International Biodeterioration & Biodegradation 65 (2011) 703e707

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International Biodeterioration & Biodegradation

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Biopesticidal value of selected essential oils against pathogenic

fungus, termites, and nematodes
Aditi Gupta, Satyawati Sharma*, S.N. Naik
Centre for Rural Development and Technology, Block III, Indian Institute of Technology, IIT Delhi, Hauz Khas 110016, India

a r t i c l e i n f o a b s t r a c t

Article history: The biopesticidal potential of six plant-derived essential oils (mint [Mentha arvensis], ajwain [Carum
Received 9 September 2010 capticum], lemongrass [Cymbopogon citrates], clove [Eugenia caryophyllata], cedarwood [Cedrus deodara],
Received in revised form and eucalyptus [Eucalyptus globulas]) was evaluated against Odontotermes obesus (termites), Fusarium
28 October 2010
oxysporum (plant pathogenic fungi), and Meloidogyne incognita (nematodes). In the case of termites,
Accepted 1 November 2010
a “no-choice” bioassay revealed that the mint oil gave the best results (100% mortality in 30 min with 10%
Available online 7 May 2011
oil and in 10 h with 0.12% oil) followed by the lemongrass and ajwain oils. The disc diffusion method was
adopted to test the anti-fungal activity of the essential oils and it was found that the clove oil gave the
Keywords:
Essential oils
maximum inhibition measured in terms of the average inhibition zone diameter (5.3
0.2 cm with 10%
Anti-termitic activity oil and 6.6
0.9 cm with 20% oil), followed by the ajwain oil. To check the anti-nematicidal activity of the
Anti-fungal activity essential oil, in-vitro growth chamber experiments revealed that eucalyptus oil was the most efficient
Nematicidal activity (100% mortality in 6 h with 1000 ml l1 oil and in 30 h with 125 ml l1 oil), followed by the ajwain oil. The
Mortality use of the crude oils at low concentrations provided satisfactory results at the laboratory level against
these pathogens, and needs further evaluation in field trials.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction to use heavy doses of chemical pesticides, which, although have an

In recent years, a significant increase in the yield of food grains and
other crops has been required to feed the world’s growing population.
However, various pests, including weeds, insects, and plant viruses,
can reduce crop production by 25e50% (Pimentel et al., 1991; Oerke,
2006). According to Levetin and McMahon (2003), about 70% of crop
diseases are caused by fungi. The extent of diseases caused by
nematodes in the agricultural systems is far from fully understood.
The yield losses by plant parasitic nematodes, in India alone, amount
to billions of rupees every year. Termites also cause a huge amount of
damage when they destroy wood and wooden products (Verma et al.,
2009). Control and repair costs due to Formosan subterranean
termites in New Orleans, for example, have been estimated to reach
$300 million annually (Suszkiw, 1998). In India, they are responsible
for the loss of 15e20% of the maize yield and about 1478 million
rupees (Joshi et al., 2005; Verma et al., 2009). Other countries as well
experience significant crop losses caused by these pests, so control-
ling them is an important economic and agricultural issue.
The green revolution introduced the use of chemical pesticides
for managing pests. To defeat plant diseases, farmers/growers have
immediate effect, are not always affordable, and may have delete-
rious effects on plants, soil, and animals. Moreover, continued use
leads to an increase in pest resistance and enhanced pest resur-
gence. In this context, efforts are being made worldwide to replace
these chemicals with biological alternatives (biopesticides), which
are less toxic to the environment.
Through the ages, plant-derived oils (essential as well as seed
oils) have evoked interest as natural products that hold promise for
their potential in pest management (Regnault-Roger, 1997; Isman,
2000; Akhtar, 2000; Oka et al., 2000; Zhu et al., 2001; Gandhi
et al., 2006; Chaieb et al., 2007; Bakkali et al., 2008). Among the
plant-derived oils, essential oils have been shown to possess
insecticidal, anti-fungal, and antibacterial properties (Burt, 2004).
The present paper describes testing the efficacy of selected plant-
derived essential oils as biopesticides against termites (Odonto-
termes obesus), plant pathogenic fungus (Fusarium oxysporum), and
plant nematode (Meloidogyne incognita).
2. Materials and methods
2.1. Essential oils

* Corresponding author. Tel.: þ91 11 2659 1116, þ91 11 2659 1121. Six different essential oils were selected based on a literature
E-mail address: satyawatis@hotmail.com (S. Sharma). survey, as well as their availability and use in traditional medicine.

0964-8305/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2010.11.018
704 A. Gupta et al. / International Biodeterioration & Biodegradation 65 (2011) 703e707
Cedarwood oil was procured from Aum Aromatics Pvt. Ltd., Mandi,
H.P., India, and eucalyptus, mint, clove, ajwain, and lemongrass oils
were procured from Gogia Chemicals, Okhla, New Delhi. These
essential oils were analyzed for their major constituents using gas
chromatographyemass spectrometry (GCeMS) (Gogia Chemicals,
Okhla, New Delhi).
2.2. Test organisms
O. obesus termites were collected from the local environment of
the Indian Institute of Technology (IIT), Delhi, and acclimatized for
about 24 h by keeping them in a BOD incubator at 28
1  C and
relative humidity of 80e85%. F. oxysporum was procured from IARI,
Delhi. M. incognita nematodes were collected from the infected
brinjal roots at the micromodel, IIT, Delhi, and incubated in a BOD
incubator at 28
1  C and 80e85% RH.
2.3. Anti-termitic assay
The “no-choice” bioassay method of Kang et al. (1990) was fol-
lowed to evaluate the anti-termitic activity of the six selected
essential oils. Petri dishes (5 cm diameter and 1 cm high) filled with
approximately 15 g of sterilized sand were used to test the toxicity
of the selected oils against the workers of the O. obesus termites.
Samples of 0.12, 0.25, 0.5, 1.0, and10% oil solutions were made in
polypropylene glycol (PPG) and 500-ml aliquots (sufficient to spread
over the whole filter paper) were applied to the filter paper samples
(Whatman No. 1, 4.5 cm in diameter). A blank filter paper and filter
paper treated with solvent only were used as the controls. After the
filter papers were placed in their respective petri plates, 15 worker
termites were added to each plate. The dishes were incubated in
the dark at 28
1  C and 80e85% RH. A few drops of water were
periodically sprinkled onto the sterilized sand in the dishes to
maintain sufficient moisture for the termites. Filter paper and water
served as the food for the termites. The whole experiment was
performed in triplicate and the mean mortality rate was counted
after specific time intervals for 72 h. The data were collected in
triplicate.
2.4. Anti-fungal assay
Screening of the oils was done by the disc diffusion method
(Bauer et al., 1966) to test the anti-fungal activity of the selected oils
against the fungal strain F. oxysporum. The fungal strain was
cultured in potato dextrose broth (PDB) at 28  C and 250 rpm for
5e7 days. The cultures were adjusted to approximately
108 CFU ml1. One hundred microliters of this solution was spread
over the plates containing PDA using a sterile spreader in order to
get uniform fungal growth on the plates. Samples of 10 and 20% oil
solution were prepared in PPG for the essential oils and a 30-ml
aliquot (sufficient to spread over the sterile disc) was pipetted onto
a 6-mm sterile disc (HIMedia Laboratories, Mumbai, India) placed
on the agar medium in the petri plates. A blank sterile disc with no
oil treatment was used as the control. The petri dishes were incu-
bated in a BOD incubator at 28
1  C and 80e85% RH for about 5
days until maximum growth of the fungus was achieved. Inhibition
zones, if any, were measured using a caliper. The experiments were
performed in triplicate.
2.5. Nematicidal assay
In-vitro growth chamber experiments were monitored to study
the effect of the selected oils on the mortality of M. incognita
second-stage juvenile nematodes. Culturing of the nematodes from
the infected soil was done according to Cobb (1918). The infected
soil was mixed with water and allowed to settle for 1e2 h. Water
was decanted and passed through a cascade of sieves of decreasing
size. Retentate from the sieve with mesh size 350 was collected.
This mesh size traps the J2 nematode larvae. Culturing of the
nematodes was done using double distilled water (DDW), as the
nematodes have a tendency to move toward fresh water. Second-
stage juveniles were then collected after 48 h in DDW. Population
counts of the nematodes, done using a stereomicroscope, gave, on
average, approximately 150 juveniles in 500 ml of the water solu-
tion. Approximately 280e300 juveniles in 1 ml were introduced in
each well of the sterilized 24-well plates. A suitable diluent that had
nil/minimum toxicity to the nematodes had to be chosen to make
the desired oil concentrations of 1000, 500, 250, and 125 ml l1.
Studies on nematodes are generally performed using water as the
solvent. Since oils are hydrophobic, a suitable surfactant had to be
added to act as a stabilizer to dissolve the oils in the water.
Therefore, the essential oils were finally diluted with water con-
taining 0.5% Tween 80 v/v, as the surfactant to obtain the desired oil
concentrations. To each well, 500 ml of the oil solution of a given
concentration was added and incubated at 28
1  C and 80e85%
RH. Each treatment had three replicates. The concentration of
Tween 80 in the control was equivalent to that in the oils. The
number of juveniles that became immotile on exposure to the oil
concentrations and on further dilution with the fresh DDW were
considered dead. The mean mortality rate measurements were
done after specific time intervals for 48 h after which the results
were subjected to a lot of error as the juveniles could die because of
aging and not because of the oil exposure. The data was collected in
triplicate.
2.6. Statistical analyses
The data collected were analyzed by one-way analysis of vari-
ance (ANOVA) using SPSS for Windows (version 18.0). The
measurement of difference was determined according to Duncan’s
multiple range test (DMRT). Values of P < 0.05 were considered
significant. Vertical bars in the figures indicate the values of
means
standard deviation (SD).
3. Results and discussion
3.1. Chemical components of the essential oils
Analyses of the essential oils using GCeMS gave their compo-
sitions, which are listed in Table 1.
3.2. Anti-termicidal activity of essential oils
The results showed that at 10% concentration of essential oils, all
the termite workers were killed within 30 min. Varying mortality
rates were observed with the oils at concentrations less than 10%.
However, essential oil concentrations as low as 0.12% were suffi-
cient to kill the termite workers within a span of 18 h (Fig. 1). Fig. 2
shows the maximum mortality rates of the termite workers with
different concentrations of the oils. It can be concluded from the bar
graph that mint oil is superior to the other oils as it takes
comparatively the least amount of time to kill the termite workers,
at all concentrations tested. This may be due to the presence of the
active component menthol present in the mint oil. After the mint
oil, lemongrass seems to be better than the other oils at 1 and 0.5%
but not at the other oil concentrations, as ajwain oil takes the upper
hand at 0.25% and at 0.12% both oils show the same results. Even
though clove oil falls next in the order, it gave the maximum
mortality, i.e. 100%, at the same time as does lemongrass at 0.5,
0.25, and 0.12%. Cedarwood and eucalyptus oil fall last in the
 A. Gupta et al. / International Biodeterioration & Biodegradation 65 (2011) 703e707
Table 1
Components of the essential oils.
Essential oil
Cedarwood oil
Mint oil
Eucalyptus oil
Clove oil
Ajwain oil
Lemongrass oil
Numbers in the brackets indicates the percentage of the components present in the plant essential oil.
efficacy order. In the present case, the mortality of the termite
workers might have been caused by the contact with the filter
paper impregnated with the essential oils (Prates et al., 1998). As
can be seen from the gas chromatography analysis, each essential
oil contains some active terpenoid among their major components
(Table 1). A number of different terpenoids in essential oils have
been known to possess anti-termitic activity (Sharma et al., 1994;
Cornelius et al., 1997; Park and Shin, 2005; Koul et al., 2008). The
toxicity of the mint and ajwain oils may be attributed to the pres-
ence of the phenolic components menthol and thymol, respec-
tively, while that of the clove oil can be attributed to eugenol
(Cornelius et al., 1997; Chen et al., 2002; Park and Shin, 2005; Koul
et al., 2008), which contains an ether linkage and a hydroxyl
attached to the benzene ring (Table 1). The compounds containing
an aldehyde group also show significantly high levels of anti-
termitic activity (Chang and Cheng, 2002), and therefore the anti-
termitic activity of the lemongrass oil can be explained due to the
presence of an aldehyde group in its major compound citral.
Cedarwood oil is known to act as a natural repellant to termite
workers due to the presence of the active components himachalol
and b-himachalene (Singh and Agarwal, 1988), while the activity of
the eucalyptus oil might be due to the presence of the oxygenated
compound cineole in it. According to Seo et al. (2009), phenolic
substances tend to exhibit stronger anti-termitic activity than the
alcohol and aldehyde groups. These results support Seo et al.
(2009), too. The activity of the individual components needs
to be evaluated to support the reasoning as there is generally
Fig. 1. Plot of percentage mortality of termite workers vs. time with 0.12% oil. Vertical
bars in the figure indicate the standard deviation (SD).
705
Components (%)
Beta-himachalene (43.9), alpha-himachalene (16.5), gamma-himachalene (11.3),
atlantone (17.5), himachalol (2.9), others
Menthol (80.1), menthone (5.9), neo-menthol (4.3), iso-menthone (2.9),
pulegol þ menthyl acetate (1.3), terpeneol (0.3)
Cineole (81.9), limonene (6.6), alpha-pinene (5.7), cymene (3.1), beta-pinene (1.5),
gamma-terpenene (0.6), myrcene (0.3), others
Eugenol (86.5), trans-caryophyllene (10.9), alpha-caryophyllene (1.5), others
Thymol (35.9), gamma-terpenene (33.6), cymene (18.8), beta-pinene (1.9),
carvacrol (1.2), alpha-pinene (0.4), others
Citral (77.8), limonene þ traces of eucalyptol (4.0), geraniol (2.7),
6-methyl-5-hepten-2-one (2.4), geranyl acetate (1.1), camphene (0.3), others
a difference between the activity of the individual component and
the crude oil, which contains many other components that might
act synergistically.
3.3. Anti-fungal activity of essential oils
The anti-fungal activity of the six selected essential oils against
F. oxysporum were measured in terms of the average inhibition zone
diameters of the fungus at 10 and 20% concentrations of each oil
tested. The results revealed that the oils had anti-fungal activity of
varying magnitudes. Among the essential oils, clove oil showed the
best results, giving the maximum diameter of the zone of inhibition
(5.3
0.2 cm with 10% oil and 6.6
0.9 cm with 20% oil) followed by
the ajwain, lemongrass, mint, cedarwood, and eucalyptus oil, in that
order (Fig. 3). Also, it can be concluded that even though clove oil gives
the maximum zone of inhibition at both 10 and 20% concentrations,
lemongrass gives the highest percentage increase in the average
inhibition zone diameter, on increasing the oil concentration from 10
to 20%, followed by the clove and then the ajwain oil. Other oils did not
show any significant increase on doubling the oil concentrations.
The greater activity of the clove oil may be attributed to its major
component, eugenol (Table 1), which is known to possess anti-
fungal activity against a variety of species (Tampieri et al., 2005;
Chaieb et al., 2007). Ajwain oil is also known to exhibit a broad
range of fungitoxic behavior (Singh et al., 2004), which may be
attributed to the presence of its phenolic component thymol. The
anti-fungal activity of the lemongrass oil may be due to the pres-
ence of its aldehyde containing the active constituent citral, while
the impact of the mint oil may be influenced by its cyclohexanol
moiety. These results are in agreement with those obtained by
Fig. 2. Bar graph showing maximum mortality rates of O. obesus workers with
different concentrations of selected essential oils.
706 A. Gupta et al. / International Biodeterioration & Biodegradation 65 (2011) 703e707
Fig. 3. Bar graph showing average inhibition zone diameters of two different
concentrations of the selected essential oils used. Vertical bars in the figure indicate
the standard deviation (SD).
Pandey et al. (2003). Lemongrass, mint, clove, ajwain, and euca-
lyptus oils have already been known to exhibit strong fungitoxic
behavior against a number of fungi (Guynot et al., 2003; Suhr and
Nielsen, 2003; Bansod and Rai, 2008; Kishore et al., 2009). The
results obtained with the mint and the eucalyptus oil can also be
supported by the work of Matan et al. (2009), in which mint and
eucalyptus oil as well as their major constituents have been found
effective against certain fungal molds. An important observation
was that the clove, ajwain, lemongrass, and mint oils, rather than
just inhibiting the growth, also tend to inhibit the fungal sporula-
tion, as evidenced by the decrease in the pink color intensity of the
fungal growth. The results are in accordance with those obtained by
Tzortzakis and Economakis (2007), in which lemongrass oil inhibits
the fungal spore production of Colletotrichum coccodes, Botrytis
cinerea, Cladosporium herbarum, and Rhizopus stolonifer. The results
obtained in these anti-fungal tests clearly indicate that the oils
containing phenolic moieties in their active constituents perform
better than the others (Manohar et al., 2001).
3.4. Nematicidal activity of the essential oils
The mortality rates of M. incognita second-stage juveniles with
different concentrations of the six selected essential oils are
summarized in Fig. 4 and Fig. 5. The results show different mortality
rates with different concentrations of oils. Comparisons show that
eucalyptus and ajwain oil are significantly better than the other oils,
as they show maximum mortality in minimum time at all the
concentrations tested, with eucalyptus oil showing slightly better
results at 1000 ml l1 and 125 ml l1 concentrations. Eucalyptus oil is
Fig. 4. Plot of percentage mortality of nematode juveniles vs. time with 1000 ml l1 oil.
Vertical bars in the figure indicate the standard deviation (SD).
Fig. 5. Plot of percentage mortality of nematode juveniles vs. time with 125 ml l1 oil.
Vertical bars in the figure indicate the standard deviation (SD).
a complex mixture of phenols, oxides, aldehydes, ketones, alcohols,
and ethers and its toxic influence may be a synergistic effect of all its
constituents. The results obtained here are in agreement with the
ones obtained by Pandey et al. (2000) and Ibrahim et al. (2006).
Ajwain oil is also known to possess good nematicidal activity (Park
et al., 2007) and again this can be attributed to its major component,
thymol. Clove oil comes next in the efficacy order, followed by mint,
cedarwood, and lemongrass oil. Some of the essential oils used in
our study have already shown potent nematicidal activity against
the pine wood nematode (Park et al., 2005). Again, it is expected that
the nematicidal activity of all these oils against M. incognita may be
due to their major components, but further studies are still neces-
sary to validate this.
4. Conclusions
From the present study, it can be concluded that oils are a good
alternative to the harmful chemical pesticides and can be effec-
tively used as an efficient biopesticide against many pathogens like
termites, fungus and nematodes. While mint oil showed compar-
atively better results in case of termites and eucalyptus oil for the
nematodes, clove oil proved to be the best for inhibiting the fungal
growth. Ajwain oil showed very good results in each case and thus
can be considered as a viable alternative for other pathogens as
well. The activities of the various oils are attributed to their major
constituents and that the oils containing phenolic moieties in them
show better toxicity than the others to the pathogens studied. Such
findings have been supported by the literature as well. Further
studies would still be required for better understanding of the
structure activity relationship as the presence of the other
constituents in the crude oil definitely modifies the influence of its
major constituent alone. We believe that the present study together
with the previous studies on oils support the biopesticidal nature of
the plant derived essential oils. These oils can be used as a cheap,
safe and efficient alternative as well as a supplement, in the
developing countries to protect the crops against the various plant
pathogens. Additional field level studies would be needed to
further validate their potential.
Acknowledgments
The authors are grateful to CSIR and NOVOD for their financial
assistance and to Gogia Chemicals, Okhla, New Delhi, for providing
the essential oils and for their GC and GCeMS analysis.
 A. Gupta et al. / International Biodeterioration & Biodegradation 65 (2011) 703e707 707

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