J Sci.Univ.Kelaniya 6 (2011) : 45-54
EFFICACY OF ESSENTIAL OILS FROM BARK AND LEAF OF CINNAMOMUM
ZEYLANICUM ON ROOT KNOT NEMATODE, MELOIDOGYNE GRAMINICOLA
IN RICE SEEDLINGS AND YOUNG RICE PLANTS
L.D. AMARASINGHE1, W.K.A.G.A. WIJESINGHE1 and
B. K. JAYAWARDHANE2
1
Department of Zoology, University of Kelaniya, Kelaniya, 11600, Sri Lanka
2
Department of Chemistry, University of Kelaniya, Kelaniya, 11600, Sri Lanka
ABSTRACT
The present study was conducted to evaluate the efficacy of essential oils from leaf
and bark of cinnamon, Cinnamomum zeylanicum in controlling Meloidogyne graminicola
and their effects on rice seedlings and young rice plants. This study was done; to determine
the LC50 of essential oils of the extracts of cinnamon leaf and bark to kill 50% of second
stage juveniles (J2) of Meloidogyne graminicola in rice root galls; to determine the efficacy
of essential oils of cinnamon; in controlling J2 in rice seedlings and young rice plants; and
on the growth of rice plants.
Cinnamon bark oil sample used in the current study contained about 72%
cinnamaldehyde and the leaf sample contained about 86% eugenol. LC50 for cinnamon leaf
oil and cinnamon bark oil for killing 50% of juveniles of M. graminicola in rice root galls
after three days of the treatment was 0.326 ppm and 0.454 ppm respectively. Number of
galls in the root system of nematode infested rice seedlings was significantly reduced when
they were treated with 0.9 ppm of cinnamon leaf oil and bark oil compared to untreated
controls. However, the nematicidal activity between the cinnamon leaf oil and bark oil was
not significantly different. The root gall index of infested young rice plants was
significantly lowered when they were treated with either cinnamon leaf oil or bark oil
compared to untreated controls. It was revealed that the mean plant height and the mean
number of roots of essential oil treated plants were positively correlated with the number
of root galls whereas, the number of dead plants and the mean percentage chlorosis were
negatively correlated. Nevertheless, the mean root length did not show any significant
correlation with number of root galls. This study concludes that essential oils of cinnamon
Corresponding author: Email: deepika@kln.ac.lk
L D Amarasinghe et. al.
leaf which contained 86% eugenol and bark which contained 72% cinnamaldehyde are
similarly effective in suppression of M. graminicola in rice seedlings and young rice
plants. According to the rates of application of essential oils of cinnamon leaf and bark,
none of them have significant negative effect on the plant growth. Hence, they can be used
as an alternative nematicide against the rice root knot nematode, M. graminicola in early
stage of rice plants.
Keywords:
Cinnamomum zeylanicum; essential oils; Meloidogyne graminicola;Oryza
sativa
INTRODUCTION
Meloidogyne graminicola (L. Golden and Birchfield) is recorded as a pest of
rice,Oryza sativa in Sri Lanka (Ekanayake & Toida, 1997, Anon, 2001; Nugaliyadde et al.,
2001; Amarasinghe et al., 2007). Nugaliadde et al., 2001 reported that there is a yield loss
due to this nematode species when more than 75% of the root system of the plant is
infested.
The characteristic damage symptoms of M. graminicola are the enlargement of
roots and formation of hooked like galls in the root tips of infested plants. They cause
arrestment of root growth. Usually, there are no distinct symptoms on foliage of affected
plants unless the nematode infestation becomes severe. In very severe cases, newly
emerged leaves appear distorted and dried up along the margins and chlorosis can be seen.
Infested plants show stunting growth resulting of a considerable yield loss (Ou, 1985).
Integrated nematode management is the widely recognized method of controlling the
Meloidogyne species (Bridge & Aploeright, 2005; Whitehead, 1998; Amarasinghe et al.,
2007) that aim to prevent further distribution of the nematode and to decrease the yield
loss. In Sri Lanka, suppression of M. graminicola has been achieved with varying success
by integrating a number of physical, chemical, and cultural measures that introduced by the
Department of Agriculture, Sri Lanka (Anon., 2001).
In later years, several authors have demonstrated to use essential oils and their
components from variety of plant species as alternative bio control agents against plant
parasitic nematodes (Abbas et al., 2009; Choi et al., 2007; Kong et al., 2007; Albuquerque
et al., 2007). The present study was carried out to determine the efficacy of essential oils of
leaf and bark of Cinnamomum zeylanicum on the second stage juveniles of M. graminicola
and their effects on rice seedlings and young rice plants.
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Efficacy of essential oils
MATERIALS AND METHODS
Source of Meloidogyne graminicola inoculum
Identification of Meloidogyne graminicola was done to the species level using the
characteristic cuticular pattern that surrounds the vulva and anus of the adult female
nematodes (Taylor & Netscher, 1974). Infested rice plants were maintained in a 36cm
diameter and 18cm deep plastic basin half filled with moisten de-faunated soil to raise the
nematode population. The root galls positive for nematodes and active juveniles collected
from them were used for experiments.
Extraction of essential oils from bark and leaf of cinnamon, Cinnamomum zeylanicum
A 40g sample of plant material, cinnamon bark and cinnamon leaf, was treated
with a 1 % solution of cellulase and 1 % pectinase enzyme and incubated with 50ml of pH
7.4, 0.05 M phosphate buffer for 14 days separately. After the incubation period, the plant
material was transferred to two flasks and essential oils were extracted separately for 3h
using steam distillation. A 1l of each sample was analyzed using Hewlett Packard gas
chromatograph (Model 5890) equipped with a FID detector (Flame Ionization Detector).
Gas chromatography profile was compared with reported values. Cinnamon bark oil
sample used in the current study contained about 72% cinnamaldehyde and the leaf sample
contained about 86% eugenol. 0.5µl of Tween-20 was used to dissolve 1µl of cinnamon
oils. Concentrations of essential oils were prepared using serial dilution with distilled
water.
Experiment I: Screening the effect of different concentrations of cinnamon leaf oil
and bark oil on J2 of Meloidogyne graminicola in the laboratory.
5ml each from 0.01, 0.03, 0.1, 0.3 and 0.9 ppm of cinnamon leaf oil and 0.1, 0.3,
and 0.9 ppm of cinnamon bark oil were added separately into cleaned petri dishes each
measuring 5cm diameter. Each concentration was replicated 10 times. 5ml each of 0.5µl
Tween-20 and distilled water were added separately into 10 petri dishes each and they
were used as two sets of controls. Three root galls each expected to contain about 70
juveniles of M. graminicola were introduced into each petri dish. Closed Petri dishes were
arranged in a complete randomized design at 26 ± 2 ° C room temperature. Three days
after inoculation, root galls were teased individually and all J2 stage nematodes were
counted on a sedgewick rafter cell held under the microscope with 10 x 10 magnification.
47
L D Amarasinghe et. al.
Nematodes were confirmed as dead if they were motionless to a mechanical pricking and
their bodies held straight after transferring them to distilled water.
Experiment II: Screening the effect of cinnamon leaf oil and bark oil on Meloidogyne
graminicola in seedling rice.
Approximately 250 rice seeds were washed well with 0.5 mgl-1 potassium
permanganate and then rinsed thoroughly with distilled water. They were equally spread
on fifteen wetted filter papers each of 9 cm diameter. They were laid on the bottom of
fifteen plastic containers each with 9 cm diameter and 6 cm height. Seeds were maintained
until they are germinated in the laboratory. After one week, well grown 10 seedlings were
allowed to remain in each pot and others were removed. Ten ml each of the following
treatments were given separately into five containers and the treatments were replicated
three times. T1 - Distilled water with 100 juveniles; T2 - 0.9 ppm cinnamon leaf oil with 100
juveniles; T3 - 0.9 ppm cinnamon bark oil with 100 juveniles; T4 - Distilled water only; T5 0.5 ppm Tween-20 only. After one week of the treatment, a soil solution was added into
each container to maintain the growth of the seedlings. Seedlings were allowed to remain
for one more week in same containers to deploy the root system. The plants were then
removed from containers and the number of root galls due to nematodes in each seedling
was counted separately.
Experiment III: Screening the effect of cinnamon leaf oil and bark oil on Meloidogyne
graminicola in potted rice plants
Twenty eight plastic containers (9 cm diameter and 6 cm height) were filled with
moisten de-faunated soil up to 1/3 each of it. Rice plants at the rate of ten plants per
container were maintained for two weeks from germination. Calculated amounts of NPK
fertilizer was added into each pot as recommended by the authority of the Department of
Agriculture, Sri Lanka. Four pots were given 10 ml of one of the following treatments. T1 Distilled water with 100 juveniles; T2 - 0.9 ppm leaf oil with 100 juveniles; T3 - 0.9 ppm
bark oil with 100 juveniles; T4 - Distilled water; T5 - 0.9 ppm cinnamon leaf oil; T6 - 0.9 ppm
cinnamon bark oil; T7
-
0.5 ppm Tween-20. Pots were maintained in a completely
randomized design outside the laboratory. The height of the plant, the number of dead
leaves, the number of roots, the length of roots, and the number of root galls in each plant
and the number of dead plants in each pot, were recorded after three weeks from
inoculation.
48
Efficacy of essential oils
Data Analysis
Data were corrected for control mortality using Abbott’s formula (Abbott, 1925).
The LC50 values were calculated by profit values of adjusted mean percentage mortality
(Minitab version 14). Data were subjected to analysis of variance including Least
Significant Difference (LSD) and Duncan’s multiple Range Test (Sokal and Rohlf, 1995).
RESULTS AND DISCUSSION
Experiment I: Screening the effect of different concentrations of cinnamon leaf and
bark oil on J2 of Meloidogyne graminicola in the laboratory
Figure 1 shows the adjusted percentage mortality of J2 stage of M. graminicola
against different concentrations of cinnamon leaf oil and bark oil. It indicates that the
highest mortality of the nematode was in 0.9 ppm of both cinnamon leaf oil and bark oil.
There was no significant difference in the nematode mortality between two concentration
levels of leaf oil namely 0.3 ppm and 0.9 ppm. However, the nematode mortality was
significantly different between 0.1 ppm and 0.9 ppm leaf oil (p = 0.001, t = 3.89, df = 19).
Figure 2 envisages that the LC50 for cinnamon leaf oil is 0.326 ppm and bark oil for 50%
mortality of J2 M. graminicola is 0.454 ppm (Figure 3). It is envisaged from LC50 values
that the concentration of cinnamon leaf oil required to kill 50% of J2 M. graminicola is
lower than that of cinnamon bark oil. However, there is no significant difference between
the percentage mortality of J2 in respective concentrations of cinnamon leaf oil and
cinnamon bark oil (p = 0.819, df = 1).
Experiment II: Screening the effect of cinnamon leaf oil and bark oil on Meloidogyne
graminicola in seedling rice
Table 1 shows the mean number of root galls in cinnamon leaf oil treated and
cinnamon bark oil treated seedlings at the end of the experiment. The number of root galls
were significantly lowered in the cinnamon leaf oil treated rice plants (p = 0.033, t = -2.38,
df = 13) and cinnamon bark oil treated rice plants (p = 0.01, t = -3.24, df = 9) compared to
untreated controls. However, the differences of gall formation are not significant between
two essential oils (p = 0.24, t = -1.24, df = 12).
49
L D Amarasinghe et. al.
Figure 1:
The adjusted percentage mortality (±SE) of second stage juveniles of M.
graminicola against the different concentrations of cinnamon leaf oil after
three days of treatment.
Figure 2: Probit values of adjusted percentage mortality of J2 against log
concentration of cinnamon leaf oil
50
Efficacy of essential oils
Figure 3:
Probit values of adjusted percentage mortality of J2 against Log
concentration of cinnamon bark oil
Table 1: Mean total root galls in cinnamon leaf oil and cinnamon bark oil treated
seedlings
Treatment
Mean total root galls (± SE)
Distilled water + juveniles (untreated)
16.0 ± 0.4a
0.9ppm leaf oil + juveniles
4.0 ± 0.2b
0.9 ppm bark oil + juveniles
1.0 ± 0.1b
Distilled water
0
0.5 ppm Tween-20
1.0 ± 0.1b
Experiment III: Screening the effect of cinnamon leaf oil and bark oil on Meloidogyne
graminicola in potted rice plants
It was observed that the percentage chlorosis and mean number of dead plants have
been increased in pots treated only with second stage juveniles (Table 2) (p = 0.003, F =
6.58). However, the mean plant height did not show and significant difference between
treatments. This is indicative that there is no significant phyto-toxicity due to cinnamon
leaf oil or Tween-20 on rice plants. The root gall formation was significantly reduced in
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L D Amarasinghe et. al.
cinnamon leaf oil treated rice plants (n = 8.5 ± 0.13; p = 0.002, t = 3.34, df = 45) and bark
oil treated plants (n= 8.3 ± 0.12; p = 0.000, t = 5.64, df = 45) compared to untreated plants
(n = 16.1 ± 1.6). However, the gall indices between cinnamon leaf oil treated rice plants
and cinnamon bark oil treated rice plants was not significant (p = 0.218, F = 1.89). An
increased number of dead plants, the lowest plantheight and the highest chlorosis were
observed in the plants treated only with nematode juveniles. However, neither the dead
plants (p = 0.441, F = 0.82) nor plant height (p = 0.054, F = 0.82) was significantly
different among treatments.
Table 2: Post-treatment measurements of cinnamon leaf oil treated young rice
plants (±SE)
Treatment
Mean %
Mean
Mean %
Mean No.
Mean
dead plants
plant
chlorosis
of roots
root
height
ab
Mean No.
of galls
length
62.5 ± 6.7a
7.2 ± 0.7
4.9 ± 0.51
16.1 ± 1.6a
15.4 ± 0.7ab
59.1 ± 4.6ab
6.9 ± 0.6
5.4 ± 0.54
8.5 ± 0.1b
25.0 0.5a
19.9 1.1ab
49.5 7.2ab
10.1 0.8
6.7 0.58
8.3 0.1b
20.0 ± 2.1ab
21.2 ± 5.4a
43.2 ± 5.4b
7.2 ± 0.3
5.1 ± 0.5
0
Distilled water + J2
20.0 ± 7.1
0.9 ppm leaf oil + J2
32.5 ± 4.7a
0.9 ppm bark oil + J2
Distilled water
c
0.9 ppm leaf oil
5.5 ± 3.7
0.9 ppm bark oil
15.0 1.4
0.5 ppm Tween-20
17.5 ± 2.7b
b
14.4 ± 1.1
b
15.4 ± 0.9
ab
42.5 ± 2.8
b
7.2 ± 0.3
4.7 ± 0.16
0
16.4 1.5
ab
44.6 2.9
b
9.2 1.3
5.5 0.34
0
28.1 ± 2.9c
8.6 ± 0.4
5.4 ± 0.39
0
17.3 ± 0.4ab
Data indicated by the same letters are not significantly different at p=0.05 according to
Duncan’s multiple range test.
These results show that the essential oils of cinnamon have shown nematicidal
activity. Studies conducted on the effectiveness of the cinnamon oil on the stored grain
insects, fungal pests and nematodes have also shown the potential of these botanicals as
replacement for synthetic insecticides (Paranagama et al., 2003, Kong et.al, 2007). In a
detailed study have shown a higher nematicidal activity of trans-cinnamaldehyde against
the Meloidogyne javanica (Oka, 2001). This study further reports that EC50 value of transcinnamaldehyde for juvenile immobilization and hatching inhibition in vitro were as low
as 15µl/l and 11.3µl/l respectively. In a micro plot experiment, soil treatment with transcinnamaldehyde (50ml/m2) has caused a reduction of the galling index and has increased
the shoot weight of tomato plants (Oka, 2001). Galling incidence and size of the gall are
dependent upon nematode density and the nematode species. In the present study, the
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Efficacy of essential oils
number of root galls has been considered as an index of the nematode population. The root
gall formation in rice seedlings and young rice plants due to M. graminicola was
significantly reduced in the treated plants with cinnamon leaf oil and cinnamon bark oil.
None of the treatments, cinnamon leaf oil, cinnamon bark oil or Tween-20 showed a
significant phytotoxicity in rice plants. Cinnamon leaf oil and cinnamon bark oil can be
used as an environmental friendly nematicide against the rice root knot nematode
Meloidogyne graminicola in rice seedlings and in young rice plants.
CONCLUSIONS
LC50 for cinnamon leaf oil for killing 50% of juveniles of M. graminicola in rice
root galls after three days of the treatment was 0.326 ppm and that for cinnamon bark oil
was 0.454 ppm.
Nematicidal activity is not significantly different between cinnamon leaf oil and
bark oil.
Concentration of 0.9 ppm of cinnamon leaf oil and bark oil is required to reduce
the formation of root galls in rice plants infested with M. graminicola.
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