Microbial Pathogenesis 59-60 (2013) 52e59

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Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Nematicidal activity of 3,4-dihydroxybenzoic acid purified from Terminalia

nigrovenulosa bark against Meloidogyne incognita
Dang-Minh-Chanh Nguyen a, b, Dong-Jun Seo a, Kil-Yong Kim a, Ro-Dong Park a, Dong-Hyun Kim c,
Yeon-Soo Han c, Tae-Hwan Kim d, Woo-Jin Jung a, *
a
Division of Applied Bioscience and Biotechnology, Institute of Environmentally-Friendly Agriculture (IEFA), College of Agricultural and Life Science, Chonnam National University,
Gwangju 500-757, Republic of Korea
b
Western Highlands Agriculture Forestry Science Institute, 53 Nguyen Luong Bang Street, Buon Ma Thuot, Viet Nam
c
Division of Plant Biotechnology, College of Agricultural and Life Science, Chonnam National University, Gwangju 500-757, Republic of Korea
d
Department of Animal Science, Institute of Agricultural Science and Technology, College of Agricultural and Life Science, Chonnam National University,
Gwangju 500-757, Republic of Korea

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

Article history: In this study, the 3,4-dihydroxybenzoic acid (3,4-DHBA) from Terminalia nigrovenulosa bark (TNB) was
Received 21 January 2013 purified and its in vitro nematicidal activity was investigated against Meloidogyne incognita. The purifi-
Received in revised form cation of 3,4-DHBA used a silica gel column and Sephadex LH-20 chromatography combined with thin-
5 April 2013
layer chromatography and high performance liquid chromatography. Structural identification of the 3,4-
Accepted 7 April 2013
Available online 18 April 2013
DHBA was conducted using 1H nuclear magnetic resonance (NMR), 13C NMR, and liquid chromatography
time-of-flight 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
Keywords:
Egg morphology
incubation. J2 mortality was increased significantly (P < 0.0001) with increasing incubation time in the
Fluorescence staining range of 54.2e94.2% from 3 to 9 h after incubation with 3,4-DHBA (1.0 mg/ml), but with no significant
Nematicidal compound difference observed where the incubation time was increased from 9 to 12 h. The 3,4-DHBA treatment
Plant extract 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.
Ó 2013 The authors. Published by Elsevier Ltd. Open access under CC BY-NC-ND license.

1. Introduction due to their toxic residues and associated environmental damage

The root-knot nematode, Meloidogyne incognita, is a major
plant-parasitic nematode species affecting the quantity and quality
of many annual and perennial crops [1]. Infected plants show
typical symptoms which include root galling, stunting, nutrient
deficiency and in particular, nitrogen deficiency [2,3]. These nem-
atodes cause billions of US$ in yield losses worldwide every year
[3e6]. Therefore the presence of this pest on cultivated crops must
be controlled [7e9].
Use of chemical nematicides is usually more effective than other
strategies, they have caused significant environmental problems
* Corresponding author. Tel.: þ82 62 530 3960; fax: þ82 62 530 2139.
E-mail address: woojung@chonnam.ac.kr (W.-J. Jung).
that resulted in severe restrictions on their use [10]. As general
awareness of the harmful effects of chemical pesticides increases
and public attitudes towards environmental pollution changes,
chemical nematicides are losing their popularity among farmers
[10e12]. In fact, plant-derived extracts have long been a
subject of research in an effort to develop alternatives to conven-
tional insecticides usually safer and with minimal residual effects
[13]. Several plant preparations have an environmentally and
toxicologically safe, selective, and efficacious nematicidal potential
[14e17]. These pesticidal compounds derived from plants are
generally considered to be non-persistent under field conditions, as
they are readily transformed by light, oxygen, or microorganisms
into less toxic products. Therefore, fewer residues are expected to
result from the use of these natural products [9,18e21].
Plant preparations can provide potential alternatives to the use
of synthetic nematicides, because they degrade to non-toxic
products, and have fewer side effects to non-target organisms
and within the broader environment [22]. Additionally, such agents

0882-4010 Ó 2013 The authors. Published by Elsevier Ltd. Open access under CC BY-NC-ND license.
http://dx.doi.org/10.1016/j.micpath.2013.04.005
 D.-M.-C. Nguyen et al. / Microbial Pathogenesis 59-60 (2013) 52e59 53

often act at multiple and novel target sites, thereby reducing the
potential of plant-parasitic nematodes becoming resistant to them
[22,23]. The nematicidal principles of plants, based on materials
such as isothiocyanates, thiophenics, glucosides, alkaloids, pheno-
lics, thianins and fatty acids, have been identified and reviewed
[9,13,24,25].
Plants in the genus Terminalia, family Combretaceae, comprising of
some 250 species, are widely distributed in tropical areas of the world
[26]. A methanol extract of Terminalia arjuna bark showed nematicidal
activity against Haemonchus contortus [27]. Also, ethanol extract of
Terminalia chebula fruit was toxic to both Meloidogyne spp. and
Cephalobus litoralis [28]. 3,4-dihydroxybenzoic acid (3,4-DHBA, pro-
tocatechuic acid), a phenolic compound, has been isolated from
Camellia sinensis [29], Hibiscus sabdariffa L. [30], Eucommia ulmoides
Oliv. [31] and Smilacis chinae [32]. Currently there are very few reports
regarding the nematicidal activity effects of 3,4-DHBA from Terminalia
nigrovenulosa bark (TNB) against root-knot nematode M. incognita. The
objectives of this study were 1) to purify the nematicidal compounds
from TNB, and 2) to evaluate the in vitro nematicidal activity of 3,4-
DHBA purified from TNB against M. incognita.

2. Materials and methods

2.1. Chemicals and materials

All solvents used for column chromatography were purchased

from Junsei Chemical Co. Ltd. (Tokyo, Japan). Silica gel plates F254
and silica gel (40e63 mm) were purchased from Merck. Sephadex-
LH 20 was purchased from SigmaeAldrich. T. nigrovenulosa bark
(TNB) used in this study was collected in DakLak province, Vietnam.
Fig. 1. Diagram of the extraction and purification of 3,4-DHBA from TNB.
2.2. Collection of eggs and M. incognita second-stage juveniles (J2)

M. incognita was collected from the roots of infected cucumber
plants. Briefly, the galled root samples were washed with water, cut
into pieces (4e8 mm long) and poured into a conical flask con-
taining a mixture of distilled water (950 ml) and 10% NaOCl
(50 ml). The flask was then shaken vigorously to dislodge the eggs
from the roots. Next, the egg suspension was passed through a
series of sieves with pore sizes of 45 and 25 mm, respectively.
Sterilized eggs were retained on a 25 mm sieve, refrigerated over-
night at 4  C in sterile distilled water, and were later used for 24 h
assays [33]. Sterilized eggs were incubated for 3e5 days using a
modified Baermann funnel method under sterile conditions, to
obtain J2 [34,35]. Next, the number of nematode juveniles was
adjusted to 250 J2/ml and the number of J2 was counted under a
microscope (40).
Elution was carried out using 200 ml of chloroform: methanol at
varying ratios (100:0 (F1), 90:10 (F2), 80:20 (F3), 70:30 (F4), 60:40
(F5) and 50:50 (F6)). Nematicidal activity assays were conducted
using all six fractions. The fraction that showed the high nemati-
cidal activity was further separated using chloroform: ethyl acetate
at varying ratios (90:10 (F4-1), 70:30 (F4-2) and 50:50 (F4-3)). The
fraction with the strongest nematicidal activity was further purified
by column (65 cm  1.8 cm) chromatography on Sephadex LH-
20 (stepwise gradient of 50e100% methanol) to yield 28 fractions
(1e28). The solvent-fractionized compounds were confirmed
by thin-layer chromatography (TLC) on silica gel plates F254
(20 cm  20 cm) [37] with chloroform: ethyl acetate: formic acid
(70:30:0.5, v/v/v), and the compound bands were observed under
ultraviolet light at 254 nm (Vilber Lourmat).

2.3. Extraction of nematicidal compound

The procedure used to isolate the pure compound from TNB is
shown in Fig. 1. The dried TNB (1 kg) was extracted in 80% methanol
(3  2 L) and the extracts were evaporated to dryness [36]. The
methanol extract was suspended in water and the suspension was
consecutively partitioned with n-hexane, chloroform, ethyl acetate,
and n-butanol. Equal volumes of each solvent and methanol extract
solution were mixed by shaking for 3 min in a separation funnel. The
organic solvent layers were concentrated to dryness by rotary evap-
oration at 40  C, and the water soluble fraction was freeze-dried.
2.5. High performance liquid chromatography (HPLC)
The purified fraction was tested for purity using an HPLC system
equipped with a C18 column (7.8 mm i.d.  300 mm, 10 mm Thermo
Hypersil), and acetonitrile: water (30:70, v/v) was used as the
mobile phase at a flow rate of 1 ml/min. A Dionex model PDA-100
photodiode array was used as the detector at multiple scan wave-
lengths [38].
2.6. Spectral identification of the nematicidal compound

2.4. Purification of 3,4-DHBA About 10 mg of the purified compound was dissolved in

methanol-d (CD3OD) and was then subjected to spectral analysis.
Ethyl acetate extract was showed the strongest nematicidal Nuclear magnetic resonance (NMR) spectra were recorded on a
activity. The ethyl acetate-dried extract (22.5 g) was subjected to Bruker DRX 500 NMR instrument, operating at 500 MHz for 1H
chromatography on a silica gel (40e63 mm) column (60 cm  5 cm). NMR, and 125 MHz for 13C NMR, both at room temperature.
54 D.-M.-C. Nguyen et al. / Microbial Pathogenesis 59-60 (2013) 52e59

Chemical shifts are reported in ppm (d), using CD3OD as the solvent
(unless otherwise indicated) and trimethylsilane as the internal
standard [32].
2.7. Liquid chromatography time-of-flight mass spectrometry
(LC-TOF-MS)
LC-TOF-MS analysis was performed as described earlier [39],
with minor modifications. Chromatographic separation was per-
formed on an Agilent 1100 trap mass spectrometer equipped with
an electrospray (ESI) interfere, a binary pump, column oven, and a
diode array detector (Agilent Technology, Santa Clara, CA, USA).
Chromatographic separation of the analyte of interest was achieved
on a C18 column (particle size 5 mm, 250  4.6 mm, Agilent Tech-
nology). The mobile phase was 5.0% acetonitrile/water at a flow rate
of 1.0 ml/min. The post-column splitting ratio was set to 3:1 before
the elution was transferred to ESI. Injection volume was 20 ml for all
analyses. TOF-MS was performed in negative mode using multiple
reactions monitoring (MRM). The optimum conditions of the
interface were as follows: ion spray voltage of 24,500 V, collision
gas (He) pressure of 1.3 m Torr and 40 psi, and flow rates of the
nebulizer gas (N2) and dry gas (N2) of 40 psi and 8.0 l/min,
respectively. The dry temperature was set to 350  C. Analysis was
detected by trap MS using MRM with a 100 ms dwell time. Data
were analyzed at the Korea Basic Science Institute, Chonnam Na-
tional University, Korea.
2.8. Nematicidal activity of 3,4-DHBA from the TNB
DHBA with 1.0 ml of nematode egg solution (approximately
250 eggs/ml) was incubated at 37  C with shaking. After 1 day,
nematode eggs were stained with Fluorescence Brightener 28
(Sigma) at a final concentration of 0.1 mg/ml for 5 min. The eggs
were washed twice with distilled water by centrifugation and then
observed under an Olympus Fluoview FV1000 confocal laser
scanning microscope with a 455-nm argon laser and appropriate
filter sets. Images were taken with confocal fluorescent microscopy
using Program FV10-ASW.
2.10. Statistical analyses
Data were compared using Tukey’s Studentized range (HSD)
test, with a P  0.05 indicating statistical significance. All data were
analyzed using Statistical Analysis System 9.1 [42] and are pre-
sented as mean  standard deviation.
3. Results
3.1. Purification of 3,4-DHBA from TNB
Of the five partitioned fractions tested, the ethyl acetate frac-
tion showed the highest nematicidal activity. Silica gel column
chromatography of the crude ethyl acetate extract yielded six
fractions (F1eF6). Among these, fraction 4 (F4) showed the
highest nematicidal activity significantly (93.3%, P < 0.0001)
compared to the remaining fractions (Fig. 2A). Further purification

A direct-contact bioassay was used to evaluate hatch inhibition

and J2 mortality of M. incognita by 3,4-DHBA. Approximately 225
eggs and 225 J2 in 900 ml of water was added, respectively to each
well of a 24-well MicrotestÔ tissue culture plate, and 100 ml of 3,4-
DHBA test solution was added at concentrations of 1.25, 2.5, 5.0 and
10 mg/ml. Therefore, the final 3,4-DHBA concentrations in the each
suspension were 0.125, 0.25, 0.5 and 1.0 mg/ml. Control samples
received 100 ml of methanol. The plate was covered with the orig-
inal solid lid and was wrapped with ParafilmÒ, and samples were
kept at room temperature (25  1  C). Hatch inhibition of samples
was based on the number of hatched juveniles 3 days after incu-
bation, as observed under a light microscope (40). Hatch inhibi-
tion (HI) was calculated from the formula:

HIð%Þ ¼ ½ðC  TÞ=C  100

where C is the control percentage hatch and T is the treatment

percentage hatch.
At 0, 3, 6, 9 and 12 h after incubation, dead and live nematodes
were counted using a light microscope (40) to evaluate mortality
rates. J2 mortality was estimated according to the mean percentage
of dead J2. Nematodes were considered dead when no movement
was observed during 2 s after mechanical prodding [40]. J2 mor-
tality was calculated from the formula:

J2 mortalityð%Þ ¼ ½number of dead J2=total number of J2

ðlive and dead J2Þ  100
Treatments were replicated five times, and the experiment was
repeated in duplicate.
2.9. Eggshell chitin fluorescence staining
Eggshell fluorescence staining was performed as described by
Ref. [41] with minor modifications. To estimate eggshell degrada-
tion by 3,4-DHBA, a solution containing a mixture of 1.0 mg of 3,4-
Fig. 2. Nematicidal activity of the six fractions were obtained from the ethyl acetate
fraction of TNB, by silica gel chromatography using chloroform/methanol [100:0 (F1),
90:10 (F2), 80:20 (F3), 70:30 (F4), 60:40 (F5) and 50:50 (F6)] (A); nematicidal activity
of the three fractions obtained from F4 by silica gel chromatography using chloroform/
ethyl acetate [90:10 (F4-1), 70:30 (F4-2), 50:50 (F4-3)] (B). Values are mean  SD of
three observations after 12 h incubation with 2 mg/ml. The different letters on the
error bars indicate significant difference based on Tukey’s Studentized range at
p  0.05.
 D.-M.-C. Nguyen et al. / Microbial Pathogenesis 59-60 (2013) 52e59 55

Fig. 3. TLC plates of 28 fractions (Fr 1e28) obtained from F4-2 by Sephadex LH-20 chromatography (A); J2 mortality of M. incognita reduced by fractions from Sephadex LH-20
chromatography (B). Values are mean  SD of three observations after 12 h incubation with 2 mg/ml. The different letters on the error bars indicate significant differences
based on Tukey’s Studentized range at p  0.05; HPLC of 3,4-DHBA purified from TNB (C).

of F4 yielded three fractions (F4-1, F4-2 and F4-3) and treatment Sephadex LH-20 chromatography, fractions 6 to14 showed a sin-
with two fractions (F4-1 and F4-2) resulted in >93% J2 mortality, gle spot and the same Rf value (0.74) on TLC plates (Fig. 3A). The
whereas treatment with F4-3 resulted in a much lower activity nematicidal activity of fractions 6e14 (98.3%) was significantly
(23.3%) (Fig. 2B). Among the 28 fractions obtained from F4-2 by higher than those of fractions 1e5 (81.7%) and fractions 15e28

Fig. 4. 1H NMR (A); 13

C NMR spectra of 3,4-DHBA purified from TNB (B). 1H- and 13
C- spectra were measured in CD3OD at 500 and 125 MHz, respectively.
56 D.-M.-C. Nguyen et al. / Microbial Pathogenesis 59-60 (2013) 52e59

Table 1
1 13
H- and C- NMR spectroscopic data of 3,4-DHBA from TNB.

Position dH (J in Hz) dC (ppm)

1 e 123.9
2 7.43 (s) 117.9
3 e 146.2
4 e 151.5
5 6.78 (d, J ¼ 8.4) 115.8
6 7.42 (d, J ¼ 8.4) 123.8
7 e 171.0

s: singlet, d: doublet.

(16.7%, Rf 0.83) (Fig. 3A and B). The HPLC chromatogram of frac-

tions 6e14 showed a single peak at 240 nm at a retention time of
7.7 min (Fig. 3C). A compound containing a mixture of fractions
from 6 to 14 was identified according by its 1H- and 13C NMR
spectra (Fig. 4). Peak data were as follows: 1H NMR (500 MHz,
CD3OD): dH 7.43 (1H, s, H-2), 7.42 (1H, d, J ¼ 8.4 Hz, H-6), 6.78 (1H,
d, J ¼ 8.4 Hz, H-5) (Table 1 and Fig. 4A). 13C NMR (125 MHz,
CD3OD): dC ¼ 171.0 (C]O), 151.5 (C-4), 146.2 (C-3), 123.9 (C-1),
123.8 (C-6), 117.9 (C-2) and 115.8 (C-5) (Table 1 and Fig. 4B). The
mass spectrum of purified compound showed a molecular ion m/z
at 153 (Fig. 5B) matched to mass spectrum of 3,4-DHBA at library
source (Fig. 5A). Based on these data along with a comparison
with published data [32,43], a nematicidal compound (fr. 6e14)
was identified as 3,4-dihyroxybenzoic acid (3,4-DHBA, proto-
catechuic acid).

3.2. Nematicidal activity of 3,4-DHBA from TNB

The 3,4-DHBA purified from TNB was evaluated for its

nematicidal activity using a direct-contact bioassay (Fig. 6). At 3
days after incubation with 3,4-DHBA, hatch inhibition was 33.3,
65.0, 76.7 and 85.0% at 0.125, 0.25, 0.5 and 1.0 mg/ml, respec-
tively, compare to the control (18.3%) (Fig. 6A). Hatch inhibition
increased significantly (P < 0.0001) with increasing concentra-
tion, indicating that hatch inhibition of the 3,4-DHBA was dose-
Fig. 6. Nematicidal activity of 3,4-DHBA purified from TNB at various concentrations
dependent. Also, evaluation of 3,4-DHBA obtained from the (0, 0.125, 0.25, 0.5, and 1.0 mg/ml). Hatch inhibition (%) of M. incognita induced by 3,4-
TNB exposed differences in toxicity against M. incognita J2 DHBA after 3 day incubations (A); Juvenile mortality induced by the 3,4-DHBA after 0,
(Fig. 6B). Briefly, J2 mortality was in the range of 7.5e54.2% after 3, 6, 9, and 12 h incubation (B). Values are mean  SD. The different letters on the error
bars indicate significant differences based on Tukey’s Studentized range at p  0.05.

Fig. 5. Liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) of 3,4-DHBA. (A) TOF-MS of 3,4-DHBA at library source. (B) TOF-MS of 3,4-DHBA purified from TNB.
TOF-MS was performed in negative mode for m/z 153.
 D.-M.-C. Nguyen et al. / Microbial Pathogenesis 59-60 (2013) 52e59 57

3 h incubation, depending on the concentration which was used. 4. Discussion

While J2 mortality was in the range of 33.3e94.2% after 12 h
incubations with 3,4-DHBA (Fig. 6B). Overall, J2 mortality
increased with increasing concentration, indicating that J2 mor-
tality was dependent on both treated concentration and incu-
bation time of 3,4-DHBA.
3.3. Effect of 3,4-DHBA on egg shape
Changes in the shape of M. incognita eggs were observed using
light and fluorescence microscopy after 1 day incubation with
3,4-DHBA (1 mg/ml) (Fig. 7). Normal eggshell shapes were
observed in the water control (Fig. 7A1-3 and B1-3). Deformation of
the egg shapes occurred after 1 day incubation (Fig. 7C1-3 and D1-
3). Eggshell disruption by 3,4-DHBA was more clearly demon-
strated by fluorescence dyeing of the eggshell surface (Fig. 7D1-3).
Staining of eggs with Fluorescence Brightener 28 resulted in a
uniform distribution of the outer-most chitin layer in the water
control (Fig. 7A), whereas the eggshell surface became slightly
uneven and deformed after 1 day incubation with 3,4-DHBA
(Fig. 7D1-3). In particular, the inside layer of the eggs appeared to
be destroyed after incubation with 3,4-DHBA (Fig. 7C2-3 and D2-3),
however, early stage juveniles were observed in the water control
(Fig. 7A3 and B3).
The nematicidal activity of plant preparations such as medicinal
plants and herb extractions against Meloidogyne spp., has been
demonstrated [1,8,17,28,33,44e47]. Aoudia et al. [9] suggested that
phenolic compounds isolated from Melia azedarach as gallic acid,
protocatechin, ferulic acid, epicatechin, p-hydroxybenzoic acid and
caffeic acid against M. incognita. 3,4-DHBA from plants has mixed
effects on normal and cancer cells in in vitro and in vivo studies [48].
A 3,4-DHBA exhibits anti-inflammatory and antitumor promotion
effects as a strong antioxidant [30,31]. In an in vitro model using
HL-60leukemia (Human promyelocytic leukemia) cells, 3,4-DHBA
showed an antigenotoxic effect and tumoricidal activity [49].
Many compounds from medicinal plants, such ethyl trans-cin-
namate and ethyl p-methoxycinnamate isolated from Kaempferia
galangal, have shown high nematicidal activity against Bursaphe-
lenchus xylophilus and M. incognita [46,50,51]. Ntalli and Caboni [17]
reviewed that botanical nematicides could be promising tools in
Meloidogyne sp. control. In our previous reports, cinnamyl acetate
purified from Cinnamomum aromaticum showed that M. incognita
J2 movement inhibition was 100% after 50 min incubation with
100 mg/ml [47]. Also, the mortality of M. incognita J2 was 100% after
12 h incubation with 1.0 mg/ml of gallic acid purified from TNB [52].
According to Sultana et al. [53], 3,5-DHBA purified from the aerial

Fig. 7. Staining of M. incognita eggs with Fluorescence Brightener 28 after various treatments. Egg shapes viewed under light microscope (A); egg shapes in the water control
stained with Fluorescence Brightener 28 and viewed under fluorescence microscope (B); egg shapes treated with 3,4-DHBA viewed under light microscopy (C); egg shapes treated
with 3,4-DHBA and stained with Fluorescence Brightener 28 viewed under fluorescence microscopy (D).
58 D.-M.-C. Nguyen et al. / Microbial Pathogenesis 59-60 (2013) 52e59
parts of Rubus niveus show strong nematicidal activity against
M. incognita, exhibiting 100% mortality of M. incognita after 48 h at
5 mg/ml. In this study, nematicidal activity was also determined for
the 3,4-DHBA purified from TNB. This is the first report of its pu-
rification from this species. Briefly, the mortality of M. incognita J2
was 94.2% after 12 h incubation with 1.0 mg/ml of 3,4-DHBA pu-
rified from TNB (Fig. 6B). The egg stage is the most resistant stage in
the nematode’s life cycle, possibly due to its three-layer shell, and
particularly, due to the inner lipo-protein layer [54]. Purified 3,4-
DHBA strongly inhibited (85.0%) hatch of M. incognita after 3 days
incubation with 1.0 mg/ml (Fig. 6A). It seems that the 3,4-DHBA
purified from TNB plays an important role for the inhibition of
egg hatching. Thus 3,4-DHBA and TNB extracts might be beneficial
as potent nematode inhibitors under specified conditions.
The morphology of eggshells covered with a chitin layer in
M. incognita was observed under light and fluorescence microscopy
after 3,4-DHBA treatment (Fig. 7). The presence of chitin in nem-
atodes has been documented in a variety of different nematode
species and is also a common structural component of the nema-
tode eggshell [55e57]. The eggs are surrounded by an eggshell, and
strength is provided by a chitinous layer [41]. Normal M. incognita
egg shapes occur at a variety of different developmental stages. The
presence of chitin in M. incognita eggs was easily detected with a
fluorescence dye, especially during the early stages of develop-
ment. Additionally, structural changes have been suggested by
disintegration of the chitin layer of the eggshells [58].
The results of this study indicate that TNB methanol extracts,
particularly 3,4-DHBA, could be useful as nematicides for use
against juveniles of M. incognita. This is a research area in which the
results of many studies can be utilized for the benefit of phytone-
matode control. Overall, the use of compounds from medicinal
plant extracts will lead to a decreased use of chemical pesticides,
and hence, to a healthier environment. However, further studies are
needed to evaluate the nematicidal effects under field conditions
for practical use of TNB extracts as novel nematicides or hatching
inhibitors. If commercial bionematicides are to be used as biological
control agents for soil-borne phytopathogens, formulations that
improve their nematicidal potency and stability must be developed
to reduce costs, and to make them practical for field use.
Acknowledgments
This research was supported by Bio-industry Technology
Development Program, Ministry for Food, Agriculture, Forestry, and
Fisheries, Republic of Korea.
References
[1] Taniwiryonoc Wiratno D, Van Den Berg H, Riksen JAG, Rietjens IMCM,
Djiwanti SR, Kammenga JE, et al. Nematicidal activity of plant extracts
against the root-knot nematode, Meloidogyne incognita. Open Nat Prod J
2009;2:77e85.
[2] Siddiqui ZA, Iqbal A, Mahmood I. Effects of Pseudomonas fluorescens and fer-
tilizers on the reproduction of Meloidogyne incognita and growth of tomato.
Appl Soil Ecol 2001;16:179e85.
[3] Adekunle OK, Akinlua A. Nematicidal effects of Leucaena leucocephala and
Gliricidia sepium extracts on Meloidogyne incognita infecting okra. J Agric Sci
2007;52:53e63.
[4] Oka Y, Koltai H, Bar Eyal M, Mor M, Sharon E, Chet I, et al. New strategies for
the control of plant-parasitic nematodes. Pest Manag Sci 2000;56:983e8.
[5] Williamson VM, Kumar A. Nematode resistance in plants: the battle under-
ground. Trends Genet 2006;22:396e403.
[6] Reynolds AM, Dutta TK, Curtis RHC, Powers SJ, Gaur HS, Kerry BR. Chemotaxis
can take plant-parasitic nematodes to the source of a chemo-attractant via the
shortest possible routes. J R Soc Interface 2011;8:568e77.
[7] Noling JW, Becker JO. The challenge of research and extension to define and
implement alternatives to methyl bromide. J Nematol 1994;26:573e86.
[8] Chitwood DJ. Phytochemical based strategies for nematode control. Annu Rev
Phytopathol 2002;40:221e49.
[9] Aoudia H, Ntalli N, Aissani N, Yahiaoui-Zaidi R, Caboni P. Nematotoxic
phenolic compounds from Melia azedarach against Meloidogyne incognita.
J Agric Food Chem 2012;60:11675e80.
[10] Harish S, Saravanakumar D, Radjacommare R, Ebenezar EG, Seetharaman K.
Use of plant extracts and biocontrol agents for the management of brown spot
disease in rice. BioControl 2008;53:555e67.
[11] Pandey R, Kalra A, Tandon S, Mehrotra N, Singh HN, Kumar S. Essential oils as
potent sources of nematicidal compounds. J Phytopathol 2000;148:501e2.
[12] Elbadri GA, Lee DW, Park JC, Yu HB, Choo HY. Evaluation of various plant
extracts for their nematicidal efficacies against juveniles of Meloidogyne
incognita. J Asia Pacific Entomol 2008;11:99e102.
[13] Ntalli NG, Menkissoglu-Spiroudi U, Giannakou I. Nematicidal activity of
powder and extracts of Melia azedarach fruits against Meloidogyne incognita.
Ann Appl Biol 2010;156:309e17.
[14] Javed N, Gowen SR, Inam Ul Haq M, Abdullah K, Shahina F. Systemic and
persistent effect of neem (Azadirachta indica) formulations against root-knot
nematodes, Meloidogyne javanica and their storage life. Crop Prot 2006;26:
911e6.
[15] Dawar S, Sattar A, Zaki MJ. Seed dressing with biocontrol agents and nema-
ticides for the control of root knot nematode on sunflower and okra. Pak J Bot
2008;40:2683e91.
[16] Ntalli NG, Menkisoglou-Spiroudi U, Giannakou IO, Prophetou-Athanasiadou DA.
Efficacy evaluation of a neem (Azadirachta indica A. Juss) formulation against
root-knot nematodes Meloidogyne incognita. Crop Prot 2009;28:489e94.
[17] Ntalli NG, Caboni P. Botanical nematicides: a review. J Agric Food Chem
2012;60:9929e40.
[18] Akhtar Y, Yeoung R, Isman MB. Comparative bioactivity of selected extracts
from Meliaceae and some commercial botanical insecticides against two
noctuid caterpillars, Trichoplusia ni and Pseudaletia unipuncta. Phytochem Rev
2008;7:77e88.
[19] Ntalli NG, Ferrari F, Giannakou I, Menkissoglu-Spiroudi U. Phytochemistry and
nematicidal activity of the essential oils from 8 Greek Lamiaceae aromatic
plants and 13 terpene components. J Agric Food Chem 2010;58:7856e63.
[20] Ntalli NG, Ferrari F, Giannakou I, Menkissoglu-Spiroudi U. Synergistic and
antagonistic interactions of terpenes against Meloidogyne incognita and the
nematicidal activity of essential oils from seven plants indigenous to Greece.
Pest Manag Sci 2010;67:341e51.
[21] Caboni P, Ntalli NG, Aissani N, Cavoski I, Angioni A. Nematicidal Activity of
(E, E)-2,4-Decadienal and (E)-2-Decenal from Ailanthus altissima against
Meloidogyne javanica. J Agric Food Chem 2012;60:1146e51.
[22] Isman MB. Botanical insecticides, deterrents, and repellents in modern agricul-
ture and an increasingly regulated world. Annu Rev Entomol 2006;51:45e66.
[23] Isman MB. Plant essential oils for pests and diseases management. Crop Prot
2000;19:603e8.
[24] Fatoki OK, Fawole B. Identification of nematicidal ingredients from neem
leaves, Siam weed leaves and roots. Afr J Plant Prot 2000;10:33e8.
[25] Cavoski I, Chami Z, Bouzebboudj F, Sasanelli N, Simeone V, Mondelli D, et al.
Melia azedarach controls Meloidogyne incognita and triggers plant defence
mechanisms on cucumber. Crop Prot 2012;35:85e90.
[26] Fabry W, Okemo PO, Ansorg R. Antibacterial activity of East African medicinal
plants. J Ethnopharmacol 1998;60:79e84.
[27] Bachaya HA, Iqbal Z, Khan MN, Jabbar A, Gilani AH, Din IU. In vitro and in vivo
anthelmintic activity of Terminalia arjuna bark. Int Agric Biol 2009;11:273e8.
[28] Zia-Ul-Haq M, Ahmad M, Akhter M. Nematicidal activity of selected flora of
Pakistan. Pak J Bot 2010;42:2119e23.
[29] Liu CL, Wang JM, Chu CY. In vivo protective effect of protocatechuic acid on
tert-butyl hydroperoxide-induced rat hepatotoxicity. Food Chem Toxicol
2002;40:635e41.
[30] Tseng TH, Hsu JD, Lo MH, Chu CY, Chou FP, Huang CL, et al. Inhibitory effect of
Hibiscus protocatechuic acid on tumor promotion in mouse skin. Cancer Lett
1998;126:199e207.
[31] Yen GC, Hsieh CL. Reactive oxygen species scavenging activity of Duzhong
(Eucommia ulmoides Oliv.) and its active compounds. J Agric Food Chem
2000;48:3431e6.
[32] Ban JY, Cho SO, Jeon SY, Bae KH, Song KS, Seong YH. 3,4-dihydroxybenzoic
acid from Smilacis chinae rhizome protects amyloid b protein (25-35)-induced
neurotoxicity in cultured rat cortical neurons. Neurosci Lett 2007;420:184e8.
[33] Zasada IA, Klassen W, Meyer SLF, Codallo M, Abdul Baki AA. Velvetbean
(Mucuna pruriens) extracts: impact on Meloidogyne incognita survival and on
Lycopersicon esculentum and Lactuca sativa germination and growth. Pest
Manag Sci 2006;62:1122e7.
[34] Bakker KR. Nematode extraction and bioassays. In: Barker KR, Carter CC,
Sasser JN, editors. An advanced treatise on Meloidogyne, vol. 2. Raleigh, NC,
USA: North Carolina State University; 1985. p. 19e35.
[35] Southey JF. In laboratory methods for work with plant and soil nematodes. 6th
ed. London, UK: Ministry of Agriculture, Fisheries and Food; 1986. p. 202.
Reference Book 402.
[36] Zhang S, Bi H, Liu C. Extraction of bio-active components from Rhodiola
sachalinensis under ultrahigh hydrostatic pressure. Sep Purif Technol 2007;57:
275e80.
[37] Wang W, Ben Daniel BH, Cohen Y. Control of plant diseases by extracts of Inula
viscosa. Phytopathology 2004;94:1042e7.
[38] Nadarassan DK, Chrystyn H, Clark BJ, Assi KH. Validation of high-performance
liquid chromatography assay for quantification of formoterol in urine samples
after inhalation using UV detection technique. J Chromatogr B 2007;850:31e7.
 D.-M.-C. Nguyen et al. / Microbial Pathogenesis 59-60 (2013) 52e59
[39] Lee HK, Ho CS, Iu YPH, Lai PSJ, Shek CC, Yun-Chuen L, et al. Development of a
broad toxicological screening technique for urine using ultra-performance
liquid chromatography and time-of-flight mass spectrometry. Anal Chim
Acta 2009;649:80e90.
[40] Cayrol JC, Djian C, Pijarowski L. Study on the nematicidal properties of the
culture filtrate of the nematophagus fungus Paecilomyces lilacinus. Rev
Nématol 1989;12:331e6.
[41] Fanelli E, Di Vito M, Jones JT, De Giorgi C. Analysis of chitin synthase function
in a plant parasitic nematode, Meloidogyne artiellia, using RNAi. Gene
2005;349:87e95.
[42] SAS Institute. SAS/STAT user’s guide, version 9.1. Cary, NC, USA 2004.
[43] Scott KN. Carbon-13 nuclear magnetic resonance of biologically important
aromatic acids. I. Chemical shifts of benzoic acid and derivatives. J Am Chem
Soc 1972;94:8564e8.
[44] Oka Y. Nematicidal activity of essential oils and their components against the
root-knot nematode. Nematology 2000;90:710e5.
[45] Ntalli NG, Vargiu S, Menkissoglu-Spiroudi U, Caboni P. Nematicidal carboxylic
acids and aldehydes from Melia azedarach fruits. J Agric Food Chem 2010;58:
11390e4.
[46] Hong TK, Kim SI, Heo JW, Lee JK, Choi DR, Ahn YJ. Toxicity of Kaempferia
galanga rhizome constituents to Meloidogyne incognita juveniles and eggs.
Nematology 2011;13:235e44.
[47] Nguyen DMC, Seo DJ, Kim KY, Kim TH, Jung WJ. Nematode-antagonistic effects
of Cinnamomum aromaticum extracts and a purified compound against
Meloidogyne incognita. Nematology 2012;8:913e24.
[48] Lin HH, Chen JH, Huang CC, Wang CJ. Apoptotic effect of 3,4 dihydroxybenzoic
acid on human gastric carcinoma cells involving JNK/p38 MAPK signalling
activation. Int J Cancer 2007;120:2306e16.
59
[49] Anter J, Romero-Jimenez M, Fernandez-Bedmar Z, Villatoro-Pulido M, Analla M,
Alonso-Moraga A, et al. Antigenotoxicity, cytotoxicity, and apoptosis induction
by apigenin, bisabolol, and protocatechuic acid. J Med Food 2011;14:276e83.
[50] Choi IH, Park JY, Shin SC, Park IK. Nematicidal activity of medicinal plant
extracts and two cinnamates isolated from Kaempferia galanga L. (Proh Hom)
against the pine wood nematode, Bursaphelenchus xylophilus. Nematology
2006;8:359e65.
[51] Kong JO, Lee SM, Moon YS, Lee SG, Ahn YJ. Nematicidal activity of cassia and
cinnamon oil compounds and related compounds toward Bursaphelenchus
xylophilus (Nematoda: Parasitaphelenchidae). J Nematol 2007;31:31e6.
[52] Nguyen DMC, Seo DJ, Nguyen VN, Kim KY, Park RD, Jung WJ. Nematicidal
activity of gallic acid purified from Terminalia nigrovenulosa bark against the
root-knot nematode Meloidogyne incognita. Nematology 2013. http://
dx.doi.org/10.1163/15685411-00002696.
[53] Sultana N, Akhter M, Khaton Z. Nematicidal natural products from the aerial
parts of Rubus niveus. Nat Prod Res 2010;24:407e15.
[54] Wharton DA. Nematode survival strategies. In: Lee DL, editor. The biology of
nematodes. New York, NY, USA: Taylor and Francis; 2002. p. 389e411.
[55] Wharton D. Nematode eggshells. Parasitology 1980;81:447e63.
[56] Veronico P, Gray LJ, Jones JT, Bazzicalupo P, Arbucci S, Cortese MR, et al.
Nematode chitin synthases: gene structure, expression and function in Cae-
norhabditis elegans and the plant parasitic nematode Meloidogyne artiellia.
Mol Gen Genet 2001;266:28e34.
[57] Harris MT, Fuhrman JA. Structure and expression of chitin synthase in
the parasitic nematode Dirofilaria immitis. Mol Biochem Parasitol 2002;122:
231e4.
[58] Bird AF, Self PG. Chitin in Meloidogyne javanica. Fund Appl Nematol 1995;18:
235e9.