Nematicidal activity of plant extracts against the root-knot nematode, Meloidogyne incognita
ABSTRACT Nematicidal activity of extracts from plants was assayed against Meloidogyne incognita. In laboratory assays extracts from tobacco (Nicotiana tabacum L), clove (Syzygium aromaticum L), betelvine (Piper betle L), and sweet flag (Acorus calamus L) were most effective in killing the nematode, with an EC50 that was 5-10 times lower than the EC50 of the synthetic pesticides chlorpyrifos, carbosulfan and deltamethrin. The shapes of the dead nematodes differed in a characteristic way, and groups of pesticides and plant extracts could clearly be distinguished based on this phenomenon, which may be an indicator for the modes of action of the tested pesticides. In a greenhouse bioassay clove bud and betelvine were tested as mulch. Experiments revealed that the total number of live nematodes on roots of pepper plants treated with mulch of the clove bud was 7% of that of the controls and did not differ significantly from that of plants treated with the recommended synthetic pesticide carbofuran. The application of clove buds as a botanical pesticide for future use against nematodes is highly promising since clove is the 6th major plant grown on Bangka Island, and the market value of clove has decreased sharply over the last years
- SourceAvailable from: Ro-Dong Park[Show abstract] [Hide abstract]
ABSTRACT: In this study, the 3,4-dihydroxybenzoic acid (3,4-DHBA) from Terminalia nigrovenulosa bark (TNB) was purified and its in vitro nematicidal activity was investigated against Meloidogyne incognita. The purification of 3,4-DHBA used a silica gel column and Sephadex LH-20 chromatography combined with thin-layer chromatography and high performance liquid chromatography. Structural identification of the 3,4-DHBA was conducted using (1)H-nuclear magnetic resonance (NMR), (13)C-NMR, and liquid chromatography-tandem mass spectrometry. Nematicidal activity bioassays revealed that 3,4-DHBA treatment resulted in 33.3, 47.5, 72.5 and 94.2% J2 mortality at 0.125, 0.25, 0.5 and 1.0 mg/ml, respectively after 12 h incubation. J2 mortality was increased significantly (P < 0.0001) with increasing incubation time in the range of 54.2-94.2% from 3 to 9 h after incubation with 3,4-DHBA (1.0 mg/ml), but with no significant difference observed where the incubation time was increased from 9 to 12 h. The 3,4-DHBA treatment resulted in 33.3, 65.0, 76.7 and 85.0% hatch inhibition at 0.125, 0.25, 0.5 and 1.0 mg/ml, respectively, 3 days after incubation. Changes in the shape of the eggs were determined after incubation for 1 day with a 3,4-DHBA concentration of 1.0 mg/ml.Microbial Pathogenesis 04/2013; · 1.97 Impact Factor
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ABSTRACT: The nematicidal, antifungal and antioxidant activities of methanol extracts from six Vietnamese native medicinal plants were evaluated by various assays in vitro. Of the plant extracts tested, Terminalia nigrovenulosa was found to possess the highest activity when compared to the others. The leaves and bark of T. nigrovenulosa showed strong inhibitory activity against Meloidogyne incognita and Fusarium solani. The DPPH (1,1-Diphenyl-2-picryl-hydrazyl) radical scavenging, reducing power and total antioxidant activities of T. nigrovenulosa bark were higher than that of the remainder plant extracts. Chitinase activity of these plants was also investigated using SDS-PAGE. The results obtained in the present study indicate that T. nigrovenulosa leaf extracts are the greatest potential source as nematicides and fungicides for the control of M. incognita and F. solani. In particularly, T. nigrovenulosa bark extracts could be used as a potential source of commercially viable levels of natural antioxidant.Journal of Applied Biological Chemistry 01/2013; 56(4).
The Open Natural Products Journal, 2009, 2, 77-85 77
1874-8481/09 2009 Bentham Open
Nematicidal Activity of Plant Extracts Against the Root-Knot Nematode,
Wiratno*,a,b, D. Taniwiryonoc, H. Van den Bergb, J.A.G. Riksend, I.M.C.M. Rietjensb,
S.R. Djiwantia, J.E. Kammengad and A.J. Murkb
aIndonesian Medicinal and Aromatic Crops Research Institute, Jl. Tentara Pelajar No. 3, Bogor, Indonesia
bSection Toxicology, Wageningen University, Postbus 8000, 6700 EA Wageningen, The Netherlands
cIndonesian Biotechnology Research Institute for Estate Crops, Jl. Taman Kencana No. 1, Bogor, Indonesia
dLaboratory of Nematology, Wageningen University, P.O. Box 8123, 6709 ES Wageningen, The Netherlands
Abstract: Nematicidal activity of extracts from plants was assayed against Meloidogyne incognita. In laboratory assays
extracts from tobacco (Nicotiana tabacum L), clove (Syzygium aromaticum L), betelvine (Piper betle L), and sweet flag
(Acorus calamus L) were most effective in killing the nematode, with an EC50 that was 5-10 times lower than the EC50 of
the synthetic pesticides chlorpyrifos, carbosulfan and deltamethrin. The shapes of the dead nematodes differed in a char-
acteristic way, and groups of pesticides and plant extracts could clearly be distinguished based on this phenomenon, which
may be an indicator for the modes of action of the tested pesticides. In a greenhouse bioassay clove bud and betelvine
were tested as mulch. Experiments revealed that the total number of live nematodes on roots of pepper plants treated with
mulch of the clove bud was 7% of that of the controls and did not differ significantly from that of plants treated with the
recommended synthetic pesticide carbofuran. The application of clove buds as a botanical pesticide for future use against
nematodes is highly promising since clove is the 6th major plant grown on Bangka Island, and the market value of clove
has decreased sharply over the last years.
Keywords: Black pepper, botanical nematicide, clove, mode of action.
White (Chitwood) (Tylenchida: Heteroderidae) is a major
plant-parasitic nematode species affecting the quantity and
quality of the crop production in many annual and perennial
crops. Infected plants show typical symptoms including root
galling, stunting and nutrient deficiency, particularly nitro-
gen deficiency . On Bangka Island, South of Sumatra,
Indonesia, this nematode is considered to be one of the major
problems in black pepper cultivation. In 2003, 4.900 ha of
the total of 52.468 ha of pepper plantations was severely
infected by this pest . Although, there is no information
about the exact impact of nematode infection on the loss of
pepper production, it is clear from visible inspection that
severely attacked plants have a reduced vitality, which pro-
duce less fruits, and finally will die. Davis and May  in-
formed that the yield losses of cotton production caused by
M. incognita in 2002 were estimated to be between 18.0-
47.3%. Therefore the presence of this pest in plantations has
to be controlled.
Root-knot nematode, Meloidogyne incognita Kofoid and
can be minimized through several approaches such as using
natural enemies [4, 5], enhancing cultural practices , cul-
tivating resistant cultivars , and applying pesticides .
*Address correspondence to this author at the Indonesian Medicinal and
Aromatic Crops Research Institute, Jl. Tentara Pelajar No. 3, Bogor,
Indonesia; E-mail: firstname.lastname@example.org
The population of plant-parasitic nematodes in the field
Since the 1950s, however, farmers have relied mainly on
synthetic pesticides rather than on other approaches. This
sometimes results in excessive and unsafe use of synthetic
pesticides . Therefore, it has become an important issue to
find alternative control strategies, which are as effective as
synthetic pesticides, safer to farmers, consumers, and the
environment and relatively easily available at low price .
One of possible alternatives is the utilization of pesticides
from plant origin, known as botanical pesticides . These
pesticides are generally considered to be non-persistent un-
der field conditions as they are readily transformed by light,
oxygen and microorganisms into less toxic products. There-
fore no residues are expected on the products or in the envi-
project in which 17 plant species were selected based on
their availability and potential use as botanical pesticide, are
tested for nematicidal activity. In the present study we evalu-
ate the nematicidal potency of extracts from these 17 plant
species, 15 of which may have nematicidal activity against
M. incognita. The results are compared to those of three syn-
thetic pesticides, namely chlorpyrifos (an organophosphate
insecticide) , carbosulfan (a carbamate insecticide) 
and deltamethrin (a pyrethroid insecticide) representing the
three groups of often used or advised pesticides to control
pests of the black pepper on Bangka Island . The 2 most
potent extracts are subsequently tested in a greenhouse ex-
periment to evaluate effectiveness of their raw materials ap-
The study reported in the present paper is part of a larger
78 The Open Natural Products Journal, 2009, Volume 2 Wiratno et al.
plied as a mulch to control the nematode attacking roots of
the pepper plant.
2. MATERIALS AND METHODS
chlorpyrifos 200 g l-1, carbosulfan 200 g l-1, deltamethrin 25
g l-1, and carbofuran 3G, purchased from the agro-chemical
shop, Sarana Tani in Bogor, Indonesia. DMSO (99.9% pure
for spectroscopy from Acros Organics), was supplied by
Sigma Aldrich (Zwijndrecht, The Netherlands), Tween 80
(synthesis grade), acetone (100%, analysis grade) and etha-
nol (absolute, analysis grade) from Merck (Darmstadt, Ger-
many) were supplied by VWR (Amsterdam, The Nether-
The synthetic pesticides that were used in this study are
2.2. Preparation of the Plant Extracts
gardens of the Indonesian Medicinal and Aromatic Crops
Research Institute (IMACRI) and extracted in the post har-
vest laboratory of the Institute. The 17 plant species and part
of the plant used for extraction are presented in Table 1. The
extraction procedures of the plant materials were based on
the method described by Yuliani and Rusli . In short 1kg
material was dried in the sun for 4-5 days then ground in a
hammer mill (Reisch Mühle made by Karl Kolb (Dreieich,
Germany)) using 3 mm grinders. To the 1 kg powder 5 l of
ethanol (96%) was added followed by 3 h mixing at 500 rpm
All plant materials were obtained from the experimental
using an electric mixer made by Karl Kolb (Dreieich, Ger-
many). Subsequently, the mixture was left overnight in the
dark at 28 ± 1ºC to allow further extraction of the active in-
gredients. After this, the mixture was filtered using Whatman
no 91 filter paper and the residues were soaked and shaken
again in 1 l of ethanol for 2 h. The solution was filtered again
and the first and second filtrate were mixed and concentrated
using a rotavapor at 45°C for approximately 3 h until all
ethanol was removed. The extracts were transferred into
brown glass bottles and stored at -4°C. Only cashew nut was
treated differently as cashew nut shell liquid (CNSL) was
prepared by pressing the shell of the cashew seed in a man-
ual presser made by the post harvest division of the
IMACRI, after which the liquid was collected and stored in a
brown glass bottle. On the following week about 10 ml of
each extract were poured into 20 ml of glass bottles and they
were stored at -20°C in the laboratory of Toxicology of
Wageningen University until further use.
2.3. Laboratory Exposure of Nematodes
the Sub Department of Nematology, Wageningen University,
the Netherlands. The tested nematode species, M. incognita,
was harvested according to the method as described by
Barker . The roots of about 3 months old tomato plants,
which had been infected with the nematode were washed in
fresh tap water. After that the roots were cut into 1-2 cm
length and put in a round filter container then gently placed
in the funnel, which had been placed in a mist chamber. Ac-
The laboratory experiment was conducted in triplicate in
Table 1. The Botanical Species and Plant Parts that were Extracted, the Yield (%), and the Density of the Final Product
Syzygium aromaticum L* Clove Bud 22.2 0.91 
Nicotiana tabacum L* Tobacco Leaf 8.1 1.07 
Piper betle L* Betelvine Leaf 8.6 0.96 
Acorus calamus L* Sweet flag Rhizome 6.5 0.92 
Chrysanthemum cinerarieaefolium L* Pyrethrum Flower 9.6 0.75 
Cymbopogon nardus L* Citronella Leaf, stem 6.2 1.06 
Derris elliptica Benth* Tuba root Root 6.3 0.96 
Azadirachta indica L* Neem Seed 5.1 1.61 
Piper nigrum L* Pepper Berries 8.6 1.03 
Andropogon zizanioides L* Vetiver Root 14.2 0.89 
Richinus communis L* Castor bean Seed 11.9 1.04 
Annona muricata L*
Graviola Seed 9.6 0.85 
Cymbopogon citratus L* Lemongrass Leaf, stem 9.5 0.86 
Anacardium occidentale L* Cashew Seed 4.1 1.00 [29, 30]
Pelargonium citrosa Van Leenii** Citrosa Leaf 9.7 0.68 
Pogostemon cablin Benth
Patchouli Leaf 12.4 0.89 -
Pachyrhizus erosus L Yam bean Seed 5.0 0.94 -
* = known to contain nematicidal properties. ** = predicted to have nematicidal effect.
Nematicidal Activity of Plant Extracts The Open Natural Products Journal, 2009, Volume 2 79
tive nematodes passed through the filter and sank to the bot-
tom of the funnel stem. After 4 days nematodes could be
harvested and used for the experiments. The average density
of nematode juveniles in the suspension thus prepared was
about 1750 ml-1.
The pesticide stocks were made in 1 ml glass vials by
diluting the extracts in a solvent mixture of DMSO:Tween
80:Acetone = 1:2:3. In a first pilot study the maximum toler-
ated total solvent concentration was determined and this
should not exceed 5% to avoid unspecific toxicity. The test
concentrations were made by adding 40?l of the plant extract
or the synthetic pesticide stocks to 460 ?l of fresh tap water
in a 12-well plate. The mixing-plate was gently shaken
manually for about 2 min to allow the pesticides to mix
properly. After that, 150 ?l of the solution was transferred
into 24 well test plates. Next, 90 ?l of the nematode suspen-
sion containing approximately 150 juveniles was added into
the wells and gently mixed for another 2 min and kept stand-
ing overnight at 24ºC. After 24 h the dead and alive nema-
todes were counted to evaluate the mortality rate. A second
pilot study was performed to determine the rough toxicities
of the pesticides in 5 mg and 31.5 mg of plant extract and
technical mixture of synthetic pesticides ml-1 exposure me-
dium, respectively. In these stock solutions, however, the
visibility of the nematodes was not enough. Therefore the
nematode solution was washed to make the nematodes com-
pletely visible. Washing was done by first adding 0.5 ml of
fresh water to the 24 well plates containing exposed nema-
todes, letting all nematodes settle again on the bottom of the
well during 3 min, and carefully removing 0.5 ml again us-
ing a micro pipette. This procedure was repeated 3 times. In
order to evaluate a possible recovery effect, the observation
of the mortality of the nematodes was conducted twice dur-
ing the pilot study. The first time was conducted immedi-
ately after washing for the second time of approximately 6 h
after the first observation. The mortality of the treated nema-
todes was determined using a stereo microscope with 10-fold
magnification. Nematodes were considered dead when no
movement was observed during two seconds even after me-
chanical prodding. As no recovery of nematodes was ob-
served, this was not further studied in the final experiment.
Washing to dilute botanical extracts before counting the
nematodes also was not needed, as in the pilot experiment
the dead nematodes were found to have a specific shape,
defined as either straight (I-shape), bent (banana-shape),
sigmoid (?-shape), and curly (?-shape) which can be used to
determine the death or live nematodes. In the final experi-
ments all pesticides were tested in at least 5 concentrations
including a solvent control. In order to make a comparison
between botanical and synthetic nematicides, lethal concen-
trations i.e. LC20, LC50 and LC90, were expressed as mg ap-
plied extract or technical mixture ml-1 exposure medium be-
cause the nature and concentration of the active ingredients
of the botanical extracts are unknown.
2.4. Greenhouse Experiment
nesian Medicinal and Aromatic Crops Research Institute,
Bogor. Nematodes for inocula were collected from the roots
of pepper plants which were grown in the Botanical Garden
of the Bangka Belitung Assessment Institute for Agricultural
Technology, which were heavily attacked by the root-knot
The greenhouse experiment was conducted at The Indo-
nematode, M. incognita. The nematodes were harvested ac-
cording to method as described by Barker .
pot containing sterilized soils, was conducted by pouring 10
ml of water containing 1000 nematode juveniles onto the soil
surface. One week after the inoculation in which the nema-
todes infest roots of the pepper plants, 10 g of carbofuran
3%, 20 g of ground clove buds, or 60 g of dried betelvine
leaves were applied evenly on the soil surface. Control con-
sisted of pots without additional application. The experi-
ments were performed with 10 replicates. Every pot was
watered three times a week with about 350 ml of fresh water.
Two months after mulching the nematodes present on the
roots of the treated plant were collected and counted. Collec-
tion was conducted according to the method described by
Barker . A 1 ml of 40 ml solution containing collected
nematodes which had been homogenized using a magnetic
stirrer was sampled using a micro pipette. The solution was
put into the 1-ml Matsunami micro slide glass and the nema-
todes were counted using a stereo microscope under 100x
Inoculation of 6 months old pepper plant grown in a 2 l
2.5. Data Analysis
groups (PO) were corrected for mortality in the solvent con-
trols (PC) using Abbott’s formula: PT (%) = [100 x (PO-PC)/
PC] . The corrected mortality (PT) was plotted against the
pesticide concentration and fitted using Slide Write Plus 6.1
(Advanced Graphics Software Inc.) to determine the LC20,
LC50 and LC90 values. Because the log scale was used for
plotting the data, the control data were included as a concen-
tration being 100x lower than the lowest test compound con-
centration. The method of 95% LSD intervals was used for
the means, and the SAS program for analysis of variance
(ANOVA) and the least significant difference (LSD) test was
used to compare the means of the bioassays. Data were
transformed into ?x+0.5.3.
The mortality rates of the nematodes in the exposure
(cashew, pressed) and 22% (clove) (Table 1). In addition to
clove also vetiver (14%), patchouli (12.4%) and castor bean
(12%) had relatively high yields. Density of the concentrated
extract (expressed as g ml-1) indicated a relatively oily con-
tent. The densities of the citrosa and the pyrethrum extracts
were 0.68 and 0.75 g ml-1 respectively, while the others had
higher densities, which were between 0.86 and 1.06 g ml-1.
In our laboratory study the average background mortality
of the nematodes in the control treatment was about 4%,
indicating good starting conditions. Application of 5 mg ex-
tract ml-1 exposure medium revealed that tobacco, clove and
betelvine were highly toxic to the nematodes, killing more
than 80% of the nematodes while the others gave quite low
mortalities (Table 2). Based on these findings, these plant
extracts were divided into 3 main groups i.e. highly toxic
(>80% mortality), consisting of clove, tobacco and betelvine,
slightly toxic (10-20% mortality) consisting of sweet flag,
pyrethrum, and citronella and not toxic (<10% mortality)
consisting of the rest of the extracts tested. The concentration
of 31.5 mg technical mixture ml-1 exposure medium of del-
tamethrin, carbosulfan, and chlorpyrifos killed 40, 73, and
The yield of the extraction procedure varied between 4%
80 The Open Natural Products Journal, 2009, Volume 2 Wiratno et al.
93% of the treated nematodes, respectively (Table 2). There-
fore, to find the LC50 values chlorpyrifos was further tested
at 0, 4, 13, 22, and 31 mg technical mixture ml-1 exposure
medium, and the other two at 0, 13, 22, 31, and 40 mg tech-
nical mixture ml-1 exposure medium.
tested at concentrations of 0, 1.2, 2.4, and 4.8 mg ml-1 expo-
sure medium, while the other groups were tested at concen-
trations of 0, 4.8, 9.6, and 19.2 mg ml-1 exposure medium.
The extracts were not tested at a higher concentration as
some of them (cashew, tuba root, and neem) did not mix
adequately at these higher concentrations. In addition, these
pesticides were not considered for possible future application
as these would require great volumes of plant material,
which would not result in a practical protocol for pesticide
use. The results revealed that tobacco, clove and betelvine
were highly toxic with LC50 values of 1.9 – 3.9 mg ml-1 ex-
posure medium. Sweet flag was moderately toxic with an
LC50 of 11.3 mg ml-1 exposure medium. The LC50 of tuba
The highly toxic group of plant extracts was further
root, citronella and pyrethrum were not reached, but their
LC20 was 5.7 – 8.9 mg ml-1 exposure medium. The remaining
10 extracts were not toxic to the nematode as the LC20 was
not reached (> 19.2 mg ml-1 exposure medium) (Table 2).
Representative dose response graphs of extracts from each of
these groups of plant extracts are given in Fig. (1). The syn-
thetic pesticides chlorpyrifos and carbosulfan both fell in the
slightly toxic group with an LC50 >19.2 mg ml-1 exposure
medium and an LC20.of 8.7-12.7 mg ml-1 exposure medium.
Deltamethrin fell into the non-toxic groups with a LC20 >
19.2 mg ml-1. This lower toxicity of the synthetic technical
pesticide mixtures compared to the plant extracts can also be
seen from the dose response curves (Fig. 2).
scope it became apparent that they had either one of four
very distinct shapes, namely: straight (I-shape), bent (ba-
nana-shape), sigmoid (?-shape), or curly (?-shape) (Table 3,
Fig. 3). The dead nematodes from the control group mostly
was straight (I shape) with only very few showing a bent
When the dead nematodes were studied under the micro-
Table 2. Mortality of M. incognita and Lethal Concentrations after 24 hrs of Exposure to Botanical Extracts or Synthetic Pesti-
cides in Aqueous Medium
Tested Compounds (Mortality (%) ± SD)a Lethal Concentrations (mg ml-1)b
Plant Extractsc LC20 LC50 LC90
Clove 98 ± 2.3 2.8 3.9 4.9
Tobacco 94 ± 3.1 1.3 1.9 3.6
Betlevine 83 ± 1.2 1.2 3.0 5.2
Sweet flag 17 ± 5.1 4.9 11.3 18.7
Pyrethrum 13 ± 3.0 8.9 > 19.2 > 19.2
Citronella 10± 3.3 5.7 > 19.2 > 19.2
Tuba root 9 ± 5.1 8.6 > 19.2 > 19.2
Neem 8 ± 3.0 > 19.2 > 19.2 > 19.2
Pepper 6 ± 9.5 > 19.2 > 19.2 > 19.2
Cashew 5 ± 3.3 > 19.2 > 19.2 > 19.2
Vetiver 4 ± 4.3 > 19.2 > 19.2 > 19.2
Castor bean 4 ± 5.0 > 19.2 > 19.2 > 19.2
Graviola 4 ± 5.0 > 19.2 > 19.2 > 19.2
Patchouli 4 ± 5.6 > 19.2 > 19.2 > 19.2
Lemongrass 4 ± 5.7 > 19.2 > 19.2 > 19.2
Yam bean 1 ± 5.9 > 19.2 > 19.2 > 19.2
Citrosa 2 ± 3.4 > 19.2 > 19.2 > 19.2
chlorpyrifos 93 ± 3.4 8.7 19.4 30.7
carbosulfan 73 ± 4.4 12.7 25.3 36.1
deltamethrin 40 ± 4.4 20.8 > 40 > 40
aPilot study, bFinal experiment, cTested in 5 mg ml-1, dTested in 31.5 mg ml-1.
Note: In the pilot study only a single concentration was tested, while in the final experiments lethality was tested for 5 exposure concentrations to evaluate LC20, LC50, and LC90..
Experiments were conducted in triplicates.
Nematicidal Activity of Plant Extracts The Open Natural Products Journal, 2009, Volume 2 81
(banana) shape. The characteristic shape of nematodes killed
by tobacco and castor bean was curly (?-shapes) with some
bent and sigmoid shapes, which was similar to those killed
by the acetylcholine esterase inhibitors chlorpyrifos and car-
bosulfan. The appearances of the nematodes killed by other
plant extracts mostly followed straight or bent shapes, simi-
lar to those killed by the pyrethroid deltamethrin. The mor-
tality and these characteristics were tested for consistency
with the highest concentrations, and all pesticides yielded
exactly the same results.
Fig. (1). Examples of plant extracts, which were highly, slightly
and not acutely toxic to M. incognita.The concentration was ex-
pressed as mg extract ml-1 exposure medium presented on a log
axis, mortality was corrected for control mortality using Abbot’s
formula, exposure was during 24 h in triplicate.
Fig. (2). Acute toxicity of three synthetic pesticides to root-knot
nematode, M. incognita, compared to that of the plant extract,
clove. The concentration of synthetic pesticides was expressed as
mg technical mixture ml-1 exposure medium presented on a log
axis, mortality was corrected for control mortality using Abbot’s
formula; exposure was during 24 h in triplicate.
Table 3. Relative Occurrence (%) of Characteristic Shapes and Percentage of Relative Occurrence Among Dead Nematodes (M.
incognita) after 24 hrs Exposure to the Highest Concentration of the Botanical Extracts or Synthetic Pesticides in Aque-
ous Medium. Number of Tested Nematodes is 150 Juveniles. The 4 Distinguished Shapes are shown in Fig. (3)
Tested Compounds Shapes of Dead Nematodes (%)
Plant Extracts Straight (I-shape) Bent (Banana-shape) Sigmoid (?-shape) Curled (?-shape)
Number of Dead Nematodes
82 The Open Natural Products Journal, 2009, Volume 2 Wiratno et al.
Fig. (3). Characteristic shapes of dead nematodes: a. straight (I-
shape), b. bent (banana-shape), c. sigmoid (?-shape), and d. curled
(?-shape). See Table 3 for percentage relative occurrence of these
shapes after exposure to the highest concentrations of the botanical
and synthetic pesticides.
potent than betelvine in reducing the total number of nema-
todes in the roots after 2 months of a single application. The
number of nematodes in the root treated with the clove bud
differed insignificantly compared to the roots treated with
the recommended synthetic pesticide, carbofuran. Although
betelvine was able to reduce the infestation of the nematodes
compared to control, this difference was not statistically sig-
nificant. In addition, the number of infected plants treated
with the clove bud was lower than that of the betelvine and
control groups. There was no plant mortality among the
clove and carbofuran treated plants. Meanwhile 1 and 3
plants died in the betelvine-treated and control groups re-
spectively (Table 4).
In the greenhouse experiments clove was 10 times more
4. DISCUSSION AND CONCLUSION
toxic against nematodes in a laboratory exposure. One of
these plants was also effective in controlling infestation of
nematodes into roots of the pepper plants during a 2 months
This study revealed that some plant extracts were highly
and betelvine, were highly toxic for the parasitic root-knot
nematode, M. incognita with LC50 values of 1.9, 3.9, and 3.0
mg extract ml-1 exposure medium, respectively. Sweet flag
was more moderately toxic (LC50 = 11.3 mg extract ml-1 ex-
posure medium). The later was still even more toxic than the
tested concentrations of the three synthetic pesticides i.e.
chlorpyrifos, carbosulfan and deltamethrin with LC50 values
of 19.4, 25.3, and >40 mg technical mixture ml-1 exposure
medium, respectively. This potent nematicidal action could
be due to the active ingredient eugenol of clove oil, which
has been shown to act as a potent nematicide . An impor-
tant active ingredient reported for sweetflag is ?-asarone,
which has also been reported to act as potent nematicide in
banana plantation in India . Clove and the sweetflag ex-
tracts have been reported to contain 88% eugenol  and
45.5% ?-asarone , respectively. Therefore, the LC50s of
clove and sweetflag extracts would be equivalent to 3.4 and
5.2 mg eugenol and ?-asarone ml-1 solvent, respectively.
Compared to the LC50’s of chlorpyrifos, carbosulfan and
deltamethrin of 19.4, 25.3, and >40 mg technical mixture
ml-1 water are equivalent to 3.8, 5.1, and > 1 mg ml-1 solvent.
The active ingredient eugenol of clove is more toxic against
M.incognita than that of the tested synthetic pesticides.
The in vivo laboratory study showed that tobacco, clove
dopholus similes has been reported before by Mustika and
Slamet , who found that on 1% concentration clove oil
administered via aqueous medium effectively killed all di-
rectly exposed nematodes within 10 minutes after applica-
tion. In addition, Meyer et al.  reported that volatiles
from 5.0% clove oil reduced nematode egg hatching in water
by 30%, and decreased viability of hatched Juvenile (J2) of
M. incognita by as much as 100%.
This finding is very promising since farmers on Bangka
Island indicate that currently there is no effective synthetic
pesticide available to control this nematode in the field .
The ineffectiveness of the pesticides used against the nema-
todes such as fenthion, lambda cyhalothrin, carbofuran, and
deltametrhin, may be the result of the low concentrations
usually applied i.e. between 1 – 4 mg technical mixture ml-1
water in combination with the limited availability of the so-
lution for the buried nematodes. These concentrations nor-
mally are used to control the above ground insect pest spe-
cies i.e. tinged bug, Dasynus piperis China, stem borer, Lo-
phobaris piperis Marsh and bug, Diconocoris hewetti Dist
. Our findings showed that the synthetic pesticides chlor-
pyrifos and carbosulfan would be effective if they could
reach the nematodes in a concentration of at least 30 mg
technical mixture ml-1 water, which is about 7-30 times
The toxic potency of clove against M. incognita and Ra-
Table 4. The Number of Infected and Dead Plants and the Number Nematodes Presents 2 Months after a Single Application of
Clove, Betelvine, and Carbofuran in a Greenhouse Experiment. The Plants were Experimentally Infected 7 Days Before
Treatments Infected Plants # of Dead Plants # of Nematodes ± SE per g Roots*
Control 10 3 335 ± 69.9b
Betelvine 8 1 274 ± 70.7b
Clove 5 0 23 ± 9.3a
Carbofuran 3 0 5.3 ± 5.1a
*Means in the same column followed by the same letter do not differ significantly (P>0.05) in the LSD test.
Nematicidal Activity of Plant Extracts The Open Natural Products Journal, 2009, Volume 2 83
higher than the concentration used by local farmers. Carbo-
furan was not tested in our study since it could not be diluted
adequately in the solvent, even after 10 min of sonification,
making it impossible to compare exposure concentrations.
The observed characteristic differences in shape of the
nematodes killed by pesticide-exposure was an interesting
finding that might be useful as an indication to analyze the
major mode of toxic action of the plant extracts of usually
very complex composition. Meanwhile, according to Akhtar
and Mahmood  the nematicidal mode of action of plant
materials still is not known. Our finding showed that the
nematodes killed by the acetyl cholinesterase inhibitors
chlorpyrifos and carbosulfan mostly had a curled shape (75-
85%) while few of them had sigmoid (7-11%) and bent (8-
In our bioassays the extract of tobacco also induced
curled (88%) and sigmoid (8%) shapes. These shapes are
similar to those of nematodes killed by the organophosphate
and carbamate pesticides, which are known to have acetyl-
choline esterase inhibiting action. This finding is in line with
the report of Nguyen et al. , who described that tobacco
has acetylcholinesterase inhibiting effects in humans, as the
neuromuscular junctions of nematodes were not fundamen-
tally different, either structurally nor functionally, from the
neuromuscular junctions of other animals  including
The pyrethroid pesticide deltamethrin and the extract of
pyrethrum, known for its pyrethroid-like action, resulted in
dead nematodes that never had curly shapes but were mostly
bent (banana-shape) (87-100%) and to some extent straight
(I-shape) (0-13%) or very few of them showing a sigmoid
shape (?-shape) (0-1%). The results shown in table 3 sug-
gested that the mechanisms of toxicity behind the curly
shape were related to that of the sigmoid shape, as their oc-
currence was related and they might just represent a gradual
difference in occurrence of the toxicity. Based on the shapes
of the dead nematodes we suggest that most of the extracts
tested had a pyrethroid-like effect on the central nervous
system of the nematodes. However, further assays in higher
concentration or longer exposure period would be helpful to
validate this finding since the mortality induced by most of
the botanical extracts still was very low. We did not have
any explanation yet for the clear relationship between shapes
of the dead nematode in relation to the pesticide exposure.
During the experiment it was observed that about five min
after exposure to organophosphate and carbamate pesticides
and a tobacco extract, nematodes showed more active
movement and most of them had formed curly shape and
stayed stable until they died. On the other hand, those ex-
posed by other treatments did not show specific shapes so
soon. Their appearances were similar as those in control.
nematodes, it is not the best candidate to be applied in prac-
tice because of its high toxicity for mammals including man
40. The other two highly toxic plants, clove and betelvine, are
more promising plants to be further developed into a botani-
cal nematicide. Of these two, clove gives the highest extrac-
tion yield (22.2%) followed by betelvine (8.6%). The extrac-
tion yield of the less toxic sweet flag is only 6.5% (Table 1).
The application of ethanol for the extraction of plant extract
as carried out in this study is not suitable for the farmers be-
Although the tobacco extract is the most toxic against the
cause it is too expensive for them while according to Zuskin
et al.  high exposure of alcohol through inhalation often
causes chronic obstructive lung disease. Therefore, easier
and simpler preparation methods must be developed before a
plant can be successfully introduced and applied as a botani-
cal pesticide for the farmers. Two promising methods are
application as an aqueous extract  and as an amendment
of organic materials as mulch [43-45]. In our study we
choose for testing clove and betelvine as a mulch because
mulching is believed to help control plant parasitic nema-
todes, as nitrate and ammonical nitrogen accumulating dur-
ing decomposition of organic matters are toxic to plant para-
sitic nematodes . The effectiveness of mulching to re-
duce the population of the nematodes will be greatly en-
hanced when the mulch also contains toxic chemicals 
such as the nematicidal compounds of clove. Amendment of
organic plant materials also increases food sources which
facilitates the population growth of bacterivorous nematodes
(Rhabditidae and Cephalobidae), fungivorous nematodes
 and predatory nematodes (Mononchidae) , which
will also lower the population density of the plant parasitic
nematodes through competition, antagonism or creating un-
The greenhouse experiment revealed that mulch from
clove bud was very potent in suppressing nematode infesta-
tion in pepper plants. After 2 months of single application
clove significantly suppressed the population of nematodes
in the pepper plant roots. The dosages of clove and betelvine
used during the greenhouse experiment were based on the
LC90 value of the laboratory bioassay, which was about 5 mg
extract ml-1 exposure medium. This value was equivalent to
about 20 g clove buds and 60 g dried betelvine leaves based
on extraction yields of 22.2 and 8.6% respectively. The dos-
age of carbofuran was 10 g which was 1/3 of what would be
recommended per plant in the field. This amount of carbo-
furan used was based on an assumption that the volume of
the soil in the pot was 1/3 of that in the field. The proposed
dosage of clove for field application therefore is 60g per
Since clove is the 6th major cultivated plant on Bangka
Island , this plant material has a good prospect to be fur-
ther developed as natural nematicide. Moreover, since the
price of clove bud dropped from about 9 US$ kg-1 in 2001
 to about 2 US$ kg-1 in 2009 , some farmers cur-
rently neglect their plantations and do not even harvest their
plants . Therefore, new additional uses of the clove bud
as nematicide would be very welcome. Although application
of 60 g clove bud per plant, with estimated costs equivalent
to about 0.12 US$, is about 4-fold more costly than the use
of 30 g carbofuran per plant, with estimated costs equivalent
to about 0.03 US$, the use of clove mulch as a nematicide
has a good prospect as it is environmentally friendly and can
be locally produced. In addition it will induce plant produc-
tion as the result of degradation of organic materials and
suppress the development of root rot disease caused by Phy-
tophthora palmivora  because clove contains eugenol,
which acts as a potent fungicide .
To allow a successful introduction, practical information
related to the use of plant materials to effectively control
pests has to be developed and made available to the farmers.
It is expected that application of clove, as a botanical pesti-
84 The Open Natural Products Journal, 2009, Volume 2 Wiratno et al.
cide will be adopted easily by local farmers on Bangka Is-
land as in the past farmers in this region used plant materials
as pesticides . The results of the present study indicate
that once a useful recipe is developed the use of clove can
help to reduce the current intensive but not so effective use
of synthetic pesticides against nematodes, and also the con-
nected risk for human and environmental health.
Medicinal and Aromatic Crops Research Institute for provid-
ing many facilities throughout this study. We acknowledge
the help of Mr. Ma’mun BSc, and Mr. Dedy Kustiwa with
the extraction of the plant materials, and of Mr. Endang Su-
gandi and Mrs. Kurniati with the greenhouse experiment.
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Received: February 09, 2009
© Wiratno et al.; Licensee Bentham Open.
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(http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the
work is properly cited.
Revised: February 12, 2009 Accepted: May 26, 2009
access article licensed under the terms of the Creative Commons Attribution Non-Commercial License