Effect Of Biofertilizers And/Or Urea On Growth, Yield, Essential Oil And Chemical Compositions Of Cymbopogon Citratus Plants.

Abstract

This investigation was conducted during two successive seasons (2010 and 2011) in the Experimental Farm Station of the Faculty of Agriculture, Minufiya University, Shebin El-Kom, to optimize the use of Biofertilizers (Nitrobin, Rhizobacteren and Microbein) with or without different doses of Urea in the cultivation of lemongrass (Cymbopogon Citratus). Data clearly showed that, nitrogen as soil application and biofertilizers had a significant effect on vegetative growth characters. On the other hand, the interaction treatments between of N and biofertilizers led to significant increment for yield of essential oil compared to control during both seasons. Using 150 kg N/fed + 1 kg Microbein/fed gave the highest values of essential oil content in both seasons. Citral A (Geranial), was found to be the first major compound in the essential oil of Cymbopogon citratus and ranged from 34.85 to 42.74 %. Its maximum content was observed in the essential oil of the herb that received 75 kg N/fed with 1 kg Rhizobacteren /fed, followed by Citral B (Neral) which ranged from its maximum content (35.62%) with75 kg N/fed without biofertilizer to its minimum relative percent (27.56%) with150 kg N/fed with 1 kg Rhizobacteren /fed and β-Myrcene was identified as the third main constituent (3.42-10.58%). applications of biofertilizers with nitrogen fertilizers resulted in a significant effect of Polyphenol and flavonoid content in both seasons.

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309
Journal of Applied Sciences Research, 9(1): 309-320, 2013
ISSN 1819-544X
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLES
Corresponding Author: El Gendy A.G. Medicinal and Aromatic Plants Department Research, National Research Center,
Giza, Egypt.
E-mail: aggundy_5@yahoo.com. Tel: +20483690146 and +201222341398
Effect Of Biofertilizers And/Or Urea On Growth, Yield, Essential Oil And Chemical
Compositions Of Cymbopogon Citratus Plants
1El Gendy A.G., 2Taghred A. Hegazy and 3El-Sayed S.M.
1Medicinal and Aromatic Plants Department Research, National Research Center, Giza, Egypt
2Horticulture Dept., Faculty of Agric., Menufiya University, Shibin El-Kom, Egypt
3Agricultural Biochemistry Dept., Faculty of Agric., Menufiya University, Shibin El-Kom, Egypt
ABSTRACT
This investigation was conducted during two successive seasons (2010 and 2011) in the Experimental Farm
Station of the Faculty of Agriculture, Minufiya University, Shebin El-Kom, to optimize the use of Biofertilizers
(Nitrobin, Rhizobacteren and Microbein) with or without different doses of Urea in the cultivation of
lemongrass (Cymbopogon Citratus). Data clearly showed that, nitrogen as soil application and biofertilizers had
a significant effect on vegetative growth characters. On the other hand, the interaction treatments between of N
and biofertilizers led to significant increment for yield of essential oil compared to control during both seasons.
Using 150 kg N/fed + 1 kg Microbein/fed gave the highest values of essential oil content in both seasons. Citral
A (Geranial), was found to be the first major compound in the essential oil of Cymbopogon citratus and ranged
from 34.85 to 42.74 %. Its maximum content was observed in the essential oil of the herb that received 75 kg
N/fed with 1 kg Rhizobacteren /fed, followed by Citral B (Neral) which ranged from its maximum content
(35.62%) with75 kg N/fed without biofertilizer to its minimum relative percent (27.56%) with150 kg N/fed with
1 kg Rhizobacteren /fed and β-Myrcene was identified as the third main constituent (3.42- 10.58%). applications
of biofertilizers with nitrogen fertilizers resulted in a significant effect of Polyphenol and flavonoid content in
both seasons.
Key words: Cymbopogon citratus, biofertilizers, mineral fertilizers, Essential Oil, Nitrobin, Rhizobacteren,
Microbein, Geranial and Neral
Introduction
Lemongrass (Cymbopogon citratus Stapf.) is cultivated in many countries around the world, e.g.
Argentina, Brazil, Cuba, Ecuador, India, Singapore, China, France, Madagascar, Haiti, Puerto Rico, Mexico,
Guatemala, Honduras and Salvador, for its essential oil rich in terpene compounds (Elizabeth et al. 2011). In
Europe, leaves are used in teas and infusions. In Mexico, lemongrass tea is used traditionally as a sleep aid,
tranquilizer, digestive, anti-influenza and antispasmodic (Rauber et al. 2005).
The volatile oil obtained from leaves of this plant is widely used by the perfume and cosmetics industries. It
has also been used in chemical synthesis, due its high contents of citral, which is a natural mixture of two
isomeric aldehydes, neral and geranial (Rauber et al. 2005)
The microbial strains (bio fertilizers) led to nitrogen fixation (N-bacteria) and availability of phosphorus
(phosphate dissolving bacteria) as well as the production of growth promoting substance (Abd-El Latif et al.,
2001).The coincident application of organic manuring and biofertilizers is frequently recommended for
improving biological, physical, and chemical properties of the soil, in addition to getting highly clean
agricultural yield (Gomaa et al., 2002).
Different organic cultivation techniques have been used to increase plant yield and essential oil in
lemongrass. Lemongrass has been inoculated with arbuscular mycorrhizal fungi (AMF), e.g. Glomus mosseae
and G. fasciculatum, and biomass yield increased by 3-10% (Elizabeth et al. 2011).
Many investigators i.e. Mona (2006) on plantago afra L, Khalid et al (2006) on Ocimum basilicum and
Abdullah, et al (2012) on Rosmarinus officinalis observed that, biofertilizers had favorable effects on growth,
yield and chemical composition.
On Foeniculum vulgare plants Mahfouz and Sharaf-Eldin (2007) found that, Application of biofertilizer,
which was a mixture of Azotobacter chroococcum, Azospirillum liboferum, and Bacillus megatherium applied
with chemical fertilizers (only 50% of the recommended dosage of NPK) increased vegetative growth compared
to chemical fertilizer treatments only. Also application by biofertilizers increased Essential oil content in the
fennel fruits compared to the half dose of chemical fertilizer. Badran and Safwat (2004) and El-Ghadban et al.

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(2006) found that, fennel responded to biofertilizer by increasing growth and oil yield as well as changing the
chemical composition.
An Iranian investigation revealed that inoculation of Ocimum basilicum roots with plant growth-promoting
Rhizobacteria (PGPR) improved growth and accumulation of essential oils. In comparison to the control
treatment, all factors were increased by PGPR treatments. The maximum Root fresh weight (3.96 g/plant), N
content (4.72%) and essential oil yield (0.82%) were observed in the Pseudomonas + Azotobacter + Azosprillum
treatment. All factors were higher in the Pseudomonas + Azotobacter + Azosprillum and Azotobacter +
Azosprillum treatments (Ordookhani et al., 2011).
Ratti et al. (2001) investigated effect of some varieties of phosphate solubilizing bacteria on the yield of
Lemon Grass and concluded that, the plant height and biomass increased compared to the control condition.
Nitrogen is most recognized in plants for its presence in the structure of the protein molecule. In addition,
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, which are essential for protein synthesis. The
porphyrin structure is found in such metabolically important compounds as the chlorophyll pigments and the
cytochromes, which are essential in photosynthesis and respiration. Coenzymes are essential to the function of
many enzymes. Accordingly, nitrogen plays an important role in synthesis of the plant constituents through the
action of different enzymes. Nitrogen limiting conditions increase volatile oil production in annual herbal.
Nitrogen fertilization has been reported to increase total oil yield in thyme [Thymus vulgaris] (Baranauskienne
et al., 2003). Also, Singh (1999) found that, Nitrogen application significantly increased crop growth values
such as plant height, leaf area index (LAI), herbage and essential oil contents lemongrass (Cymbopogon
flexuosus)
The objective of the present investigation was to optimize the use of Biofertilizers (Nitrobin, Rhizobacteren
and Microbein) with or without different doses of Urea in the cultivation of lemongrass. Plant yield in terms of
herb weight, essential oil yield and its composition were monitored.
Materials And Methods
2.1. The studied location:
This investigation was conducted during two successive seasons (2010 and 2011) in the Experimental Farm
Station of the Faculty of Agriculture, Minufiya University, Shebin El-Kom, as the clay loam soil. The
mechanical and chemical properties of the used soil in this study were determined according to Jackson (1973)
and Cottenie et al (1982) and were as follows.
pH 7.9, EC 1.73 ds/m; 2.84% organic matter, 44.34% silt, 3.84% coarse sand, 27.39% fine sand 23.20%
clay, i.e. the texture was a clay loam soil. The available Macro nutreients were 250 ppm N, 125 ppm P and 320
ppm K.
2.2. Plant material:
Uniform plants of lemongrass (30cm in height) introduced from medicinal and Aromatic plant section,
Agriculture Research Center, Ministry of agriculture were planted during the 1st week of March during both
seasons. The transplants were individually placed at 60 cm apart from each other and rows 40 cm apart. Each
plot contained about 70 plants (20000 plant/fed). Plants were irrigated immediately after transplanting and later
as required to maintain vigorous growth.
2.3. Field Trials:
The experimental trials included various levels of Urea 46% N (0, 75 and 150 kg N/Fed) as a source of
chemical fertilizers combined with one of Nitrobin, Rhizobacteren or Microbein at recommended dose (1 kg
/fed) that were individually used as a source of bio fertilizers. Urea was added after one month from cultivation.
So, the studied treatments were 12 treatments.
The biofertilizers such as Nitrobin, Rhizobacteren and Microbein were obtained from General Organization
for Agricultural Equalization Fund (GOAEF), Agricultural Research Center, Giza, Egypt.
- Nitrobein is the commercial name of nitrogen fixing bacteria containing Azotobacter spp. (Azotobacter
chroococcum). and Azospirilum spp. (Azopirillum lipoferum).
- Rhizobacterin a commercial product that contains a specific strain of Rhizobiusm spp which fixes
atmospheric N.
- Microbein is a commercial product that contains a wide range of microbes such as Bacillus polymixa,
Bacillus megatherium, Pseudomonas fluorescence, Azospirilum spp and Azotobacter spp. The strains of the
biofertilizer were mixed and added to the experiment soil, in both seasons.

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2.4. Data recorded:
Plants were harvested (cutting) twice near of the soil where, the first cut after 6 months from cultivation in
August and the 2 nd one after three months later in the November in both seasons and the following parameters
were recorded:
1- Plant height (cm)
2- Number of tillers
3- Fresh and dry weight of herbage (g/plant and ton/fed)
The harvested plants were air dried and stored in the laboratory to analyse. Dried and separated leaves
samples were kept in plastic bags at room temperature until analysis for essential oil.
2.5. Chemical constituents determination:
2.5.1. Essential oil production:
The volatile oil was isolated from (mean of two cuts) leaves during both seasons by hydrodistillation for 3
hr in order to extract the essential oils according to Guenther (1961). The isolated volatile oil was dehydrated
over anhydrous sodium sulphate and stored in refrigerator until GC-MS analysis.
2.5.2. GC-MS analysis:
The GC-Ms analysis of the essential oil samples was carried out in the second season using gas
chromatography – mass spectrometry instrument stands at the Laboratory of medicinal and aromatic plants,
National Research Center with the following specifications. Instrument: a TRACE GC Ultra Gas
Chromatographs (THERMO Scientific Corp., USA), coupled with a THERMO mass spectrometer detector (ISQ
Single Quadrupole Mass Spectrometer). The GC-MS system was equipped with a TG-WAX MS column (30 m
x 0.25 mm i.d., 0.25 μm film thickness). Analyses 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: 40 C for 1 min; rising at 4.0 C/min to
150 C and held for 6 min; rising at 4 C/min to 200 C and held for 1min. The injector and detector were held at
200 and 200 C, respectively. Diluted samples (1:10 hexane, v/v) of 0.2 μL of the mixtures were always injected.
Mass spectra were obtained by electron ionization (EI) at 70 eV, using a spectral range of m/z 40-450. Most of
the compounds were identified using two different analytical methods: (a) KI, Kovats indices in reference to n-
alkanes (C9-C22) (National Institute of Standards and Technology 2009); and (b) mass spectra (authentic
chemicals and Wiley spectral library collection). Identification was considered to be tentative when it was based
on mass spectral data only.
2.5.3. Polyphenol content:
Total Polyphenol content (mg/100g) in dry leaves was determined based on the method described by
Singleton et al. (1965).
2.5.4. Flavonoid content:
The content of total flavonoids (mg/100g) in dry leaves was determined according to the method given by
Meda et al. (2005).
2.6. Statistical analysis:
Data of the present study were statistically analyzed according to Cochran and Cox (1987) in where data for
each season were analyzed separately. The differences between means of the treatments were considered
significant and highly significant when they were more than least significant differences (LSD) at 5%,
respectively.
Results:
3.1. Growth characteristics:
3.1.1. Effect of nitrogen application:

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Data presented in Tables (1, 2 and 3) clearly showed that, nitrogen as soil application had a significant
effect on vegetative growth characters i.e. plant height (cm), tillers number/plant, as well as fresh and dry
weights (g/plant or ton/fed). The vegetative growth parameters were increased significantly as a result of urea
fertilizer. The maximum mean values of vegetative growth characters were recorded with soil application at 150
Kg N/ fed in both seasons. The stimulation effects of applying nitrogen on vegetative growth may be attributed
to the well known functions of nitrogen in plant life, as described in the Introduction. Moreover, nitrogen is
involved in many organic compounds of the plant system. A sufficient supply of various nitrogenous
compounds is, therefore, required in each plant cell for its proper functioning. Generally, the enhancing effect of
N-fertilization on plant growth may be due to the positive effects of nitrogen on activation of photosynthesis and
metabolic processes of organic compounds in plants which, in turn, encourage plant vegetative growth. The
increase in the fresh herbage yield with nitrogen fertilization is in agreement with results reported were recorded
by Agamy (2004) on Foeniculum vulgare; Said-Al Ahl (2005) on Anethum graveolens; Mauyo et al. (2008) on
Cleome gynandra, Said-Al Ahl et al (2009) on Origanum vulgare, Aziz and El Ashiry (2009) on Melissa
officinalis, El Gendy, (2010) on Artemisia annua and Ordookhani et al., (2011) on Ocimum basilicum.
3.1.2. Effect of biofertilizer application:
biofertilizer application (Nitrobin, Rhizobacteren or Microbein) had a significant effect on plant height
(cm), number of tillers/plant, herb fresh and dry weight (g/plant) as well as fresh and dry yield of herb (ton/fed),
in two cuts during both seasons. The highest vegetative growth characters were recorded with the addition of
Microbein compared to the control in two cuts during both seasons. The microbial strains (bio fertilizers) led to
nitrogen fixation (N-bacteria) and availability of phosphorus (phosphate dissolving bacteria) as well as the
production of growth promoting substance (Abd-El Latif et al., 2001)
The positive effect of Biofertilizers (Nitrobin, Rhizobacteren and Microbein) on growth characteristics in
general may be due to organic matter which leads to a clean product of plants, continuous supply of nutrients,
improve some physical and chemical properties of soil, and increase water retention than that for chemical
fertilizers (Das 2008). Also these results may be due to the role of Azotobacter and Azospirillum in nitrogen
fixation. In addition, they provide growth promoting substances such as indole acetic acid and gibberellins
(Fayez et al., 1985).
These results are in agreement with those of Gad (2001) for fennel (Foeniculum vulgare) and dill (Anethum
graveolens) also, Mahfouz and Sharaf-Eldin (2007) on Foeniculum vulgare plants, who reported that
biofertilizers on these plants increased growth and yield. Amin (1997), who studied coriander (Coriandrum
sativum), fennel (Feoniculum vulgare) and caraway (Carum carvi), showed that the growth was influenced by
seed inoculation (Azotobacter and Azospirillum) with a half dose of inorganic fertilizer. Plant growth was nearly
equal to that obtained when the plants were fertilized with a full dose of inorganic fertilizer. Tehlan et. al.
(2004) reported that plant growth and seed yield of fennel varied according to the strain applied. Mahfouz and
Sharaf-Eldin (2007), also Ordookhani et al., (2011) on Ocimum basilicum found the same results.
3.1.3. Effect of the interaction:
Results presented in Tables (1, 2 and 3) indicated that, plant height (cm), fresh weight (g/plant) and fresh
yield (ton/fed) were significantly affected by the interaction between urea and bio fertilizer in two cuts during
both seasons. Number of tillers/plant was significantly affected by the interaction in the second season only and
the first cut of the first season. On the other hand, dry weight (g/plant) and dry yield (ton/fed) were significant
increased in the second cut of both seasons and insignificant in the first cut of both season. The maximum mean
value of plant height (cm), number of tillers/plant, fresh weight (g/plant), dry weight (g/plant), fresh yield
(ton/fed) and dry yield (ton/fed) were recorded with plants received 150 kg N / fed + Microbein in the two cuts
for both seasons. The lowest mean values of vegetative growth characters were obtained as a result of the
control treatment during the two cuts during both seasons.
Generally with different treatments under study, the mean values at the second season were higher than
those of the first season. The difference in the means of vegetative growth characters of both seasons may be
attributed to varying environmental factors, i.e. temperature (air and soil), light levels and moisture conditions.
[The synthesis of secondary metabolites has been related to the capture of light energy (Omer et al., 1994,
Ozguven et al., 2008, Said Al Ahl, 2005 and Hellal et al 2011)].

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Table 1: Effect of Bio and /or Urea fertilization on Plant height (cm) and Number of tillers/plant of lemon grass plant during the two growing seasons of 2010/2011.
Treatments
Plant height (cm)
N
umber of tillers/plant
1st season 2010
1 st Cut 2 nd Cut 1 st Cut 2 nd Cut
0 75 150 Mean A 0 75 150 Mean A 0 75 150 Mean A 0 75 150
M
ean A
Control 71.30 74.40 77.63 74.44 82.83 82.16 91.21 85.40 16.11 21.88 22.90 20.29 22.54 26.85 35.87 28.42
Ni 81.80 85.76 92.35 86.36 92.31 101.90 107.30 100.50 23.98 31.60 37. 26 30.94 33.30 38.33 47.00 39.54
Rh 105. 06 119.63 129.83 118.17 113.90 129.90 134.23 126.01 31.02 41.33 56.23 42.86 45.30 50.00 63.48 52.92
Mi 120.93 132.30 138.33 130.52 130.06 138.63 148.56 139.08 56.03 60.53 73.23 63.26 57.60 63.00 77.06 65.88
Mean B 94.77 103.02 109.53 102.37 113.14 120.32 25.79 38.83 47.40 39.68 44.54 55.85
LSD
at 5%
A
B
AB
2.573
2.432
4.427
2.749
4.050
4.763
2.590
1.950
4.492
2.530
1.826
ns
2 nd season 2011
1 st Cut 2
nd Cut 1 st Cut 2 nd Cut
Control 73.30 76.57 77.96 75.94 84.90 84.66 92.63 87.39 16.96 25.74 26.53 23.07 25.53 36.06 41.23 34.27
Ni 83.40 88.50 95.63 89.17 95.03 105.53 116.80 105.78 28.63 32.96 41.06 34.21 38.56 39.20 49.10 42.28
Rh 109. 06 122.13 136.30 122.49 118.13 139.10 143.90 133.71 42.15 48.56 54.13 48.28 47.73 55.61 66.10 56.48
Mi 130.83 136.60 149.60 139.01 132.93 144.93 150.66 142.84 72.16 72.03 75.0 73.06 74.36 72.30 79.03 75.23
Mean B 99.14 105.95 114.87 107.70 118.55 125.99 39.97 44.82 49.18 46.36 50.79 58.86
LSD
at 5%
A
B
AB
2.179
2.986
3.763
2.566
4.349
4.444
2.129
1.518
3.688
2.956
4.473
5.121
Ni = Nitrobin, Rh= Rhizobacteren, Mi = Microbein A= Bio fertilizers, B= Urea levels 0, 75 150 kg/fed AB= Interaction
Table 2: Effect of Bio and /or Urea fertilization on Fresh and Dry weight (g/plant) of lemon grass plant during the two growing season of 2010/2011.
Fresh weight (g/plant) Dry weight (g/plant)
1st season 2010
1 st Cut 2 nd Cut 1 st Cut 2 nd Cut
0 75 150 Mean A 0 75 150 Mean A 0 75 150 Mean A 0 75 150 Mean A
Control 236.93 335.60 403.93 325.48 256.03 389.63 407.10 350.92 70.17 74.60 75.90 73.55 77.20 83.26 82.70 81.05
Ni 408.30 510.90 596.30 505.18 533.30 602.33 762.60 632.74 83.26 84.90 85.26 84. 47 87.56 88.40 96.75 90.90
Rh 611.41 788.67 791.96 730.66 882.16 892.23 925.56 899.98 82.03 92.23 96.00 90.08 87.23 93.56 97.56 92. 78
Mi 895.63 910.60 996.30 934.17 911.33 940.30 1073.30 974.97 95.60 108.06 106.90 103.52 93.20 98.23 97.60 100.66
Mean B 538.06 636.44 697.12 645.70 706.12 792.14 82.76 89.94 91.01 86.29 90.86 110.56
LSD
at 5%
A
B
AB
14.42
16.75
24.99
22.46
25.35
38.91
5.826
4.914
ns
2.763
3.98
4.786
2 nd season 2011
1 st Cut 2 nd Cut 1 st Cut 2 nd Cut
Control 238.13 338.13 404.10 326.78 259.60 403.06 407.76 356.80 69.86 76.53 77.46 74.61 76.33 83.93 84.23 81.49
Ni 408.70 477.26 594.06 493.34 535.13 591.96 735.56 620.88 82.86 83.70 87.90 84.82 81.36 90.63 96.06 89.35
Rh 612.60 790.50 793.60 732.23 883.96 889.00 902.50 891.82 84.90 91.36 94.73 90.33 88.56 91.30 94.36 91.40
Mi 922.90 911.93 993.40 942.74 905.96 942.03 1039.56 962.49 95.90 101.43 107.46 101.59 94.30 96.90 110.90 100.70
Mean B 545.58 629.45 696.29 646.16 706.50 771.33 83.38 88.25 91.88 85.13 90.69 96.38
LSD
at 5%
A
B
AB
16.86
19.335
29.21
20.145
30.726
34.89
2.780
3.252
ns
3.017
1.913
5.22
Ni = Nitrobin, Rh= Rhizobacteren, Mi = Microbein A= Bio fertilizers, B= Urea levels 0, 75 150 kg/fed AB= Interaction
Table 3: Effect of Bio and /or Urea fertilization on Fresh and Dry weight (ton/fed) of lemon grass plant during the two growing season of 2010/2011.
Fresh weight (ton/fed) Dry weight (ton/fed)
1st season 2010
1 st Cut 2 nd Cut 1 st Cut 2 nd Cut
0 75 150 Mean A 0 75 150 Mean A 0 75 150 Mean A 0 75 150 Mean A
Control 4.74 6.71 8.08 6.51 5.12 7.79 8.14 7.02 1.40 1.49 1.52 1.47 1.54 1.67 1.65 1.62
N
i 8.17 10.22 11.93 10.10 10.67 12.05 15.25 12.65 1.67 1.70 1.71 1.69 1.75 1.77 1.94 1.82
Rh 12.23 15.77 15.84 14.61 17.64 17.84 18.51 18.00 1.64 1.85 1.92 1.80 1.75 1.87 1.95 1.86
Mi 17.91 18.21 19.93 18.68 18.23 18.81 21.47 19.50 1.91 2.16 2.14 2.07 1.86 1.97 2.21 2.01
Mean B 10. 76 12.73 13.94 12.91 14.12 15.84 1.66 1.80 1.82 1.73 1. 82 1.94
LSD
at 5%
A
B
AB
0.288
0.335
0.500
0.449
0.507
0.778
0.116
0.098
ns
0.055
0.079
0.095
2 nd season 2011
1 st Cut 2 nd Cut 1 st Cut 2 nd Cut
Control 4.76 6.76 8.08 6.54 5.19 8.06 8.16 7.14 1.40 1.53 1.55 1.49 1.53 1.68 1.69 1.63
N
i 8.17 9.55 11.88 9.87 10.70 11.84 14.71 12. 42 1.66 1.67 1.76 1.70 1.63 1.81 1.92 1.79
Rh 12.25 15.81 15.87 14.64 17.68 17.78 18.05 17.84 1.70 1.83 1.90 1.81 1.77 1.83 1.89 1.83
Mi 18.46 18.24 19.87 18.85 18.12 18.84 20.79 19.25 1.92 2.03 2.15 2.03 1.89 1.94 2.22 2.01
Mean B 10.91 12.59 13.93 12.92 14.13 15.43 1.67 1. 77 1.84 1.70 1.82 1.93
LSD
at 5%
A
B
AB
0.337
0.387
0.584
0.403
0.615
0.698
0.055
0.065
ns
0.060
0.038
0.104
Ni = Nitrobin, Rh= Rhizobacteren, Mi = Microbein A= Bio fertilizers, B= Urea levels 0, 75 150 kg/fed AB= Interaction
3.2. Essential oil production:
3.2.1. Effect of nitrogen application:
Data recorded in Table (4) indicate that, nitrogen fertilization significantly affected the essential oil
percentage (mean of two cuts) in both seasons. Nitrogen treatments significantly increased essential oil (%)
during both seasons. The maximum mean value of essential oil (0.79 and 0.78%) was recorded with plants
fertilized with 150 kg N /fed during first and second season, respectively. On the other hand, nitrogen fertilizers
had a significant increment on essential oil yield (L. /fed) comparing with control. The maximum mean value of
essential oil yield (L/fed) were resulted from plants received 150 kg N/fed (14.62 and 14.60 L/fed) in first and

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second season, respectively. The increment of herb yield may be due to weight of herb and /or essential oil
percentage.
Nitrogen fertilization might enhance the essential oil biosynthesis processes through its direct or indirect
role in plant metabolism resulting in more plant metabolites. The response of volatile oil content to nitrogen
fertilization might be attributed to de novo meristemic cell metabolism in building dry matter with essential oil
production. These finding are in agreement with those of Omer (1998), Omer et al. (2008) and Said-Al Ahl et al
(2009) who found that, nitrogen fertilizer was effective in increasing essential oil of Origanum syriacum,
Ocimum americanum and Origanum vulgare respectively .
3.2.2. Effect of biofertilizer application:
Concerning the effect of biofertilizer on the content of essential oil (%) in both seasons was recorded in
Table (4). Application of 1 kg/fed Nitrobin or Rhizobacteren significantly decreased essential oil percentage in
both seasons comparing to control. While, application of 1 kg/fed Microbein significantly increased essential oil
percentage in both seasons comparing to control. The maximum mean value of essential oil % was recorded
with plants fertilized with 1 kg/fed Microbein (0.75 %) in both seasons. On the other hand, biofertilizer had a
significant increment on essential oil yield (L. /fed) comparing with control. The maximum mean value of
essential oil yield (L/fed) were resulted from plants received1 kg/fed Microbein (14.95 and 15.26 L/fed) in first
and second season, respectively.
Biofertilizers (Nitrobin, Rhizobacteren and Microbein) are a rich in organic matter which leads to a clean
product of plants, continuous supply of nutrients, improve some physical and chemical properties of soil, and
increase water retention than that for chemical fertilizers (Das 2008). Similar results were found by Badran and
Safwat (2004), El-Ghadban et al. (2006), Mahfouz and Sharaf-Eldin (2007) and Ordookhani et al., (2011).
3.2.3. Effect of the interaction:
Concerning the effect of interaction treatments on essential oil (%), data recorded in table (4) pointed out
that, these treatments affected significantly of essential oil (%). The maximum essential oil percentage was
determined in plants fertilized with 150 kg N combined with 1kg/fed Nitrobein (0.88 and 0.80 in first and
second season, respectively.
Table 4: Effect of Bio and /or Urea fertilization on essential oil %, essential oil yield (L/ Fed), Polyphenol (mg/100 g herb) and Total Flavonoid (mg/100 g herb) of lemon grass plant
during the two growing season of 2010/2011.
Essential oi l % essential oil yi eld L/ Fed Polyphenol mg/100 g herb Total Flavonoid mg/100 g herb
1st seaso
n
0 75 150 Mean
A 0 75 150 Mean
A 0 75 150 Mean
A 0 75 150 Mean
A
Control 0.60 0.62 0.84 0.69 8.84 9.79 13.32 10.65 386.49 406.90 433.76 409.05 324.89 351.79 386.11 354.26
Ni 0.48 0.70 0. 88 0.69 8.20 12.13 16.02 12.12 308.67 384.20 422.87 371.91 251.33 331.60 372.44 318.46
Rh 0.54 0.50 0.66 0.57 9.14 9.29 12.77 10.40 330.34 342.28 356.27 342.96 260.49 274.31 305.28 280 .03
Mi 0.70 0.74 0.80 0.75 13.22 15.27 16.36 14.95 402.29 448.70 517.74 456.24 303.81 387.32 434.14 375.09
Mean B 0.58 0.64 0.79 9.85 11.62 14.62 356.95 395.52 432.66 285.13 336.26 374.49
LSD at
5% A 0.02 0.82 10.37 6.78
B 0.01 0.44 7.18 4.78
AB 0.12 1.42 17.96 12.47
2nd season
0 75 150 Mean
A 0 75 150 Mean
A 0 75 150 Mean
A 0 75 150 Mean
A
Control 0.72 0.72 0.76 0.73 10.53 11.55 12.29 11.46 448. 24 485.44 504.12 479.27 356.70 395.90 420.50 391.03
Ni 0.59 0.66 0. 80 0.68 9.69 11.51 14.72 11.97 420.88 449.09 460.13 443.37 351.50 385.40 410.00 382.30
Rh 0.61 0.64 0.76 0.67 10.58 11.69 14.37 12.21 409.20 438.16 513.07 453.48 327.60 365.30 448.30 380.40
Mi 0.74 0.74 0.78 0.75 14.07 14.68 17.03 15.26 478.24 497.69 575.87 517.27 392.50 424.60 508.10 441.73
Mean B 0.67 0.69 0.78 11.22 12.36 14.60 439.14 467. 60 513.30 357.08 392.80 446.73
LSD at
5% A 0.012 0.42 7.70 7.71
B 0.012 0.44 8.70 6.60
AB 0.063 2.30 13.34 14.08
Ni = Nitrobin, Rh= Rhizobacteren, Mi = Microbein, A= Bio fertilizers, B= Urea levels 0, 75, 150 kg/fed And AB= Interactio
On the other hand, the interaction treatments between nitrogen and biofertilizers led to significant increment
for yield of essential oil (L/fed) compared to control in both seasons. From the data tabulated in Table (4) it is
obvious that, using 150 kg N/fed + 1 kg microbein/fed gave the highest values of oil content (16.36 and 17.03
L/fed), followed by the application of 150 kg N/fed + 1 kg Nitrobin/fed (16.02 and 14.72 L/fed) in first and
second season, respectively.
The mean values of essential oil at the second season were higher than those of the first season. The
difference in the means of essential oil of both seasons may be attributed to varying environmental factors, i.e.
temperature (air and soil), light levels and moisture conditions. The environmental factors appear to expert a
greater influence on the accumulation of total oil rather than on the chemical composition of plants (Morales et
al., 1993). The synthesis of secondary metabolites has been related to the capture of light energy (Omer et al.,
1994 and Ozguven et al., 2008).

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J. Appl. Sci. Res., 9(1): 309-320, 2013
3.3. Essential oil composition:
Gas liquid–Mass spectrum analyses (table 5) indicated that, the identified compounds were 36 for essential
oil of Cymbopogon citratus. Citral A (Geranial), was found to be the first major compound in the essential oil of
Cymbopogon citratus and ranged from 34.34 to 42.75 %. Its maximum content was observed in the essential oil
of the herb that received 75 kg N/fed with 1 kg Rhizobacteren /fed, while the minimum content was recorded
with plants that received 150 kg N/fed with 1 kg Rhizobacteren /fed., The second main compound was identified
as the Citral B (Neral) which ranged from its maximum content (35.62%) with75 kg N/fed without biofertilizer
to its minimum relative percent (27.56%) with150 kg N/fed with 1 kg Rhizobacteren /fed. The quality of C.
citratus essential oil was related to its citral content, i.e. a mixture of isomeric aldehydes, neral and geranial.
Essential oils with a citral content > 75% are considered of high quality (Combrinck et al. 2011), β-Myrcene
was identified as the third main constituent and ranged from 3.42 to 10.95%. Elizabeth et al 2011 reported that,
citral was the most important component of the essential oil, with concentrations ranging from 59.7 to 64.0%.
Another factor that affects the quality of the essential oil of C. citratus is β-myrcene. Oils with β-myrcene are
poorly soluble in ethanol and form opalescent solution, this property is a limitation for their use in the perfume
industry (Chingin et al. 2008). Nerol was found to be the 4 th main compound (4.06 – 5.77 %) followed by
Linalool (1.37 – 3.23%) followed by 6-methyl-5-hepten-2-one (0.60-2.47%). The oxygenated compounds
accounted for (85.64- 93.72 %) and the non-oxygenated compounds were (5.43-13.07 %), Monoterpene
compounds reached (97.84-98.93 %) and the sesquiterpenoids compounds were (0.52-1.31 %).
These results agreed with Aziz and El-Ashry (2002), Koffi1 et al., (2009) and Aziz et al., (2010) reported
that, the main components of C. citratus oil are neral, geranial and citronellol represented about 80 % of the
essential oil. More over the essential oil of lemongrass is characterized by a high content of citral (>45%)
(Khanuja et al., 2005) as well as the quality is generally determined by its content of citral (Negrelle and
Gomes, 2007).
Correlation coefficient between Citral B (Neral) and Cital A (Geranial) percentage in various fertilizer
treatments was R2= 0.924 (Fig. 1).
Fig. 1: Correlation between Citral B (Neral) and Cital A (Geranial) in various treatments.
3.4. Polyphenol content
3.4.1. Effect of nitrogen application:
Data presented in Table (4) explain that, nitrogen application significant increased mean value of
polyphenol content (mg/100 g herb) of the lemon grass leaves. The Polyphenol content (mg/100 g herb) was
increased significantly as a result of increment of urea fertilizer. The mean value of Polyphenol content showed
significant increment due to 75 kg N/fed and 150 kg N/fed compared to control. The highest Polyphenol content
were recorded with soil application of 150 Kg N/ fed (432.66 and 513.30 mg GAE/100 g dry herb) in first and
second seasons, respectively. The increment of Polyphenol due to nitrogen application was found by (Mudau et
al., 2005 and 2007).
y=0.922x+8.920
R²=0.924
28
30
32
34
36
38
40
42
44
27 29 31 33 35 37
CitalA(Geranial)%
CitralB(Neral)%
CorrelationbetweenCitralB(Neral) andCitalA(Geranial)

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J. Appl. Sci. Res., 9(1): 309-320, 2013
3.4.2. Effect of biofertilizer application:
biofertilizer application (Nitrobin, Rhizobacteren or Microbein) had a significant effect on Polyphenol
content (mg/100 g herb). Polyphenol content showed significant decreased due to application of Nitrobin and
Rhizobacteren in both seasons compared with control, while Microbein showed significant increment in
Polyphenol content (mg/100 g herb) in both season. Higher mean content of polyphenol was obtained with 1 kg
Microbein/ fed (456.24 and 517.27 mg GAE/100 g dry herb) in the first and second seasons, respectively. The
positive effect for Microbein may be due to the effect of all strains specially Bacillus spp, Azotobacter spp. and
Azospirillum spp. In addition, phosphate-dissolving bacteria secrete organic acids, which lead to a transfer of
fixed phosphate to available phosphate. Many investigators have explained the role of Bacillus megatherium,
which increases the availability of nitation in the soil (Mahfouz and Sharaf-Eldin 2007). Azospirillum strain and
Azospirillum is a free living, plant growth-promoting bacterium, capable of affecting growth and yield of
numerous plant species, as it is capable of producing various phytohormones that improve growth (Yoav et al.,
2004 and Abdelhamid et al 2010).
3.4.3. Effect of interaction treatments:
The interaction between nitrogen applications with biofertilizers application resulted in a significant
increment of polyphenol content in both seasons (Table, 4). The maximum plant Polyphenol content (mg
GAE/100 g dry herb) values were recorded from the combination of nitrogen 150 kg N/fed with 1 kg
microbein/fed (517.74 and 575.87 mg GAE/100 g dry herb) in the first and second seasons, respectively, while,
the minimum Polyphenol content (mg GAE/100 g dry herb) values were resulted from the plants received 1 kg
Nitrobein without any treatment (308.67 mg GAE/100 g dry herb) in the first and 1 kg Rhizobactrein without
any treatment (409.2 mg GAE/100 g dry herb) in the second seasons. In other words, as mentioned before, the
positive interactions between the applied of biofertilizer and nitrogen application on polyphenol content may be
due to the promoting effects of both Biofertilizers and nitrogen together on the accumulated polyphenol lemon
grass leaves. These results are in agreement with those of Gomaa and Abou-Aly (2001) on anise (Pimpinella
anisum) and Mahfouz and Sharaf-Eldin (2007) on fennel plants.
3.5. Flavonoid content
3.5.1. Effect of nitrogen application:
The flavonoids content (mg/100 g dry herb) in lemon grass was significantly increase with soil nitrogen
application as shown in table 4. The flavonoid accumulations in lemon grass plants were higher and showed the
positive correlation. However, at higher nitrogen level (150 kg N/ fed) gave the maximum mean amount of
flavonoids in dry leaves (374.49 and 446.73 mg/100 g dry herb) in the first and second seasons, respectively.
Similar data was obtained by those of Nirwan (2007), Khan et al. (2010) and Hanudin et al (2012) reported that
application of 3 g urea, 0.6 g MgSO4 and 60% paranets shading could improve the plant growth, N-Mg uptake,
total flavonoid and phyllanthin yield of Phyllanthus niruri.
The biosynthesis of secondary metabolites varies among plants, even in different organs of plants and their
biosynthesis depends on the environmental factors in which they grow. Differences in biosynthesis can result
from both genetic and phenotypic variations. Phenotypic variation is especially pronounced in the physiological
responses of a plant under growth conditions (Khan et al., 2010). The variation in biosynthesis of secondary
metabolites in different parts of plant may be due to expression or activity of genes or enzymes (Jiang et al.,
2006).
Hanudin et al 2012 reported that, Optimum N fertilization can support the formation of plant vegetative
organs, for example, leave, stem and branch; so, can increase the plant biomass and also increase the total
flavonoid and phyllanthin yield, because the value of total flavonoid and phyllanthin yield was computed by
multiplying the dry weight and total flavonoid and phyllanthin content.
3.5.2. Effect of biofertilizer application:
biofertilizer Microbein 1 kg /fed showed a positive effect on flavonoid content (mg/100 g dry herb) in
lemon grass leaves at both seasons. While biofertilizers Nitrobin and Rhizobacteren at the same concentration
showed a significant decreased flavonoid content (Table, 4) at both seasons compared with control. Higher
mean content of flavonoid was obtained with 1 kg Microbein/ fed (434.14 and 508.10 mg /100 g dry herb) in the
first and second seasons, respectively. Free living nitrogen fixing bacteria such as Azotobacter and Azospirillum
have the ability not only to fix nitrogen but also to release certain phytohormons of GA3 and IAA nature which

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J. Appl. Sci. Res., 9(1): 309-320, 2013
could stimulate plant growth, absorption of nutrients and photosynthesis process (Fayez et al., 1985 and Abdel-
Latif et al., 2001).
These results may be due to the converting of the unavailable forms of nutrient elements to available forms
by the microorganisms in biofertilizer. The microorganisms also produce growth promoting substances resulting
in more efficient absorption of nutrients, which main components of photosynthetic pigments and consequently
the chlorophyll content as well as N, P and K percentages were increased (Gomaa and Abou-Aly, 2001). These
results agree with those of Badran and Safwat (2004), El-Ghadban et al. (2006) and Ordookhani et al., 2011
3.5.3. Effect of interaction treatments:
Table 4 demonstrated the effect of interaction between biofertilizers and different concentrations of
nitrogen, on the flavonoid content of the lemon grass leaves. The highest flavonoid content (534.14 and 608.10
g/100g) was found in the plants received 1 kg microbein with 150g N /fed. in the first and second seasons,
respectively , while treatment 1kg Nitrobein and 1 kg Rhizobectrein without any treatment showed the lowest
value from the flavonoid content (351.33 and 407.60mg/100 g dry herb)in both seasons. These results agree
with those of (Said Al Ahl, 2005 and Hellal et al 2011), who reported that the combined application of 60 kg N/
fed with biofertilizer resulted in the highest fresh and dry weight of different plant parts, oil percentage,
chlorophyll, total flavonoids and NPK contents of dill plant.
Table 5: Effect of Bio and /or Urea fertilization on chemical constituents of essential oil detected with GC/MS from Cymbopogon citrates plants.
KI*
Control
Urea fertilizers Biofertilizers Interaction
75 N 150 N Ni Rh Mi
Ni +
75N
Ni +
150 N
Rh+
75N
Rh +
150 N
Mi +
75N
Mi +
150 N
1 Sabinene 1206 0.3 - - 0.36 0.56 1.04 0.92 0.8 0.83 - 0. 83 0 .56
2 β- myrcene 1249 9.09 4.78 7.96 10.58 7.84 7.21 5.35 9.09 3.24 10.95 6.76 7.84
3 α- terpinene 1258 - - - - - 0.28 - 0.25 - - 0.24 -
4 Limonene 1276 0.47 - - 0.59 0.68 1.49 0.75 1.04 0.76 - 1.11 0.68
5 β- ocimene 1318 0.4 - 0.45 0.33 0.43 0.32 - 0.48 - 0.41 - 0.43
6 γ- terpinene 1323 0.28 - - 0.34 0.37 1 0.21 0.68 - - 0.81 0.37
7 Rothrockene 1333 0.17 - 0.19 - - - - - - - - -
8 Cymene 1349 0.31 0.35 0.63 1.14 1.44 0.61 2.39 0.78 0.63
9
6-methl-5-
hepten-2-one 1429 2.47 1.79 1.93 2.06 1.74 1.44 0.99 1.64 0.6 2.44 1.63 1.74
10
Aretimisa
ketone 1433 - - - - - - - - 0.22 - - -
11 Verbenol 1484 0.37 0.12 0.41 0.43 0.28 0.35 - 0.35 - 0.46 0.32 0.28
12 Perllene 149 8 1.13 0.92 1.09 1.8 1 1.41 0.85 1.18 - 1.84 1.47 1
13 β- citronellal 1565 0.25 0.15 0.21 0.24 0.21 - - 0.23 0.37 0.25 - 0.21
14
2,2-Dimethyle-
3,4-Octadienal 1591 0.18 - - 0.37 - - - - - 0.34 0.35 -
15 Linalool 1535 2.71 1.83 2.53 3. 23 2.48 2.35 1.83 2.45 1.37 2.34 3.02 2. 48
16
Trans-γ-
bisabolene 1552 0.28 0.25 0.35 - 0.19 0.25 0.2 0.27 0.52 0.21 0.22 0.19
17 Cis-vebenol 1559 1.42 1.09 1.11 1.07 1.09 1.17 0.69 1.26 0.89 1.12 0.72 1.09
18 2-undecanone 1582 0.24 0.25 0.26 0. 46 0.21 0.35 0.21 0.31 0.61 0.48 0.21
19 Terpinene-4-ol 1588 - 0.12 - - 0.18 0.35 0.26 - - - - 0.18
20
Rose furan
epoxide 1594 0.93 0.73 1.02 1.03 0.71 1.63 0.42 0.8 - 1.26 0.87 0.71
21 Anethol 1653 0.22 0.12 0.16 0. 44 0.25 0.43 0.32 0.27 - 0.3 0.49 0.25
22 Citral B (Neral ) 1680 31.69 35.62 33.31 29.09 32.04 29.82 34.38 31.35 35.27 27.56 29.89 32.04
23 Geranyl formate 1691 - 0.13 0.13 0.33 - 0.27 0.19 - 0.43 0.33 0.31 -
24 Piperitone 1716 - - 0.15 - - - - - 0.26 - - -
25
Cital A
(Geranial) 1733 37. 17 41.44 39.19 35.85 38.84 37.18 40.3 36.94 42. 75 34.34 36.8 38. 84
26 Geranial acetate 1745 1.53 0.92 1.11 1.27 1.36 1.7 1.43 1.86 1.69 2.43 2.43 1.36
27 β- citronellol 1752 0.56 0.52 0.58 0.7 0.64 0.73 0.53 0.58 0.28 0.81 0.69 0.64
28 (z)-geraniol 1787 - 0. 11 - 0.29 - - - - - 0.37 0.32 -
29 2-tridecanone 1791 0.33 0.26 0.33 0.45 0.25 0.3 0.37 0.23 0.34 0.51 0.42 0.25
30
N
erol 1836 5.32 4.8 4.8 5.77 4.53 4.97 4.71 5.05 4.06 6.42 6.8 4.53
31
Caryophlene
oxide 1955 0.29 0.25 0.31 0.41 0.33 0.41 0.37 0.39 0.47 0.49 0.52 0.33
32 yomogi alcohol 204 4 0.38 1.14 0.46 0.32 0.7 0.58 0.65 0.39 0.49 0.55 0.3 0.7
33 Junipercamphor 2139 0.28 0.24 0.38 0.31 0.26 0.36 - 0.3 0.22 0.32 0.32 0.26
34 Globulol 2204 - 0.17 - - - - 0.23 - 0.28 - - -
35 Neric acid 2272 0.17 0.32 0.2 0. 24 0.32 - 0.34 0.22 0.37 0.27 - 0.32
36 Geranic acid 2310 0.64 1.08 0.71 0.74 1.26 0.92 1.12 0.71 1.13 1.78 0.53 1.26
Total identified 99.58 99. 15 99.33 99.45 99. 38 99.09 99. 2 99.63 99.54 98. 71 99.43 99.38
Oxygenated
compounds 88.48 93.72 90.38 87.2 88.68 86.36 91.82 86.21 91.1 85.64 88.68 88.68
Non-
Oxygenated
compounds 11.1 5.43 8. 95 12.25 10.7 12.73 7.38 13.42 8.44 13.07 10.75 10.7
Monoterpene 98.73 97.84 98.29 98.93 98.6 98.07 97.89 98.47 98.15 98.19 98.37 98.6
Sesquiterpenoid
s
0.85 1.31 1.04 0.52 0.78 1.02 1.31 1.16 1.39 0.52 1.06 0.78
KI* = Confirmed by Comparison with Kovats Indices on TG-WAX MS
Concluding Remarks:
In conclusion, the results of current experiment show that Biofertilizers (Nitrobin, Rhizobacteren and
Microbein) and Urea fertilizer increased the vegetative growth parameter, growth yield and yield of essential oil

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J. Appl. Sci. Res., 9(1): 309-320, 2013
of lemongrass leaves. Biofertilizers (Nitrobin, Rhizobacteren and Microbein) can be used in organic cultivation
of crops with higher plant yield and essential oil content.
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