Journal of Medicinal Plants Research Vol. 6(21), pp.

3793-3799, 9 June, 2012

Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR12.370
ISSN 1996-0875 ©2012 Academic Journals

Full Length Research Paper

Effects of the application of vermicompost and nitrogen

fixing bacteria on quantity and quality of the essential
oil in dill (Anethum graveolens)
1 1 2
Mohammad Taghi Darzi *, Mohammadreza Haj Sayed Hadi and Farhad Rejali
1
Department of Agronomy, Faculty of Agriculture, Islamic Azad University, Roudehen Branch, Roudehen, Iran.
2
Department of Biology, Institute of Soil and Water Research, Karaj, Iran.
Accepted 22 March, 2012

The main objective of this study was to determine the effects of vermicompost and nitrogen fixing
bacteria on the quantity and quality of essential oil in dill essential oil content in seed, essential oil yield
and carvone content, limonene content and dillapiole content in essential oil. The experiment was
carried out at the Hamand Research Station of Damavand in 2011. Vermicompost (0, 4, 8 and 12 ton/ha)
and nitrogen fixing bacteria, mixture of Azotobacter chroococcum and Azospirillum lipoferum (non-
inoculated, inoculated seeds and inoculated seeds + spray on the plant base at stem elongation stage)
were used as the effecting parameters. The present results have shown that the highest essential oil
content in seed and carvone content in essential oil and the minimum dillapiole content in essential oil
were obtained after applying 4 ton/ha vermicompost. The maximum essential oil yield was obtained
after applying 8 ton/ha vermicompost. Nitrogen fixing bacteria also showed significant effects on
essential oil content, essential oil yield and carvone content in essential oil. The maximum essential oil
content and carvone content were obtained by using the nitrogen fixing bacteria once. The highest
essential oil yield was obtained by using the nitrogen fixing bacteria twice.

Key words: Dill, vermicompost, Azotobacter, Azospirillum, essential oil.

INTRODUCTION

The intensive use of chemical fertilizers has side effects provides many microsites for the microbial activity and
in polluting underground water, destroying strong retention of nutrients. It is rich in microbial
microorganisms and reducing soil fertility. At present, population and diversity, particularly, fungi, bacteria and
using organic manures and biofertilizers, such as actinomycetes. It contains plant growth regulators and
vermicompost and nitrogen fixing bacteria have led to a other growth-influencing materials produced by
decrease in the application of chemical fertilizers and microorganisms (Edwards, 1998; Atiyeh et al., 2002;
have provided high quality products free of harmful Arancon et al., 2005). Free-living nitrogen fixing bacteria,
agrochemicals for human safety (Mahfouz and Sharaf such as, Azotobacter chroococcum and Azospirillum
Eldin, 2007; Malik et al., 2009). Vermicompost is the lipoferum, were found to have not only the ability to fix
product of the degradation of organic matter through nitrogen, but also the ability to release phytohormones
interactions between earthworms and microorganisms. similar to gibberellic acid and indole acetic acid, which
Vermicompost contains most nutrients in plant available could stimulate plant growth, absorption of nutrients and
forms, such as nitrates, phosphates and exchangeable photosynthesis (El Ghadban et al., 2006; Mahfouz and
calcium and soluble potassium (Atiyeh et al., 2002). Sharaf Eldin, 2007). Production of medicinal plants is
Vermicompost has large particulate surface area that mainly under the circumstances of sustainable
agricultural system. In this system, management of
environmental parameters is very critical. By using
correct nutritional sources through organic manures and
*Corresponding author. E-mail: [email protected]. Tel: biofertilizers, quantity and quality of active substances,
+982176503197. Fax: +982176503197. such as, essential oil in medicinal plants can be
3794 J. Med. Plants Res.

maximized. Anethum graveolens, commonly known as replications. Dill seeds were directly sown by hand into the field at a
dill, is an herbaceous annual plant, which is native to rate of 12 kg ha−1 to a depth of 2 cm. There was no incidence of
pest or disease on dill during the experiment. Weeding was done
mediterranean countries and Southeastern Europe. Dill manually and the plots were irrigated weekly. All necessary cultural
seed and leaves are used as flavouring in sauces, practices and plant protection measures were followed uniformly for
vinegars, pastries and soups. The dill seeds have all the plots during the entire period of experimentation.
essential oil as an active substance, while carvone and Twenty plants were randomly selected from each plot and the
limonene are the most important constituent of dill, which observations were recorded. In this study, the quantitative and
is used in pharmaceutical industry as a diuretic, stimulant qualitative traits of essential oil in dill essential oil content in seed,
essential oil yield and carvone content, limonene content and
and a carminative (Bailer et al., 2001; Singh et al., 2005; dillapiole content in essential oil were evaluated.
Callan et al., 2007).
Some studies have reported that vermicompost can
increase the quantity and quality of essential oil in a few Essential oil extraction and analysis
medicinal plants, such as, basil (Singh and Ramesh,
2002; Anwar et al., 2005; Geetha et al., 2009), coriander In order to determine the essential oil content (%), a sample of 100
g of dill seeds from the each treatment were crushed in electric
(Singh et al., 2009), fennel (Darzi et al., 2009; Moradi et grinder and were mixed with 500 ml distilled water and then were
al., 2011), chamomile (Haj Seyed Hadi et al., 2011) and subjected to hydro-distillation for 3 h using a Clevenger-type
cumin (Saeid Nejad and Rezvani Moghaddam, 2011). apparatus. The essential oil content was measured after
Several other studies have reported that nitrogen fixing dehydrating water by anhydrous sodium sulfate (Sephidkon, 2002;
bacteria, such as A. chroococcum and A. lipoferum could Kapoor et al., 2004). Essential oil yield also was calculated by using
essential oil content. For identifying the essential oil components,
cause increased quantity and quality of essential oil of
essential oil fraction was collected and subjected to gas
some medicinal plants, such as celery (Migahed et al., chromatography (GC) and gas chromatography-mass spectrometry
2004), fennel (Abdou et al., 2004; Mahfouz and Sharaf (GC/MS) analysis. For GC analysis from a Younglin Acm600,
Eldin, 2007; Azzaz et al., 2009; Moradi et al., 2011), equipped with HP-5 MS capillary column (30 m × 0.25 µm) and for
lemon balm (Harshavardhan et al., 2007), turmeric GC/MS analysis from an Agilent 6890 GC and Agilent 5973 MS,
(Velmurugan et al., 2008), hyssop (Koocheki et al., equipped with HP-5 MS capillary column (30 m × 0.25 µm) was
used.
2009), cumin (Saeid Nejad and Rezvani Moghaddam, Authentic reference substance of carvone, limonene and
2010) and black cumin (Valadabadi and Farahani, 2011). dillapiole were used to establish the retention times (Sephidkon,
Therefore, the main objective of the present field 2002).
experiment was to investigate the effects of
vermicompost and nitrogen fixing bacteria on quantity
and quality of the essential oil in dill (A. graveolens). Statistical analysis

All the data were subjected to statistical analysis (one-way ANOVA)

MATERIALS AND METHODS using SAS software (SAS Institute, version 8, 2001). Differences
between the treatments were performed by Duncan’s Multiple
Range Test (DMRT) at 5% confidence interval. Transformations
Field experiment
were applied to the data to ensure that the residuals had normal
distribution (Zar, 1996).
A 4 × 3 factorial experiment, arranged in a randomized complete
blocks designed with three replications, was conducted in the
Experimental Station of the Research Institute of Forest and
Rangeland, Damavand, Iran during the growing season of 2011. RESULTS
The geographical location of the experimental station was 35° 39´ N
and 52° 5´ E with the altitude of 1800 m. The treatments consisted Essential oil content in seed
of different concentrations of vermicompost (0, 4, 8 and 12 ton/ha)
and nitrogen fixing bacteria, with different mixture inoculation
conditions of A. chroococcum and A. lipoferum (non-inoculated,
The present results have indicated that essential oil
seed inoculated and seed inoculated + spraying on the plant base content in seed was significantly affected by the
at stem elongation stage). Inoculation was carried out by dipping application of vermicompost (Table 3). The most
the dill seeds in the cells suspension of 108 CFU/ml for 15 min. The significant essential oil content (2.21%) was obtained by
vermicompost was prepared from cow dung by employing epigeic applying 4 ton vermicompost per hectare. Nitrogen fixing
species of Eisenia foetida. The required quantities of vermicompost bacteria, had also, a significant effect on essential oil
were applied and incorporated to the top 5 cm layer of soil in the
experimental beds before the plantation of dill seeds. Several soil
content (Table 3), as higher essential oil content (2.19%)
samples (0 to 30 cm depth) were taken for the nutrient and trace was recorded by using nitrogen fixing bacteria once
element analysis prior to land preparation. Chemical and physical (inoculated seeds).
properties of the experimental soil and vermicompost are presented
in Tables 1 and 2. Nitrogen (20 kg/ha) was applied to the plots,
based on the soil and vermicompost analysis, in the stem Essential oil yield
elongation stage.
Each experimental plot was 3 m long and 2 m wide with the
spacing of 10 cm between the plants and 40 cm between the rows. The results presented in Table 3 have demonstrated that
There was a space of 1 m between the plots and 2 m between essential oil yield was influenced by the application of
 Darzi et al. 3795

Table 1. Physical and chemical properties of soil on the experimental site.

Cu Zn Mn Fe Mg Ca K P N O.C EC
pH Texture
(mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (%) (ds/m)
0.46 1.2 8.8 7.4 - - 550 40 9.0 0.8 0.70 7.1 Loamy-clay
*Available form of nutrients was measured.

Table 2. Chemical characteristics of vermicompost.

K (%) P (%) N (%) O.C (%) O.M (%) EC(ds/m) pH

3.9 0.67 11.3 26.1 45 1.8 8.5

Table 3. Mean comparison of the quantitative and qualitative traits of essential oil in dill at various levels of vermicompost and nit rogen fixing
bacteria.

Essential oil
Essential oil Carvon content in Limonene content in Dillapiole content
Treatment content in seed
yield (kg/ha) essential oil (%) essential oil (%) in essential oil (%)
(%)
Vermicompost (ton/ha)
b c c a a
v1 2.00 26.1 50.7 37.9 2.31
a b a a b
v2 2.21 43.0 55.6 36.0 1.81
a a a a b
v3 2.20 48.6 54.6 36.6 1.89
a ab b a a
v4 2.14 46.1 52.3 37.1 2.58

Nitrogen fixing bacteria

b c b a a
n1 2.04 35.5 52.6 36.9 2.16
a b a a a
n2 2.19 41.4 53.8 36.3 2.15
a a a a a
n3 2.17 45.8 53.6 37.5 2.12
Means, in each column for each factor followed by at least on letter in common, are not significantly different at 5% probability level using Duncans’
Multiple Range Test. v1, v2, v3 and v4 represent 0, 4, 8 and 12 ton/ha vermicompost, respectively. n1, n2 and n3 represent non-inoculated,
inoculated seeds and inoculated seeds + spraying on the plant base at stem elongation stage, respectively.

vermicompost, significantly (Table 3). Among various Carvone content in essential oil
treatments, the application of 8 ton vermicompost per
hectare has indicated maximum increase in essential oil The results indicated that carvone content in essential oil
yield (48.6 kg/ha). Nitrogen fixing bacteria showed was significantly affected by the application of
significant effect on essential oil yield (Table 3), as the vermicompost (Table 3). The highest carvone content
highest essential oil yield (45.8 kg/ha) was obtained in was obtained with applying 4 ton/ha vermicompost
the third treatment level of biofertilizer (a two-times (55.6%). Nitrogen fixing bacteria showed significant effect
application of Azotobacter + Azospirillum). on carvone content in essential oil (Table 3), as the
The present results show that the interaction of highest carvone content (53.8%) was obtained in the
vermicompost and nitrogen fixing bacteria was significant second treatment level of biofertilizer (once application of
(Figure 1). The highest essential oil yield (60.9 kg/ha) nitrogen fixing bacteria).
was obtained after the integrated application of 8 ton/ha The present results show that the interaction of
vermicompost and twice using nitrogen fixing bacteria. vermicompost and nitrogen fixing bacteria was significant
The interaction of vermicompost and nitrogen fixing (Figure 2). The highest carvone content (56.0%) was
bacteria, on the essential oil yield, revealed that the obtained after the integrated application of 4 ton/ha
application of 4 and 8 ton/ha vermicompost successively vermicompost and the use of nitrogen fixing bacteria
in the level of twice application of nitrogen fixing bacteria, once. The interaction of vermicompost and nitrogen fixing
resulted in a significant increase in essential oil yield. bacteria, on the carvone content, revealed that the
3796 J. Med. Plants Res.

Figure 1. Mean comparison for essential oil yield after the intraction of different factors.

Figure 2. Mean comparison for carvone content in essential oil after the intraction of different factors.

application of 4 ton/ha vermicompost in the level of one various levels of vermicompost had significant effects on
time using of nitrogen fixing bacteria, which resulted in a the dillapiole content in essential oil. The minimum
significant increase in carvone content. dillapiole content (1.81%) was obtained by using 4 ton/ha
vermicompost. Nitrogen fixing bacteria did not show
significant effect on dillapiole content (Table 3).
Limonene content in essential oil The present results show that the interaction of
The results indicated that limonene content in essential vermicompost and nitrogen fixing bacteria was significant
oil was not affected by vermicompost and nitrogen fixing (Figure 3). The maximum dillapiole content (3.54%) was
bacteria (Table 3). obtained after the integrated application of 12 ton/ha
vermicompost and twice application of nitrogen fixing
bacteria. The interaction of vermicompost and nitrogen
Dillapiole content in essential oil fixing bacteria, on the dillapiole content, revealed that the
application of 4 and 8 ton/ha vermicompost, successively
The results presented in Table 3 have revealed that in the level of twice application of nitrogen fixing
 Darzi et al. 3797

Figure 3. Mean comparison for dillapiole content in essential oil after the intraction of different factors.

bacteria, which resulted in a significant decrease in (2009) on Artemisia pallens, Koocheki et al. (2009) on
dillapiole content. Hyssopus officinalis, Saeid Nejad and Rezvani
Moghaddam (2010) on Cuminum cyminum and
Valadabadi and Farahani (2011) on Nigella sativa. These
DISCUSSION results likely show that the positive and synergistic effect
of interaction between two factors is highly dependent on
According to the present analysis, vermicompost the effect of organic matter contained in vermicompost,
application through increase of the mineral uptake, such on the activity of nitrogen fixing bacteria. Many reports
as, nitrogen and phosphorus (Arancon et al., 2006; have shown that the interaction between organic
Zaller, 2007), has a positive effect on proper biomass manures and biofertilizers can be beneficial for essential
production, and subsequently, the enhanced essential oil oil yield (Koocheki et al., 2009; Moradi et al., 2011;
content in seed. Improved essential oil content of Valadabadi and Farahani, 2011).
medicinal plants have previously been reported in the Vermicompost has significantly influenced the carvone
presence of optimal amounts of vermicompost (Anwar et content in essential oil. On the other hand, vermicompost
al., 2005; Darzi et al., 2009; Geetha et al., 2009; Singh et application through the improvement of biological
al., 2009; Haj Seyed Hadi et al., 2011). Nitrogen fixing activities of soil and mineral element absorption (Zaller,
bacteria also have increased essential oil content by 2007), caused more biomass production and flowering
enhancing the nitrogen absorption (Migahed et al., 2004). promotion and subsequently seed ripening which leads to
The present result were derived from the improvement of improvement of the essential oil quality (carvone
nitrogen fixing bacteria’ activities in soil at the second and content). These findings are in accordance with the
the third treatments levels (inoculated seed and observations of Anwar et al. (2005) on Ocimum basilicum
inoculated seed + spraying on plant base at stem and Darzi et al. (2009) and Moradi et al. (2011) on F.
elongation stage), which are in agreement with the vulgare. The present results suggest that the influence of
previous studies carried out on the fennel and turmeric nitrogen fixing bacteria on the carvone content in
(Mahfouz and Sharaf Eldin, 2007; Velmurugan et al., essential oil was due to increased nitrogen uptake
2008; Azzaz et al., 2009). (Kalyanasundaram et al., 2008). The finding of this
Increased essential oil yield in vermicompost investigation is in agreement with the reports of
treatments can be owing to the improvement of yield Harshavardhan et al. (2007) on Mellisa officinalis,
components, such as, essential oil content and seed Mahfouz and Sharaf Eldin (2007) and Moradi et al.
yield. Our findings are in accordance with the (2011) on F. vulgare and Velmurugan et al. (2008) on
observations of Singh and Ramesh (2002), Pandey Curcuma longa. Many studies have reported that the
(2005), Anwar et al. (2005), Saeid Nejad and Rezvani interaction between organic manures and biofertilizers
Moghaddam (2011) and Moradi et al. (2011). Nitrogen caused an increase in essential oil quality
fixing bacteria, promoted essential oil yield through the (Harshavardhan et al., 2007; Padmapriya and Chezhyian,
enhancement of yield attributes. These result are in 2009).
agreement with the investigation of Abdou et al. (2004) Applying proper amount of vermicompost, through
and Mahfouz and Sharaf Eldin (2007) on Foeniculum increase of the carvone content in essential oil, has a
vulgare, Swaminathan et al. (2008) and Kumar et al. negative effect on other components of essential oil, such
3798 J. Med. Plants Res.

as, dillapiole content and subsequently have decreased (Foeniculum vulgare Mill.). Egypt. J. Agric. Res., 84(3): 977-992.
Geetha A, Rao PV, Reddy DV, Mohammad S (2009). Effect of organic
dillapiole content in essential oil. The present result is in
and inorganic fertilizers on macro and micro nutrient uptake, oil
agreement with the report of Anwar et al. (2005) on O. content, quality and herbage yield in sweet basil (Ocimum basilicum).
basilicum and Darzi et al. (2009) and Moradi et al. (2011) Res. Crops., 10(3): 740-742.
on F. vulgare. Haj Seyed Hadi MR, Darzi MT, Ghandehari Z, Riazi GH (2011). Effects
of vermicompost and amino acids on the flower yield and essential oil
production from Matricaria chamomile L. J. Med. Plants. Res., 5(23):
5611-5617.
Conclusion Harshavardhan PG, Vasundhara M, Shetty GR, Nataraja A, Sreeramu
BS, Gowda MC, Sreenivasappa KN (2007). Influence of spacing and
integrated nutrient management on yield and quality of essential oil in
Conclusively, the results of current experiment show that lemon balm (Mellisa officinalis L). Biomed., 2(3): 288-292.
vermicompost and nitrogen fixing bacteria have Kalyanasundaram B, Kumar TS, Kumar S, Swaminathan V (2008).
stimulatory effects on the quantity and quality of the Effect of N, P, with biofertilizers and vermicompost on growth and
Essential oil in dill and thus have considerable potential physiological characteristics of sweet flag (Acorus calamus L.). Adv.
Plan. Sci., 21(1): 323-326.
for providing nutritional elements in essential oil
Kapoor R, Giri B, Mukerji KG (2004). Improved growth and essential oil
production of dill, especially for the sustainable yield and quality in Foeniculum vulgare Mill on mycorrhizal
production systems. inoculation supplemented with P-fertilizer. Bioresource. Technol., 93:
307-311.
Koocheki A, Tabrizi L, Ghorbani R (2009). Effect of biofertilizers on
agronomic and quality criteria of Hyssop (Hyssopus officinalis).
ACKNOWLEDGEMENT Iranian J. Agron. Res., 6(1): 127-137.
Kumar TS, Swaminathan V, Kumar S (2009). Influence of nitrogen,
The authors wish to thank the Research Division of phosphorus and biofertilizers on growth, yield and essential oil
constituents in ratoon crop of davana (Artemisia pallens Wall.).
Islamic Azad University, Roudehen Branch for providing Electronic J. Environ. Agric. Food. Chem., 8(2): 86-95.
financial support to undertake this research project. Mahfouz SA, Sharaf Eldin MA (2007). Effect of mineral vs. biofertilizer
on growth, yield, and essential oil content of fennel (Foeniculum
vulgare Mill). Int. Agrophisics., 21(4): 361-366.
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