh weight of Satureja hortensis L. plants as a result of planting at narrow spacing is in agreement with the findings of EL-Gendy et al. (2001) on Ocimum basilicum L. cv. Grande Verde, Badi et al. (2004) on Thymus vulgaris, Verma et al. (2008) on Indian basil, Ocimum basilicum L. and Yousefzadeh and Sabashnia (2016) on Dracocophalum moldavica L. plants, who found that the narrow plant spacing significantly increased the herb fresh yield per unit area. Regarding the interaction between the effect of nitrogen fertilization and plant spacing on the herb fresh yield / feddan, data presented in Tables (9 and 10) show that significant differences were recorded between these two factors at both cuts in the two growing seasons. In the two seasons at the first cut, the highest herb fresh weight (10.20 and ton/feddan) were obtained from the plants fertilized with N2 (80 kg N/fed.) and planted at the narrowest spacing S1 (15 50 cm) in both seasons, respectively, whereas the lowest herb fresh yield (3.095 and ton/fed) were resulted from the unfertilized plants and planted at the widest spacing treatments, respectively. In the two seasons at the second cut, the highest herb fresh weights (9.937 and ton/fed.) were recorded in plants fertilized with the highest nitrogen level N3 (120 kg N/fed.) and cultivated at 66

110 narrowest spacing (15 50 cm) treatment, whereas the lowest herb fresh yield (1.984 and ton/fed.) resulted from the unfertilized plants (N0) and planted at the widest spacing treatment in both seasons, respectively, this effect was noticed at both cuts in the first season. d. Herb dry weight (g / plant) and (ton / feddan) 1. Herb dry weight (g / plant) Data presented in Tables (11 and 12) show the effect of N fertilization, plant spacing and interaction between them on dry of herb weight g / plant of Satureja hortensis L. plants in the two growing seasons, data indicated that: Nitrogen fertilization at all concentrations N1, N2 and N3 (40, 80 and 120 kg N/fed.) significantly increased the herb dry weight / plant at both cuts in the two seasons compared to unfertilized plants N0 (control). Nitrogen fertilization at the highest level (120 kg N/fed.) had a maximum increase on herb dry weight (g / plant) compared to 40 and 80 kg N/fed. at the first cut in the two growing seasons, these increases were insignificant between 40 and 80 kg / fed. treatments. In the second cut in both seasons, N3 (120 kg N/fed.) treatment significantly increased the herb dry weight (g / plant) compared to N1 (40 kg N/fed.) in the two seasons, whereas, the plants fertilized with N2 (80 kg N/fed.) treatment had a different response according to the growing season, i.e in the first season, the increase was significant while in the second season was insignificant as compared to the highest level of N. 66

111 Table 11. Effect of nitrogen fertilization, plant spacing and their interaction on dry weight (g / plant) of Satureja hortensis L. plant in the first season, 2014 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N 3.26 S 5.89 N x S 5.65 Second cut S S S Mean L.S.D at 0.05 for N 3.56 S 3.22 N x S 6.16 N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm Table 12. Effect of nitrogen fertilization, plant spacing and their interaction on dry weight (g / plant) of Satureja hortensis L. plant in the second season, 2015 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N 4.98 S 4.12 N x S 8.63 Second cut S S S Mean L.S.D at 0.05 for N 3.01 S 5.28 N x S 5.21 N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm 60

112 The mean values were and in the first season and and g/plant in the second season resulted from plants treated by 80 and 120 kg N/ fed., respectively. The highest plant dry weights g / plant were obtained with the widest spacing S3 (45 50 cm) compared to the other plant spacing (S1 and S2), these increases were significant as compared to the narrowest spacing S1 (15 50 cm) at both cuts in the two growing seasons., while S2 (30 50 cm) spacing, the increase was insignificant in the first season and significant in the second season at the first cut, compared to S3 (45 50 cm). The opposite trend was recorded in the two seasons in the second cut. The increase in herb dry weight (g / plant) as a result of wide spacing may be attributed to an increase in metabolic activity by greater availability of water, nutrients, sunlight and other resources. Similar increases in plant dry weight as a result of wide spacing have been reported by Gopichand et al. (2013) on peppermint, Mentha peperita L. and Yousefzadeh and Sabaghnia (2016) on dragonhead, Dracacephallum moldavica L. plants. The interaction between the effect of nitrogen fertilization and plant spacing on the herb dry weight (g / plant), data presented in Tables (11 and 12) show that significant differences were recorded between the values obtained from the plants receiving the various combinations of these two factors at both cuts in the two growing seasons. The highest values for dry weights / plant were obtained from in the plants receiving the highest level of N (120 kg 66

113 N/fed.) and planted at the wide spacing (N3S3) at both cuts in both seasons, the mean values were 68.44, and 78.92, g / plant, respectively. On the other hand, the lowest dry weights / plant were recorded in unfertilized plants (N0) and planted at narrow spacing treatment at the first cut in both seasons and at the second cut in the first season, while at the second cut of the second season, the lowest value (26.50 g/plant) was obtained when the plants unfertilized and planted at medium spacing (N0S2). 2. Herb dry weight (ton / feddan) Data presented in Tables (13 and 14) show that, the dry weight ton/feddan of summer savory plants was considerably affected by nitrogen fertilization, plant spacing and their interaction at both cuts in the two growing seasons. Nitrogen fertilization at all levels of N1, N2 and N3 (40, 80 and 120 kg N/fed., respectively) significantly increased the herb dry weight compared to unfertilized plants (N0) at both cuts in the two seasons. Application of nitrogen fertilizer at N3 level (120 kg N/fed.) significantly increased the herb dry weight (ton/fed.) in most cases compared to N1 and N2 levels (40 and 80 kg N/fed.), with only exception at the first cut in the first season, the mean values were 1.722, and ton/fed. for the plants fertilized by N3, N1 and N2 levels, respectively without significant differences between them. 66

114 Table 13. Effect of nitrogen fertilization, plant spacing and their interaction on dry weight (ton/feddan) of Satureja hortensis L. plant in the first season, 2014 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N S N x S Second cut S S S Mean L.S.D at 0.05 for N S N x S N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm Table 14. Effect of nitrogen fertilization, plant spacing and their interaction on dry weight (ton/feddan) of Satureja hortensis L. plant in the second season, 2015 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N 0150 S N x S Second cut S S S Mean L.S.D at 0.05 for N S N x S N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm 66

115 Recently, nitrogen application at the highest level (120 kg N/fed.) significantly increased the herb dry yield (ton/fed.) compared to other N applications (40 and 80 kg N/fed.) and control plants (N0) in both two second cut of the first and second seasons respectively. The favorable effect of N at high level (N3) on the herb dry yield/fed. confirms the role of nitrogen in increasing the yield of summer savory plants. Similar results were found on some aromatic plants by Abd EL-Azim (2003) on Salvia officinalis, Alizadeh et al. (2010), Mumivand et al. (2011) and Jalili (2015) on Satureja hortensis L. plants. Cultivating plants at the narrowest spacing significantly increased the herb dry weight (ton/fed.) in comparison to plants cultivated at medium and widest spacings at both cuts in the two growing seasons. In the first season (2014), the mean values were 2.431, and ton/fed. at the first cut and 2.537, and ton/fed. at the second cut obtained from narrow, medium and wide spacings, respectively. Moreover, the mean values were 3.006, and ton/fed. at the first cut and 2.047, and ton/fed. at the second cut resulted from narrow, medium and wide spacing, respectively in the second season (2015). Significant differences in herb dry weights were thus recorded at different plant spacings, this effect may be attributed to, at narrowest spacing the number of plants/ unit area was increased than medium and widest spacing. In this experiment the number of plants / feddan were 66

116 53,333, 26,666 and 17,777 plants / feddan when cultivated at narrow, medium and wide spacing, respectively. These results agreed with those obtained by Verma et al. (2008) on Indian basil, Ocimum basilicum L., AL-Kiyyam et al. (2008) on Origanum syriacum L. and Yousefzadeh and Sabaghria (2016) on Dracocephalum moldavica L. plants, they found that the narrow spacing (closer plant spacing) recorded maximum dry herb yield/unit area. Regarding the interaction between the effects of nitrogen fertilization and plant spacing on the herb dry yield (ton/feddan), the data presented in Tables (13 and 14) show that significant differences were recorded between the values obtained from plants receiving the different treatments of these two factors at both cuts in the two investigated seasons. The highest herb dry weights (ton per feddan) were obtained from the plants fertilized with N2 level (80 kg N/fed.) and planted at the narrowest spacing S1 for the two cuts in the second season (2015). Meanwhile, the plants cultivated at the narrowest spacing and fertilized with low level of nitrogen N1 (40 kg N / fed.) at the first cut and high level of nitrogen at the second cut in the fiest season (2014), produced the highest dry herb (ton / feddan). The lowest values of dry yield / feddan were obtained from the unfertilized plants (N0) and planted at wide spacing treatments at the first and second cuts in two growing seasons (2014 and 2015). 66

117 3. Essential oil production a. Essential oil percentage in fresh herb The data in Tables (15 and 16) show the effect of different nitrogen levels N0, N1, N2 and N3 (0.0, 40, 80 and 120 kg N / feddan), respectively on the essential oil percentage of summer savory plants in the two seasons. Increasing the nitrogen fertilizer application caused a gradual steady increase in the essential oil percentage at the first and second cuts in the first season (Table 15). The favorable effect of nitrogen fertilizer on essential oil percentage was particularly evident at the levels of 80 and 120 kg N / feddan, which gave significantly higher essential oil percentages, with the mean values of and 0.612% at the first cut and and 1.129% at the second cut, respectively compared with N1 (40 kg N/feddan) and control treatments. Data presented in the second season in Table (16) indicate that increasing the nitrogen fertilizer levels (0-120 kg N/feddan) caused gradual increases with insignificant differences between N treatments at the first cut. At the second cut the essential oil percentages of summer savory plants increased gradually with increasing N fertilization levels, i.e. all nitrogen fertilizer levels (N1, N2 and N3) significantly increased the essential oil percentage with mean values of 0.946, and 0.992%, respectively compared with 0.858% for the control plants N0 (without nitrogen fertilizer). Similar results were recorded at both cuts of the first season. These 66

118 Table 15. Effect of nitrogen fertilization, plant spacing and their interaction on essential oil % of Satureja hortensis L. plant in the first season, 2014 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N S N x S Second cut S S S Mean L.S.D at 0.05 for N S N x S N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm Table 16. Effect of nitrogen fertilization, plant spacing and their interaction on essential oil % of Satureja hortensis L. plant in the second season, 2015 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N N.S. S N x S Second cut S S S Mean L.S.D at 0.05 for N S N x S N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm 66

119 results agreed with EL-Gohary et al. (2015), Jalili (2015) and Babalar et al. (2016) on summer savory, Satureja hortensis L. they found that with increasing the nitrogen levels, increased the essential oil percentage compared to control. Values of essential oil percentage in the fresh herb of summer savory (Satureja hortensis L.) plants in the two seasons (Tables 15 and 16) show variation with different plant spacing in both seasons. The narrowest spacing S1 (15 50 cm) gave the highest significant volatile oil percentage in comparison with the medium S2 (30 50 cm) and widest S3 (45 50 cm) spacing at the two cuts in both seasons, the mean values were 0.637, and 0.547% at the first cut, and 1.065, and 0.909% at the second cut in the first season for the narrowest, medium and widest spacings, respectively Also, the mean values showed the same trend at the first and second cuts of the second season. Concerning the interaction between the two factors of this experiment were observed in the resulting. Significant effects essential oil percentages of summer savory plant at both cuts in both seasons. The highest volatile oil percentages were recorded when the plants were treated with the highest level of nitrogen (N3) and planted at S1 (15 50 cm) spacing at first cut in first season and two cuts in the second season, the mean values were and 0.787, 1.130%, respectively. Meanwhile, the medium spacing S2 and high level of nitrogen N3 gave the highest essential oil percentage at the second cut in the first season the mean value was The lowest 66

120 essential oil percentages resulted from unfertilized plants and planted at S3 (45 50 cm) spacing, the values were and % at the two cuts in the first season and and % at both cuts in the second season, respectively. In general, data in Tables (15 and 16) show that, the essential oil percentage in all treatments at the first cut (June cut) were lower than those at the second cut (August cut) in both seasons. Also, the general means of the essential oil percentages were and 0.727% in the first cut and and 0.936% in the second cut in the first and second seasons, respectively, this effect presumably due to longer light duration, higher temperature and higher light intensity previously dominated during summer growing season. This can be the most suitable condition for oil synthesis and accumulation in the leaves. These results agreed with EL-Sayed (1979) on geranium, Pelargonium graveolens L., Hiekal (2005) on Thymus vulgaris, EL-Sayed et al. (2009) on Artemisia dracunculus L. and Ali (2016) on Coriandrum sativum L. plants. b. Essential oil yield (ml / plant) and (L / feddan) 1. Essential oil yield (ml / plant) The results presented in the two seasons Tables (17 and 18) show the effect of nitrogen fertilizer, plant spacing and their interaction on the essential oil yield / plant of Satureja hortensis L. plants in the growing seasons. Nitrogen fertilization at all rates (N1, N2 and N3, 40, 80 and 120 kg N/fed., respectively) significantly increased the essential oil 66

121 yield / plant compared to the unfertilized control plants at the two cuts in the two growing seasons. The favorable effect of nitrogen fertilizer (ammonium sulfate 20.5% N) on the essential oil yield / plant was particularly evident at the highest rate (120 kg N/fed.) which gave significantly higher essential oil yield / plant (1.448 and ml / plant, respectively) at both cuts in the first season than any other fertilization treatments (40 and 80 kg N/fed.). The differences between the essential oil yield of plants receiving these two treatments was significant, the mean values were and or and ml / plant at both cuts, respectively. Similar results were noticed at both cuts in the second growing season. The increases in the essential oil yield / plant as a result of applying nitrogen fertilization is in agreement with findings of Ali (2009) on Foeniculum vulgare, Alizadeh et al. (2010) and Babalar et al. (2016) on summer savory, Satureja hortensis L. plants, they found that increasing ammonium sulfate rates significantly increased the essential oil yield/plant. The essential oil yield / plant of summer savory plants was significantly affected by the plant spacing at both cuts in the first season. The widest spacing significantly increased the essential oil yield/plant than the medium and narrowest spacings, the mean values were 1.322, and ml / plant at the first cut and were 1.643, and ml / plant at the second cut, respectively. Similar result was recorded at the first cut, in the second season (2015). Changing plant spacings at the second cut in 60

122 Table 17. Effect of nitrogen fertilization, plant spacing and their interaction on the essential oil yield (ml / plant) of Satureja hortensis L. plant in the first season, 2014 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N S N x S Second cut S S S Mean L.S.D at 0.05 for N S N x S N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm Table 18. Effect of nitrogen fertilization, plant spacing and their interaction on the essential oil yield (ml / plant) of Satureja hortensis L. plant in the second season, 2015 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N S N x S Second cut S S S Mean L.S.D at 0.05 for N S N.S. N x S N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm 66

123 the first season, had no significant effect on the essential oil yield / plant. Generally, the widest plant spacing (45 50 cm) resulted in the highest essential oil yield / plant than medium and narrow spacing treatments. These results agreed with those of Gopichand et al. (2006) on Curcuma arometica and Hussien et al. (2006) on Dracocephalum moldavica L. plants. Regarding the interaction between the effect of nitrogen fertilization and plant spacing on the essential oil yield/plant, data presented in Tables (17 and 18) show significant differences were recorded in the essential oil yields of plants receiving the various combinations of these two factors at both cuts of the two seasons. The highest essential oil yield / plant was obtained by the plants fertilized with highest level of N (120 kg N/fed.) and planted at widest spacing S3 (45 50 cm), the mean values were and ml / plant at the first cut in the two seasons, respectively, meanwhile, the lowest essential oil yield / plant was recorded with unfertilized plants and planted at the narrowest spacing, the mean values were and ml / plant at both seasons, respectively. In the second cut, the highest essential oil yield / plant was obtained from the plants fertilized with N3 and planted at medium and narrow spacing, which gave the values were and ml / plant in both seasons, respectively. The lowest essential oil yield / plant resulted from the unfertilized plants and planted at narrow 66

124 spacing at the second cut in both seasons, the mean values were and 0.490% respectively. 2. Essential oil yield (L/feddan) The data presented in Tables (19 and 20) show the effect of N fertilization, plant spacing and their interaction on the essential oil yield L/feddan of Satureja hortensis L. plants in the two growing seasons. Nitrogen fertilization at the different levels (N1, N2 and N3; 40, 80 and 120 kg N/fed., respectively) significantly increased the essential oil yield / feddan compared to the unfertilized (N0) plants at both cuts in the two seasons. The essential oil yield / fed. have been gradually increased with increasing the nitrogen level at both cuts in the first season and at the second cut in the second season. A maximum increase in the essential oil yield / fed. was recorded in the plants fertilized with the highest N level (120 kg N/fed.) compared to plants receiving nitrogen fertilizer at the rates of 40 and 80 kg N / fed. at both cuts in the first season, the mean values were 45.31, and L / fed. at first cut and 74.08, and L / fed. at the second cut, respectively. Similar results were noticed at the second cut in the second season. On the other hand the essential oil yield / fed. at the first cut, was slightly lower in the plants fertilized with N3 in comparison to those fertilized with N2, giving the mean values were and L / fed., respectively without significant differences between them. 66

125 Table 19. Effect of nitrogen fertilization, plant spacing and their interaction on the essential oil yield (L / feddan) of Satureja hortensis L. plant in the first season, 2014 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N 1.80 S 2.07 N x S 3.11 Second cut S S S Mean L.S.D at 0.05 for N 3.97 S 2.29 N x S 6.87 N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm Table 20. Effect of nitrogen fertilization, plant spacing and their interaction on the essential oil yield (L / feddan) of Satureja hortensis L. plant in the second season, 2015 Plant Spacing Nitrogen fertilization N0 N1 N2 N3 Mean First cut S S S Mean L.S.D at 0.05 for N 3.84 S 4.23 N x S 6.64 Second cut S S S Mean L.S.D at 0.05 for N 2.46 S 3.21 N x S 4.27 N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm 66

126 Generally, the essential oil yield/fed. significantly increased with different levels of nitrogen fertilization. This effect may be attributed to the important role of nitrogen in biosyntheses of volatile oil in the aromatic plants. These results agree with Jalili (2015) and Babalar et al. (2016) on summer savory, Satureja hortensis L. plant, they found that increasing nitrogen fertilization rates significantly increased the essential oil yield / fed. The plant spacings that were used for planting summer savory plants had a significant effect on the essential oil yield / fed. at both cuts of the two growing seasons, planting at the narrowest spacing S1 (15 50 cm) gave the highest essential oil yield / fed. with mean values of and L/fed. at the first season and and L / fed. in the second season at the first and second cuts, respectively, whereas the widest spacing S3 (45 50 cm) gave the lowest oil yields. The favorable effect of narrow plant spacing on the essential oil yield / fed. may be attributed to the large number of plants per area unit (53,333 plants/fed.) and an increase in the oil yield/plant. The increase in the essential oil yield / fed. as a result of narrow plant spacing is in agreement with the findings of Badi et al. (2004) on Thymus vulgaris, Verma et al. (2008) on Indian basil, Ocimum basilicum L. and Raina et al. (2013) on Ocimum sanctum L. plants, they found that the maximum essential oil yield was obtained at the narrow spacing / unit area. Regarding the interaction between the effect of nitrogen fertilization and plant spacing on the essential oil yield / fed., data 66

127 presented in Tables (19 and 20) show that, significant differences were recorded on the values of the various combinations of these two factors at both cuts in the two growing seasons. The lowest values of the essential oil yield / fed. were recorded in unfertilized plants (N0) that were planted at widest spacing S3 (45 50 cm) at both cuts of the two seasons. On the other hand, the highest essential oil yields /fed. were obtained from the plants fertilized with the highest N level N3 (120 kg N/fed.) and planting at narrowest spacing in most cases (at both cuts in the first season and at second cut in the second season). The only exception was notcied in the plants fertilized with N2 (80 kg N/fed.) and planting at narrowest spacing S1 (15 50 cm) at the first cut in the second season, the mean value was L/fed. C. Essential oil components by Gas Chromatography (GC). The data presented in Tables (21, 22, 23 and 24) and illustrated in Figures (1-24) show the main constituents of the essential oil of summer savory, Satureja hortensis L. of all treatments at both cuts in the two seasons as identified by GC. Seven components had been identified in the essential oil. The mean values of major components were carvacrol (47.26 and 42.90%) and (45.61 and 40.39%) and γ- terpinene (38.30 and 39.10%) and (39.41 and 37.20%) at the first and second cuts, respectively, followed by ρ-cymene (3.91 and 4.02%) and (4.16 and 3.94%), terpinolene (2.48 and 4.40%) and (2.47 and 8.06%), α-terpinene (2.65 and 2.82%) and 66

128 Table 21. Effect of nitrogen fertilization and plant spacing and their interactions on the essential oil components (%) of Satureja hortensis L. plant at the first cut in the first season, The components (%) of the essential oil Nitrogen fertilization (N) Plant spacing (S) N0 N1 N2 N3 Mean α-thujene S S S Mean α-pinene S S S Mean α-terpinene S S S Mean ρ-cymene S S S Mean γ-terpinene S S S Mean Terpinolene S S S Mean Carvacrol S S S Mean Seven components representing = (identified) 97.01% Other components representing = (unidentified) 2.99% N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm 66

129 Table 22. Effect of nitrogen fertilization and plant spacing and their interactions on the essential oil components (%) of Satureja hortensis L. plant at the second cut in the first season, The components (%) of the essential oil Nitrogen fertilization (N) Plant spacing (S) N0 N1 N2 N3 Mean α-thujene S S S Mean α-pinene S S S Mean α-terpinene S S S Mean ρ-cymene S S S Mean γ-terpinene S S S Mean Terpinolene S S S Mean Carvacrol S S S Mean Seven components representing = (identified) 96.28% Other components representing = (unidentified) 3.72% N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm 66

130 Table 23. Effect of nitrogen fertilization and plant spacing and their interactions on the essential oil components (%) of Satureja hortensis L. plant at the first cut in the second season, The components (%) of the essential oil Nitrogen fertilization (N) Plant spacing (S) N0 N1 N2 N3 Mean α-thujene S S S Mean α-pinene S S S Mean α-terpinene S S S Mean ρ-cymene S S S Mean γ-terpinene S S S Mean Terpinolene S S S Mean Carvacrol S S S Mean Seven components representing = (identified) 97.07% Other components representing = (unidentified) 2.93% N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm 66

131 Table 24. Effect of nitrogen fertilization and plant spacing and their interactions on the essential oil components (%) of Satureja hortensis L. plant at the second cut in the second season, The components (%) of the essential oil Nitrogen fertilization (N) Plant spacing (S) N0 N1 N2 N3 Mean α-thujene S S S Mean α-pinene S S S Mean α-terpinene S S S Mean ρ-cymene S S S Mean γ-terpinene S S S Mean Terpinolene S S S Mean Carvacrol S S S Mean Seven components representing = (identified) 96.25% Other components representing = (unidentified) 3.75% N. fertilization: N0= 0.0, N1= 40, N2= 80 and N3= 120 Kg N/feddan Plant spacing: S1= 15cm, S2= 30cm and S3= 45cm 60

132 97 Fig.1.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N0S1 treatment at the first cut in the first season, 2014.

133 98 Fig.2.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N0S2 treatment at the first cut in the first season, 2014.

134 99 Fig.3.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N0S3 treatment at the first cut in the first season, 2014.

135 100 Fig.4.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N1S1 treatment at the first cut in the first season, 2014.

136 101 Fig.5.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N1S2 treatment at the first cut in the first season, 2014.

137 102 Fig.6.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N1S3 treatment at the first cut in the first season, 2014.

138 103 Fig.7.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N2S1 treatment at the first cut in the first season, 2014.

139 104 Fig.8.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N2S2 treatment at the first cut in the first season, 2014.

140 105 Fig.9.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N2S3 treatment at the first cut in the first season, 2014.

141 106 Fig.10.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N3S1 treatment at the first cut in the first season, 2014.

142 107 Fig.11.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N3S2 treatment at the first cut in the first season, 2014.

143 108 Fig.12.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N3S3 treatment at the first cut in the first season, 2014.

144 109 Fig.13.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N0S1 treatment at the second cut in the first season, 2014.

145 110 Fig.14.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N0S2 treatment at the second cut in the first season, 2014.

146 111 Fig.15.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N0S3 treatment at the second cut in the first season, 2014.

147 112 Fig.16.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N1S1 treatment at the second cut in the first season, 2014.

148 113 Fig.17.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N1S2 treatment at the second cut in the first season, 2014.

149 114 Fig.18.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N1S3 treatment at the second cut in the first season, 2014.

150 115 Fig.19.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N2S1 treatment at the second cut in the first season, 2014.

151 116 Fig.20.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N2S2 treatment at the second cut in the first season, 2014.

152 117 Fig.21.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N2S3 treatment at the second cut in the first season, 2014.

153 118 Fig.22.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N3S1 treatment at the second cut in the first season, 2014.

154 119 Fig.23.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N3S2 treatment at the second cut in the first season, 2014.

155 120 Fig.24.GC chromatogram of Satureja hortensis L. essential oil distilled from plants treated by N3S3 treatment at the second cut in the first season, 2014.

156 (2.77 and 3.11%), α-pinene (1.57 and 1.86%) and (1.70 and 2.10%) and α- thujene (0.84 and 1.18%) and (0.95 and 1.45%) in the first and second cuts in both seasons, respectively. The total identified components in the essential oil (97.01 and 96.28%) and (97.07 and 96.25%) at the first and second cuts in both seasons, respectively. These results agree with results obtained by Zahedifar and Najafian (2015), EL-Gohary et al. (2015) and Babalar et al. (2016) on summer savory (Satureja hortensis L.). Carvacrol content (the first major component): The recorded data in both seasons were presented in Tables (21, 22, 23 and 24) and Figs (1-24) show the effect of nitrogen fertilizer treatments on the carvacrol content in both cuts in the essential oil extracted from summer savory plants. In the first season the highest mean of carvacrol contents and 44.34% were obtained in the oil of unfertilized plants (N0) at both cuts, respectively compared to fertilized plants with different nitrogen levels (N1, N2 and N3). This effect may be due to carcavrol synthesis and accumulation in essential oil of summer savory plants not need to more nitrogen uptake from the soil medium. In the second season the highest carvacrol contents in the essential oil extracted from plants fertilized with N3 (120 kg N/fed.) were and 42.10% at the first and second cuts, respectively compared to other N treatments (N1 and N2) and unfertilized plants. On the other hand at the first cut, the lowest carcavrol content was 44.86% was obtained in essential oil extracted from plants treated with N2 (80 666

157 kg N/fed.) compared to the other N levels and control plants, while in the second cut of the second season the lowest carvacrol (37.22 %) was obtained from the essential oil control plants. The plants grown at widest spacing (45 50 cm) contained the slightly higher carvacrol contents in the oil, which gave and 43.43% at both cuts, respectively compared to medium and narrow spacings (30 50 cm and cm), the mean values were 45.83, 47.16% and 41.97, 43.31% at both cuts, respectively in the first season. The plants grown at narrowest spacing S1 (15 50 cm) at the first cut and the plants grown at medium spacing S2 (30 50 cm) at the second cut had higher carvacrol contents in essential oil (46.06 and 41.83%, respectively) than the other plant spacing S3 (45 50 cm) in the second season. Regarding the interaction between the effect of nitrogen levels and plant spacing on carvacrol percentage in the essential oil, data presented in Tables (21, 22, 23 and 24) and Figs (1-24) show that the highest carvacrol percentages were recorded in the essential oil extracted from plants fertilized with nitrogen at N1 and planted at a spacing of S3 (45 50 cm) at the first cut (52.50%) and in the essential oil extracted from unfertilized plants when transplanting at a spacing of S3 (45 50 cm) at the second cut (47.04%) in the first season. In the second season, the highest carvacrol contents were found in the essential oil extracted from plants treated with nitrogen at N3 level and planted at narrowest spacing S1 and in oil of plants fertilized with N3 level and planted at medium spacing S2 666

158 treatments, with mean values of and 44.43% at the first and second cuts, respectively compared to the other interaction treatments. Gamma-terpinene content (the second major component) Gamma-terpinene percentage was the lowest mean content in the essential oil obtained from unfertilized plants (N0), the values were and 37.85% in both cuts of the first seasons, respectively compared to the plants fertilized by different nitrogen levels of N1, N2 and N3 (40, 80 and 120 kg N/feddan, respectively) which recorded the highest values of γ-terpinene contents, the means values were 39.34, and 38.82% at the first cut and 39.94, and 38.55% at the second cut, respectively in the first season. Similar results were noticed in γ-terpinene contents in the essential oil extracted from plants fertilized with most nitrogen levels at both cuts in the second season. Generally, nitrogen at all levels (N1, N2 and N3) increased the content of γ-terpinene in the essential oil of summer savory plants compared to the unfertilized plants (N0) at both cuts in both seasons. The highest mean content of γ-terpinene was recorded when the plants cultivated at medium spacing (39.03 and 39.17%) in both cuts of the first season compared to the widest and narrowest spacings, the values were 37.66, 38.20% and 39.13, 38.87% at both cuts, respectively in the first season. In the second season, the highest mean content of γ-terpinene was recorded when the plants 666

159 cultivated at widest spacing S3 (45 50 cm), with mean values of and 37.43% at both cuts compared to medium and narrowest spacings which slightly decreased, the values were 39.23, 39.07% and 36.92, 37.23% at both cuts, respectively. Concerning the interaction effect between nitrogen levels and plant spacing on γ-terpinene percentage in the volatile oil of summer savory, data presented in Tables (21, 22, 23 and 24) and Fig (1-24) reveal that, the highest content (41.77%) was recorded in the oil of the plants fertilized with N1 (40 kg N/fed.) and planted at S2 (30 50cm) at the first cut. Also, plants fertilized by N1 and planted at wide spacing S3 (45 50 cm) resulted the highest content of γ- terpinene (43.01%) compared to the other interaction treatments at the second cut in the first season. In the second season, the highest γ-terpinene content in the essential oil was 41.31% obtained in the plants fertilized with 80 kg N/fed. and planted at widest spacing (S3) at the first cut, moreover, the unfertilized plants (N0) and transplanted at widest spacing (S3) contained the highest content of γ-terpinene (41.92%) in essential oil than the other interaction treatments at the second cut. Other components of α-thujene, α-pinene, α-terpinene, ρ- Cymene and Terpinolene contents Generally, these components mentioned before in the essential oil of summer savory, Satureja hortensis L., the percentages had no detectable trend as affected by N levels fertilizer and plant density as well as interaction between them at the first and second cuts in the first season. Similar trend was recorded at the 666

160 first cut in the second season. On the other hand, the percentages of components mentioned before in the essential oil (α-thujene, α- Pinene, α-terpinene, ρ-cymene and Terpinolene) were higher from unfertilized plants than in essential oil extracted from plants treated with nitrogen levels N1, N2 and N3 at the second cut in the second season. The plant spacing and/or interaction treatments had no detectable effect on the percentage of these components. These results agree with Kandil et al. (2009) on Genovese basil, Runyoro et al. (2010) on Ocimum basilicum and EL-Ziat (2015) on Ocimum species. 3. Anatomical studies a. Shoot anatomy using light microscope As inferred earlier throughout the morphological investigations of vegetative growth and herb yield of summer savory as affected by different levels of nitrogen fertilizer under plant spacing of cm, increasing level from nitrogen fertilizer induced significant gradual increase in plant height and yield of fresh and dry herbs of summer savory plants cultivated at cm at the two cuttings of the two studied seasons. This may justify a further study on the internal structure of the main stem and leaves of plants which were affected by different levels of nitrogen fertilizer under plant spacing of cm. Investigations were carried out in the second season on plants of the first cut at the age of 14 weeks from sowing date. 666

161 126 Table 25. Measurements in micro-meter (µm) of certain histological characters transverse sections through the median portion of the main stem of summer savory plant aged 14 weeks from sowing date, just prior the first cutting, as affected by different levels of nitrogen fertilizer (Means of three sections from three specimens) Histological characters 100 kg ammonium sulphate (20.5% N) / feddan (t1) 200 kg ammonium sulphate (20.5% N) / feddan (t2) Treatments ±% to t1 300 kg ammonium sulphate (20.5% N) / feddan (t3) ±% to t2 Stem diameter Epidermis thickness Cortex thickness Phloem tissue thickness Xylem tissue thickness Vessel diameter Pith diameter

162 Epidermis Cortex Phloem Xylem Vessel A pith Figure 25. Transverse sections through the median portion of the main stem of summer savory plant, at the age of 14 weeks from sowing date, as affected by different levels of nitrogen fertilizer A. From plant received 100 kg ammonium sulphate (20.5% N) / feddan.. (x 125) (Cont.) 721

163 Epidermis Cortex Phloem Cambium zone Xylem B Vessel Pith Figure 25. Cont. B. From plant received 200 kg ammonium sulphate (20.5% N) / feddan. (Cont.) 721

164 Epidermis Cortex Phloem Cambium zone Xylem Vessel C Pith Figure 25. Cont. C. From plant received 300 kg ammonium sulphate (20.5% N) / feddan. 721

165 Microscopical characters were examined through specimens of the median portion of the main stem and its corresponding leaf. This surely highlights the effect of studied treatments on microscopical characters of these organs. Anatomy of the main stem Microscopical measurements of certain histological characters in transverse sections through the median portion of the main stem of summer savory aged 14 weeks from sowing date, just prior the first cut of the second season, as affected by different levels of nitrogen fertilizer, under plant spacing of cm, are presented in Table (25) Likewise, microphotographs illustrating these treatments are shown in Figure (25). Data presented in Table (25) and illustrated in Figure (25) clearly show that increasing level from nitrogen fertilizer induced prominent increase in stem diameter of summer savory at its median portion and this effect was reflected, at least, on most of included tissues. It is noted that raising nitrogen fertilizer from 100 kg ammonium sulphate (20.5% N) / feddan to 200 kg (the first dose) induced prominent increase in stem diameter by 51.9% over the plants which were treated by 100 kg ammonium sulphate. The increase in stem diameter could be attributed mainly to the increase induced in all included tissues. The thickness of epidermis, cortex, phloem tissue and xylem tissue as well as mean diameter of vessel and pith diameter were increased, due to raising the level of nitrogen fertilizer from 100 to 200 kg ammonium 666

166 sulphate (20.5% N) / feddan, by 1.9, 41.3, 98.1, 51.5, 23.9 and 50.1 % ; respectively. Likewise, increasing level of nitrogen fertilizer from 200 to 300 kg ammonium sulphate in stem diameter by 28 % due to the increase induced in all included tissue except the thickness of epidermis which showed negligible decrease of 3.6 % below the thickness of plants epidermis of which were treated with 200 kg ammonium sulphate (20.5% N) / feddan. The slight decrease in epidermis thickness could be attributed mainly to the progressive increase in secondary growth which makes the vascular tissues forces the cortex outward and this forces the epidermis which being suppressed in its thickness. Such treatment increased the rest tissues by 51.5, 43.2, 18.2, 21.1 and 30.5 % for thickness of cortex, phloem tissue, xylem tissue, vessel diameter and pith diameter; respectively. As far as the author is aware, previous information about the effect of nitrogen fertilizer on the anatomical structure of the main stem of summer savory are not available. Anatomy of the leaf blade: Certain microscopical characters of a leaf from the middle portion of the main stem of summer savory as affected by different levels of nitrogen fertilizer were followed up in from of measurements being given in Table (26). These characters are further shown as microphotographs illustrated in Figure (26). It is realized that increasing level of nitrogen fertilizer resulted in thicker leaves. This effect was attributed to increase in 666

167 132 Table 26. Measurements in micro-meter (µm) of certain histological characters transverse sections through the blade of the leaf developed on the median portion of the main stem of summer savory plant aged 14 weeks from sowing date, just prior the first cutting, as affected by different levels of nitrogen fertilizer (Means of three sections from three specimens) Histological characters 100 kg ammonium sulphate (20.5% N) / feddan (t1) 200 kg ammonium sulphate (20.5% N) / feddan (t2) Treatments ±% to t1 300 kg ammonium sulphate (20.5% N) / feddan (t3) ±% to t2 Lamina thickness Palisade tissue thickness Spongy tissue thickness Dimensions of midvein bundle: Length Width

168 Upper epidermis Palisade tissue Spongy tissue Lower epidermis Midvein bundle A B Trichome Figure 26. Transverse sections through leaf blade developed on the median portion of the main stem of summer savory plant, at the age of 14 weeks from sowing date, as affected by different levels of nitrogen fertilizer. (x 125) A. From plant received 100 kg ammonium sulphate (20.5% N) / feddan. B. From plant received 200 kg ammonium sulphate (20.5% N) / feddan. (Cont.) 311

169 C Figure 26. Cont. A. From plant received 300 kg ammonium sulphate (20.5% N) / feddan. 311

170 thickness of lamina and in dimensions of midvein bundle. It is obvious that raising the level of nitrogen fertilizer from 100 to 200 kg ammonium sulphate (20.5% N) / feddan induced prominent increase in thickness of lamina by 47.8% due mainly to the prominent increase induced in thickness by 51.6 and 37.5%; respectively. Also, dimensions of midvein bundle were increased by 68.4 % in length and by 44.8 % in width. In this respect, increasing the level of nitrogen fertilizer from 200 to 300 kg ammonium sulphate (20.5% N) /feddan increased all included tissue of summer savory leaf blade. The increments were 10.1, 3.9, 17.7, 8.3 and 10.5 % for thickness of lamina, palisade tissue, spongy tissue, length of midvein bundle and with of midvein bundle; respectively. As far as the author is aware previous information about the effect of different levels from nitrogen fertilizer on the anatomical structure of summer savory leaves are not available. b. Number of glandular hairs using SEM: Data presented in Table (27) and Figures (27-34) show the effect of different nitrogen levels on the number of glandular hairs per unit surface area (0.04 cm 2 ) and per leaf of Satureja hortensis L. at the first cut in the second season (2015). Generally, the number of glandular hairs per unit (0.04 cm 2 ) and per leaf was higher on the lower epidermis of mature leaf than on the upper epidermis leaf of different fertilization concentrations of N0, N1, N2 and N3 as 0.0, 40, 80 and 120 kg N/fed., respectively, the mean values of glandular hairs per 0.04 cm 2 were 666

171 136 Table 27. Effect of nitrogen fertilization levels on the number of glands per unit surface area (0.04 cm 2 ) and per leaf of Satureja hortensis L. plants at the first cut in the second season, (2015). Treatments Numbers of glands Essential oil yield Leaf area ml/plant* cm 2 ** Upper surface Lower surface Per (0.04 cm 2 ) Per leaf Per (0.04 cm 2 ) Per leaf Total leaf N0S N1S N2S N3S N0S2 = 0.0 kg N/fed. at (30 50 cm) N1S2 = 40 kg N/fed. at (30 50 cm) N2S2 = 80 kg N/fed. at (30 50 cm) N3S2 = 120 kg N/fed. at (30 50 cm) * = from Table (18) ** = mean of mature leaves (cm 2 )

172 A B Fig. 27. Scanning electron microscope (SEM) micrographs of the glandular hairs on the upper epidermis of mature leaf of Satureja hortensis L. plant treated with N0S2 treatment at the first cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair. 137

173 A B Fig. 28. Scanning electron microscope (SEM) micrographs of the glandular hairs on the lower epidermis of mature leaf of Satureja hortensis L. plant treated with N0S2 treatment at the first cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair. 138

174 A B Fig. 29. Scanning electron microscope (SEM) micrographs of the glandular hairs on the upper epidermis of mature leaf of Satureja hortensis L. plant treated with N1S2 treatment at the first cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair. 139

175 A B Fig. 30. Scanning electron microscope (SEM) micrographs of the glandular hairs on the lower epidermis of mature leaf of Satureja hortensis L. plant treated with N1S2 treatment at the first cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair. 140

176 A B Fig. 31. Scanning electron microscope (SEM) micrographs of the glandular hairs on the upper epidermis of mature leaf of Satureja hortensis L. plant treated with N2S2 treatment at the first cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair. 141

177 A B Fig. 32. Scanning electron microscope (SEM) micrographs of the glandular hairs on the lower epidermis of mature leaf of Satureja hortensis L. plant treated with N2S2 treatment at the first cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair. 142

178 A B Fig. 33. Scanning electron microscope (SEM) micrographs of the glandular hairs on the upper epidermis of mature leaf of Satureja hortensis L. plant treated with N3S2 treatment at the first cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair. 143

179 A B Fig. 34. Scanning electron microscope (SEM) micrographs of the glandular hairs on the lower epidermis of mature leaf of Satureja hortensis L. plant treated with N3S2 treatment at the first cut in the second season, A. Total glandular hairs (0.04 cm 2 ). B. Glandular hair. 144