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Crop Protection 81 (2016) 138e144

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Crop Protection
journal homepage: www.elsevier.com/locate/cropro

Cis- and trans-protopinium, a novel nematicide, for the eco-friendly


management of root-knot nematodes
Ishrat Naz a, *, Saifullah Abdulka a, I. Munir b, M. Ahmad a, A. Ali a, A. Sultan c,
J.E. Palomares-Rius d, S. Ali e, I. Ahmad a
a

Department of Plant Pathology, The University of Agriculture, Peshawar, 25130, Khyber Pakhtunkhwa, Pakistan
Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar, 25130, Khyber Pakhtunkhwa, Pakistan
Department of Poultry Sciences, The University of Agriculture, Peshawar
d
Department of Crop Protection, Institute for Sustainable Agriculture (IAS), Consejo Superior de Investigaciones Cientcas (CSIC), Apartado 4084, 14080
rdoba, Spain
Co
e
Phytopharmaceutical and Nutraceutical Research Lab, Institute of Chemical Sciences, University of Peshawar, Peshawar, Pakistan
b
c

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 25 February 2015
Received in revised form
28 November 2015
Accepted 15 December 2015
Available online 5 January 2016

Plant-derived nematicides are being increasingly used while select synthetic nematicides are phased out
for environmental concerns. This is the rst report on the in vitro nematicidal activity of cis- and transprotopinium isolated from roots of Fumaria parviora and its in vivo effect on the southern root-knot
nematode Meloidogyne incognita. Cis- and trans-protopinium was isolated from the methanolic fraction FM2.1, and its structure elucidated using 13C and 1H nuclear magnetic resonance (NMR). The NMR
spectra were characterized using deuterated dimethyl sulfoxide (DMSO) at temperatures of 25 and 80  C.
In an in vitro study, over 120 h of incubation, the area under cumulative percent hatch inhibition and
mortality of cis- and trans-protopinium reached 100% at a concentration of 200 mg mL1. In the greenhouse and eld settings, cis- and trans-protopinium was evaluated against M. incognita on tomato at a
concentration of 100, 200, and 300 mg mL1 for two consecutive seasons, that is, spring and autumn, in
2010. At a concentration of 300 mg mL1 in the greenhouse and eld trials during spring and autumn, cisand trans-protopinium signicantly reduced the nematode galling index, the number of females per
gram of root, and the reproduction factor, as well as increased plant height, fresh and dry shoot weights,
and root length. Therefore, cis- and trans-protopinium can be used as an effective and safe nematicide
against M. incognita on tomato in an organic and sustainable agricultural production system. Phytochemicals have various agricultural applications, especially to control economically important nematode
pests.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Cis- and trans-protopinium
Fumaria parviora
Nematicidal activity
Meloidogyne incognita
Integrated disease management
Greenhouse
Tomato

1. Introduction
Tomato (Solanum lycopersicum L.) is an important crop in tropical and subtropical countries, and root-knot nematodes of the
genus Meloidogyne are pests that reduce tomato yields signicantly
(Luc et al., 2005). The southern root-knot nematode M. incognita
causes extensive damage to and signicantly reduces the yield of
tomato in Pakistan, especially in the Khyber Pakhtunkhwa province
(Naz et al., 2013a). The indiscriminate use of synthetic nematicides
has caused environmental and health problems, leading to a ban on
most of these nematicides (Backman, 1997).

* Corresponding author.
E-mail address: ishratro@hotmail.com (I. Naz).
http://dx.doi.org/10.1016/j.cropro.2015.12.006
0261-2194/ 2015 Elsevier Ltd. All rights reserved.

Nematologists continue to develop control methods, particularly focusing on endoparasitic nematodes such as root-knot and
cyst nematodes (Roberts, 1995). Plant-derived secondary metabolites and their derivatives have been proposed as novel chemicals
for eliminating nematode pests; alkaloids, avonoids, glycosides,
tannins, saponins, steroids, phenols, and many essential oils have
been investigated in this respect (Chitwood, 2002). More than
30,000 secondary plant metabolites have been reported (Harborne,
1998); further, a variety of phytochemicals from several families
(such as Meliaceae, Asteraceae, Myrtaceae, Lauraceae, Lamiaceae,
and others) have been used for insecticidal, acaricidal, antibacterial,
antifungal, and anti-nematodal applications (Chitwood, 2002).
Many plants with known anti-nematodal properties may be used as
green or dry amendments or plant-derived natural products (biopesticides) to minimize the populations of root-knot nematodes in

I. Naz et al. / Crop Protection 81 (2016) 138e144

agriculture (Chitwood, 2002). Most of these plants can be safely


used in integrated disease management (IDM) and integrated pest
management (IPM), as phytochemicals are relatively safer and
more eco-friendly, pose lesser risks to human and animal health,
have a selective mode of action, and will prevent the buildup of
resistance in pests (Isman, 2006).
We have already reported the nematicidal activity of extracts
from roots and stem of Fumaria parviora Lam., identifying seven
classes of bioactive compounds. We found that the n-hexane and
methanolic (MeOH) root extracts showed the strongest nematicidal
activity against egg-hatching and juvenile (J2) viability in both the
in vitro and in planta experiments at different concentrations (Naz
et al., 2013a). The secondary metabolites of F. parviora are reported
to affect bacteria, fungi, viruses, and gastrointestinal nematodes
(Orhan et al., 2007; Al-Shaibani et al., 2009). To the best of our
knowledge, alkaloids of F. parviora have not been investigated for
their nematicidal activity in the laboratory, greenhouse, and more
importantly the eld setting on tomato. We report on the following
points in the present paper: a) the isolation of cis- and trans-protopinium from the MeOH root extracts and the elucidation of its
structure using spectra obtained at 25 and 80  C, b) the in vitro
nematicidal effect of the alkaloid cis- and trans-protopinium of F.
parviora against M. incognita, and c) the in planta nematicidal
effect against M. incognita on tomato under articially inoculated
greenhouse and naturally infested eld conditions.

2. Materials and methods


2.1. Chemicals
The solvents used were chloroform, ethyl acetate, and methanol
after distillation for column chromatography. The dried methanolic
root fractions/compound was dissolved in dimethyl sulfoxide
(DMSO, 99.9%, Merck, Germany).

2.2. General techniques for extraction, isolation, and identication


of the compound
All fractions were ltered through Whatman No. 1 (10-mm) lter
paper (Sigma-Aldrich) and dried at 60  C in a rotary evaporator
(Heidolph, Schwabach, Germany). For column chromatography
(CC), the stationary phase was silica gel (Si 60, 200 mesh, Sigma
Aldrich) and the mobile-phase solvents were MeOH (methanol),
EtOAC (ethyl acetate), and CHCl3 (chloroform). For thin-layer
chromatography (TLC), TLC plates (Merck Silica gel 60 F254) and
glass plates (20  20 cm, 10-mm pore size, Merck, Darmstadt, Germany) were used. The alkaloids in MeOH fractions were detected
by spraying TLC plates with Dragendorff's reagents (solution of
bismuth nitrate potassium iodide). The retardation factor values
were obtained. The melting points were determined in a glass
capillary (M-560, BCHI, Flawil, Switzerland), which were left uncorrected. Mass spectra (by electron ionization mass spectrometry
(EIMS)) were obtained at the James Hutton Institute (JHI), Scotland,
on JEOL MSRoute, using a direct insertion probe. Spectroscopic data
(1H and 13C NMR spectra) were recorded in deuterated DMSO at 25
and 80  C on a Bruker AVANCE 400 instrument, and the J values
were measured in Hertz. Carbon atoms were assigned by a combination of DEPTq135 and heteronuclear single-quantum coherence (HSQC) experiments. A combination of 1-D and 2-D NMR
experiments was conducted, and the spectra were processed by
Bruker TOPSPIN software. 1He1H and 1He13C NMR correlations
were established by standard procedures (Naz et al., 2013b).

139

2.3. Plant collection, column chromatography, and activity-guided


fractionation
Mature F. parviora plants were collected in February and March
of 2009 from wheat elds at the Agriculture Research Farms,
Peshawar. The plants were identied and deposited in the herbarium (voucher specimen; No. ISH-1732) of the Weed Science
Department, the University of Agriculture (Naz et al., 2013a). Roots
(1 kg) of full-grown F. parviora plants were washed in tap water
and freeze-dried at 80  C for 24 h. The roots were lyophilized and
crushed in a grinder to around 1 mm, and then extracted with
methanol for 4 h in a Soxhlet apparatus. The methanol extract from
22.0 g of roots was dried in vacuo and extracted with 10% aqueous
hydrochloric acid. This was further extracted with EtOAC to give
two layers, that is, EtOAC (FM1, 3.0 g) and aqueous layers. The
aqueous layer was basied with ammonia hydroxide (NH4OH) to a
pH of 8.0, followed by extraction with EtOAC to give FM2 (4.0 g,
alkaloids) and the aqueous layer FM3 (120.0 mg). The FM1 and FM2
layers were combined and dissolved in CHCl3, then subject to column chromatography over silica gel, and eluted with solvents of
increasing polarity to afford a major fraction FM2.1. The elutes from
MeOHeCH3Cl (1:17) of FM2.1 produced an alkaloid after crystallization from methanol as a colorless amorphous powder. Its structure was determined as cis- and trans-protopinium based on
spectroscopic data and comparisons with the literature (Tousek
et al., 2005). A nematicidal bioassay was conducted using microwell plates, as described by Naz et al. (2013a).
2.4. Maintenance of M. incognita and preparation of stock solutions
Females of M. incognita were harvested from roots of susceptible
tomato (S. lycopersicum) cv. Moneymaker 2e4 months after inoculation. The eggs used in the bioassay were extracted using a 1%
NaOCl solution (Naz et al., 2013a), and the concentration of the egg
suspension was adjusted to 1000 50 eggs mL1. For the bioassay,
J2s from freshly extracted eggs were separated after 48 h and
concentrated to a nal optimized concentration of 200 J2s mL1,
according to the reported procedures (Naz et al. 2013a). The J2 larvae
and eggs of M. incognita were subjected to a microwell bioassay
following the standard procedures (Naz et al., 2013a). The stock
solution (5 mg mL1) was prepared by reconstituting cis- and transprotopinium in DMSO (1% v/v), and further dilutions were made in
distilled water. A total of four nal concentrations (i.e., 50, 100, 150,
and 200 mg mL1) of cis- and trans-protopinium were prepared (Naz
et al., 2013b). The nematode eggs were disinfected with 1% NaOCl for
5 min and rinsed on an aperture sieve (36 mm) with distilled water
(Meyer et al., 2004). In an assay, the eggs (1000 50) and J2s (200) of
M. incognita were used in a nal volume of 1 mL at nal concentration. Simple distilled water (SDW) dissolved in 1% DMSO was
used as the control. The experiments were performed twice under
identical conditions in a completely randomized design and replicated ve times. Finally, the microwell plates were incubated under
humidied conditions at 25  C (2  C) for 3 days in the dark. Unhatched eggs and J2 larvae in each well were recorded after a range
of incubation hours (i.e., 24, 48, 72, 96, and 120 h) (Naz et al., 2013b).
Both the eggs and larvae were transferred to SDW for 24 h to observe
their reclamation. Motionless juveniles with straight posture were
declared as dead (Naz et al., 2013a). The percentage of unhatched
eggs and J2 mortality in each well was calculated (Naz et al., 2013b).
Data were statistically analyzed by analysis of variance (ANOVA)
using Statistix (NH Analytical Software, Roseville, MN, USA). The
area under cumulative number of nematode percentage hatch inhibition (AUCPHI) and mortality (AUCPM) were estimated by trapezoidal integration (Campbell and Madden, 1990) for in vitro
experiments on the hatching and mortality of J2s exposed to

140

I. Naz et al. / Crop Protection 81 (2016) 138e144

different concentrations of cis- and trans-protopinium.


2.5. In planta green house experiments using various
concentrations of cis- and trans-protopinium during the spring and
autumn of 2010
Separate pot experiments were conducted (at 24e28  C) under
greenhouse conditions at the Department of Plant Pathology, the
University of Agriculture, Peshawar, during the spring and autumn
of 2010. The properties of greenhouse soils are listed in Table 1. Airdried greenhouse soil (100 g) was crushed to a size of ~1 mm and
analyzed for its chemical and physical properties. The electrical
conductivity (EC) and pH were measured using an EC meter (HM
digital EC3) and a soil pH meter (Lutron pH-212), respectively, in
soil/water in a ratio of 1:10 (Rhoades, 1996). Soil organic matter and
physical analyses were conducted at the Pesticide Residue Laboratory at the Agricultural Research Institute (ARI), Tarnab, Peshawar. For the greenhouse experiments, 15-cm clay pots were used,
each carrying 1 kg of steam-sterilized and air-dried soil. The soil
used was a mixture of sand and clay loam in a ratio of 1:1 (v/v). For
the greenhouse experiments, nematode eggs were obtained from
the roots of 70-day-old susceptible tomato (cv. Moneymaker)
plants as mentioned earlier. Two-week-old tomato seedlings were
inoculated with 5000 10 eggs J2s of M. incognita using a sterilized micropipette. A stock solution of cis- and trans-protopinium
(1 mg mL1 initially reconstituted in DMSO; 1% v/v) was used to
prepare the three concentrations of 100, 200, and 300 mg mL1.
Each concentration (50 mL) was applied as a soil drench to the
rhizosphere of tomato root 2 days post inoculation. The plants
irrigated with 45 mL of distilled water and 5 mL of DMSO (1.0%)
were used as controls (Check plants). The experiments were
completely randomized and replicated 10 times, each replicate
being a single potted plant. The experiment was conducted once in
the spring and autumn of 2010. All pots were watered with 350 mL
of fresh water three times a week (Naz et al., 2013a), and they were
fertilized weekly with the recommended doses of nitrogen, phosphorous, and potassium fertilizers (Heitz et al., 2011). The experiments were terminated 65 days after inoculation, when all plants
were grown and exhibited typical aboveground disease symptoms
(i.e., yellowing and stunting). The following variables were
assessed: root galling index (GI), number of females per gram of the
root, reproduction factor (Rf nal population/initial population),
plant height (cm), fresh and dry shoot weights (g), and fresh root
weight (g), and length (cm). The root GI was assessed under a
stereomicroscope using a 0e5 galling scale (Taylor and Sasser,
1978). Females and eggs were extracted from 1 g of roots (Naz
et al., 2013b).
2.6. Bare-root dip of tomato using various concentrations of cisand trans-protopinium during the spring and autumn of 2010 in
Dargai elds
Field experiments were conducted in Dargai in the spring and
autumn of 2010 under naturally infested eld conditions (M.

incognita). The chemical and physical properties of eld soils were


determined in the Pesticide Residue Laboratory at the ARI, Tarnab,
Peshawar, as described earlier (Table 1). The seedling bare-root dip
technique (Ahmad and Alam, 1997) was used to evaluate the efcacy of cis- and trans-protopinium at three different concentrations
against M. incognita with nematode population levels of 3e4 juveniles g1 of soil during the spring and autumn of 2010 in naturally infested soil. Nematodes were speciated in terms of female
perineal patterns; morphology; and morphometric measurements
of juveniles, males, and females obtained from previous tomato
crops. Three-week-old susceptible tomato seedlings (cv. Moneymaker) raised in sterilized (100  C for 6 h) nursery beds were
dipped in different concentrations (i.e., 100, 200, and 300 mg mL1)
of trans-protopinium for 6 h (Vadhera et al., 2000). Tomato seedlings used as controls were dipped in SDW for the same duration.
Each treatment was replicated 10 times in a randomized complete
block design. The treated seedlings were transplanted in Dargai
eld plots, each measuring 2  4 m. The eld consisted of 15 plants/
plot with a plant (PeP) and row (ReR) spacing of 30 and 60 cm,
respectively. Routine agronomic practices such as irrigation and
earthing up were performed during the experiments, and all weeds
were managed manually. Farm yard manure at 8 ton ha1 and
recommended doses of nitrogen, phosphorus, and potassium (NPK)
were added at 100, 50, and 50 kg ha1, respectively (Vadhera et al.,
2000). Forty-ve days post transplanting, the tomato roots were
randomly collected from a depth of 10e15 cm from 15 plants in
each plot using a garden spade. The roots were washed gently to
remove soil, and nematode females were counted with the aid of a
stereoscopic microscope. The following variables were assessed: GI,
Rf, females per gram of root, plant height (cm), fresh and dry shoot
weights (g), fresh root weight (g), root length (cm), and yield (kg/
plot). Yield data were recorded using a total of 12 pickings per
season. GI was assessed under a stereomicroscope using a 0e5
galling scale, as described by Taylor and Sasser (1978).
2.7. Statistical analysis
The recorded data were analyzed by ANOVA using Statistix (NH
Analytical Software, Roseville, MN, USA). Fisher's protected least
signicant difference test (LSD) at P 0.05 was used to compare the
treatment means (Gomez and Gomez, 1984).
3. Results
3.1. Characterization of cis- and trans-protopinium
The obtained alkaloid was a mixture of cis- and trans-protopinium, which was characterized using deuterated DMSO at 25 and
80  C. Cis- and trans-protopinium appeared at a retardation factor
value of 0.36 on TLC. It was obtained as a white amorphous powder
(MeOH) with a melting point of 207e208  C (Lit. m.p. 207  C)
(Fig. 1). Molecular formula C20H19NO5, ESI m/z: 354.1329. UV lmax
nm (MeOH) 240, 290; IR n cm1 3424 (OH str.), 2911 (CH str.
saturated), 1605 (C]C str. aromatic). 1H NMR (at 80  C for trans-

Table 1
Physicochemical characteristics of the greenhouse of the University of Agriculture, Peshawar and eld soils of Dargai, Khyber Pakhtunkhwa.
Soil

pH

Organic matter

1
(g kg of soil)

EC (dSm1 at 25  C)

Silt (%)

Clay(%)

Sand (%)

Green house

Spring, 2010
Autumn, 2010

Field
Spring, 2010
Autumn, 2010

7.2
7.3

9.9
9.8

0.19
0.18

29.9
29.0

23.0
22.5

47.1
48.5

7.4
7.3

11.0
10.2

0.19
0.20

28.9
28.6

22.0
22.3

49.1
49.1

I. Naz et al. / Crop Protection 81 (2016) 138e144

Fig. 1. Structure of the cis- and trans-protopinium (an isoquinoline alkaloid) isolated
from methanolic root fraction of Fumaria parviora.

protopinium) d ppm: 8.73 (1H, br. S, eOH at C-14), 7.28 (1H, br. s, H1), 6.97 (1H, d, J 8.0 Hz, H-11), 6.89 (1H, s, H-4), 6.80 (1H, d,
J 8 Hz, H-12), 6.10 (2H, s, H-16), 6.07 (2H, s, H-15), 4.55 (2H, br. s,
H-8), 3.81 (1H, br. s, Ha-6), 3.73 (1H, br. s, Hb-6), 3.23 (1H, br. s, Ha5), 3.11 (1H, br. s, Hb-5), and 2.95 (3H, s, NCH3). 13C NMR (at 80  C
for trans-protopinium) d ppm: 148.6 (C-3), 148.0 (C-2), 146.1 (C-10),
144.1 (C-9), 127.1 (C-14a), 125.3 (C-4a), 123.7 (C-12a), 122.6 (C-12),
109.5 (C-8a), 108.9 (C-4 and C-11), 106.5 (C-1), 102.2 (C-16), 102.0
(C-15), 55.0 (C-8), 53.7 (C-6), 42.7 (NCH3), and 23.9 (C-5). These
spectral data were comparable with the reported values for transprotopinium.
1
H NMR (at 25  C for trans-protopinium) d ppm: 8.65 (1H, br. S,
eOH at C-14), 7.33 (1H, s, H-1), 6.97 (1H, d, J 8 Hz, H-11), 6.89 (1H,
s, H-4), 6.81 (1H, d, J 8 Hz, H-12), 6.09 (2H, s, H-16), 6.06 (2H, s, H15), 4.53 (2H, br. s, H-8), 4.04 (2H, br. s, H-13), and 2.91 (3H, s,
NCH3). 13C NMR (at 25  C for trans-protopinium) d ppm: 125.5 (C4a), 124.6 (C-12a), 122.2 (C-12), 108.6 (C-4), 108.5 (C-11), 106.2 (C1), 101.8 (C-16), 101.6 (C-15), 91.6 (C-14), 54.8 (C-8), 53.4 (C-6), 42.2
(NCH3), 34.6 (C-13), and 23.4 (C-5).
1
H NMR (at 25  C for cis-protopinium) d ppm: 8.70 (1H, s, eOH
at C-14), 7.08 (1H, s, H-1), 6.97 (1H, d, J 8 Hz, H-11), 6.88 (1H, s, H4), 6.74 (1H, d, J 8 Hz, H-12), 6.09 (1H, s, H-16), 6.06 (1H, s, H-15),
4.67 (2H, br. s, H-8), 3.67 (2H, br. s, H-13), and 3.02 (3H, s, NCH3). 13C
NMR (at 25  C for cis-protopinium) d ppm: 127.4 (C-4a), 123.3 (C12a), 121.5 (C-12), 109.2 (C-4), 108.2 (C-11), 105.6 (C-1), 101.8 (C-16),
101.6 (C-15), 92.4 (C-14), 54.2 (C-8), 52.6 (C-6), 44.0 (NCH3), 38.5 (C13), and 22.9 (C-5).
3.2. In vitro nematicidal effect of cis- and trans-protopinium on
percent hatch inhibition and J2 mortality of M. incognita
The AUCPHI (Fig. 2a) increased with the concentration of the
compound. The AUCPHI ranged between 8.6% and 100% over 120 h
of incubation. The four concentrations showed highly signicant
differences with Fisher's protected LSD test. The AUCPM was the
highest (100%) at 200 mg mL1. The lowest mortality (6.8%) was
observed in controls (Fig. 2b). The four concentrations differed
signicantly on conducting Fisher's protected LSD test. The nal
percent hatch inhibition and mortality (Fig. 2c and d) showed a
polynomial-type increase with R2 of 0.98 and 0.93, respectively.
This graph showed a saturation point on hatch inhibition and
mortality for 200 mg mL1.
3.3. In planta effect of cis- and trans-protopinium on M. incognita
on tomato and other plant growth parameters
In the greenhouse experiments conducted in the spring and
autumn of 2010, the application of cis- and trans-protopinium at
three concentrations (i.e., 100, 200, and 300 mg mL1) signicantly
reduced the nematode infestation and enhanced the plant growth
parameters (Table 2). Data from the two seasons showed that cis-

141

and trans-protopinium exhibited high toxicity at all concentrations,


with the highest concentration of 300 mg mL1 being the most
effective. Root GI (1.5 and 1.4), females per gram of root (17.3 and
15.6), and Rf (0.24 and 0.20) signicantly (P < 0.05) reduced during
the growing seasons (spring and autumn) of 2010, compared to
treatment with distilled water as the control (Table 2) and from
100 mg mL1. The plant growth parameters were signicantly
(P < 0.05) enhanced. The plants showed clear growth at concentrations of 200 and 300 mg mL1 for all parameters studied. The
highest concentration of the tested compound did not exert any
phytotoxic effect on above- and belowground plant parts. The fresh
root weight was the highest in the untreated controls (68.3 and
69.3 g) in both the spring and autumn of 2010, whereas this was
gradually reduced to a minimum of 30.6 and 29.6 g at a concentration of 300 mg mL1 of cis- and trans-protopinium, respectively
(Table 2).
3.4. Bare-root dip of tomato using different concentrations of cisand trans-protopinium under naturally infested eld conditions
The results presented in Table 3 show that treatment with cisand trans-protopinium at three levels signicantly (P < 0.05)
reduced the GI, females per gram of root, and Rf. GI (1.5 and 1.7),
females per gram of root (38.3 and 30.0), and the Rf (0.23 and 0.20)
were minimum at the highest concentration of cis- and trans-protopinium, both in spring and in autumn, respectively (Table 3). The
plant growth parameters of tomato were found to increase in all
treated plots. The maximum plant height (63.0 and 76.6 cm), fresh
(73.3 and 82.0 g) and dry shoot weight (38.0 and 51.3 g), and root
length (52.9 and 65 cm) were recorded in the plots treated with
300 mg mL1 of cis- and trans-protopinium (Table 3). The fresh root
weight was highest in the untreated control plots. The maximum
increase in the fruit yield per plot was observed in all treated plots.
The yield increase was signicantly high in all treated plots, ranging
from 19.3 to 23.5 and from 19.2 to 23.6 kg/plot, in spring and
autumn of 2010, respectively (Table 3).
4. Discussion
Phytochemicals or botanical extracts have been used to control
the population of Meloidogyne spp. due to the rising environmental
pollution from the use of persistent nematicides (Chitwood, 2002).
Many plant species produce different allelochemicals with great
nematicidal potential (Chitwood, 2002). In the present study, both
the eggs and J2s of M. incognita were killed by the alkaloid cis- and
trans-protopinium. The hatch inhibition and J2 mortality of M.
incognita were found to be dose dependent. A literature survey
showed that three alkaloids (i.e., wuchuyamide I, rutaecarpine, and
evodiamine) isolated from the fruits of Evodia rutaecarpa had a
similar toxic potency against M. incognita (Liu et al., 2013). Alkaloid
extracts in either the crude or pure form have nematicidal properties, as is evident from the methanolic root extracts of Pulsatilla
koreana (LC50 92.8 mg mL1) (Li et al., 2013) and the crude alkaloid
extract (ED50 76.3 mg mL1) of, for example, drupacine against M.
incognita (Yanhua et al., 2013).
We identied and isolated the compound cis- and trans-protopinium from the methanolic fraction of F. parviora with column
chromatography. This compound appeared dark gray in color during TLC when exposed to UV light and bright orange when sprayed
with Dragendorff's reagent. The isolated alkaloid displayed the
spectra of two compounds: one major (trans-protopinium) and one
minor (cis-protopinium) compound in a ratio of 2:1. From the NMR
spectra, measured in DMSO at 25  C, we were unable to deduce the
structure as the H-NMR signals were overlapped and broad. The
NMR spectra of the alkaloid were then obtained at 80  C using the

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I. Naz et al. / Crop Protection 81 (2016) 138e144

Fig. 2. Nematcidal effect of the cis- and trans-protopinium against Meloidogyne incognita eggs and J2s in vitro tests. Cumulative hatch inhibition of M. incognita cis- and transprotopinium over 120 h incubation at 25  C over a series of concentrations. b) Cumulative mortality of M. incognita juveniles using cis- and trans-protopinium over 120 h incubation
at 25  C over a series of concentrations. Each point represents the average of two experiments with ve replicates. Curves from each treatment combination followed by the same
lower case (rst experiment) or upper-case letters (second experiment), do not differ signicantly (P > 0.05) according to Fisher's protected LSD test. c) Relationship between pure
compound concentration (50, 100, 150 and 200 mg mL1) and nal cumulative hatch inhibition. d) Relationship between pure compound concentration (50, 100, 150 and
200 mg mL1) and nal J2s mortality of M. incognita. The lines represent the predicted function explained by polynomial function models.

Table 2
Effect of different concentrations of cis- and trans-protopinium against Meloidogyne incognita on tomato cv. Moneymaker and plant growth parameters under greenhouse
conditionsa.
Cis- and trans- protopinium (mg
mL1)

GIb

Females g1 of
root

Rf (Pf/
Pi)

Plant height
(cm)

Fresh shoot weight


(g)

Dry shoot weight


(g)

Fresh root weight


(g)

Root length
(cm)

0 (Control)c

4.8
ad

63.3 a

0.84 a

39.0 c

40.0 d

20.3 c

68.3 a

12.3 c

e
4.7 A

100

200
300
LSDf (P < 0.05)
a
b

2.7 b
48.6
c
2.0 c
2.0 C
1.5 d
1.4 D
0.45
0.18

65.0 A

0.83 A

3.0 B
48.6 C

35.6 b
24.3 b

41.3 C
36.0 B
24.3 BC

43.0 C
0.51 b
42.3 b

21.6 C
0.51 B
41.0 B

69.3 A
49.0 b
13.2 c

13.6C
49.6 B
14.3C

28.3
28.3
17.3
15.6
9.7
13.5

0.38
0.38
0.24
0.20
0.17
0.14

52.3
52.0
57.6
61.0
3.7
6.1

58.3
57.3
69.3
73.0
4.9
6.9

25.6
27.3
31.6
32.3
3.4
3.0

39.6 b
38.6 BC
30.6 c
29.6C
5.9
9.4

16.8
19.3
22.6
28.0
1.7
2.1

b
B
c
C

bc
C
c
D

b
B
a
A

b
B
a
A

Data are means of 10 replicated plants per treatment using the combination of two experiments.
Galling index: 0c no gall on roots;1 12; 2 310; 3 1130; 4 31100; 5 >100 galls per root (Taylor and Sasser,

c The treatment with 0

mg mL1 was HO (control) mixed with DMSO (1%, v/v).

2
d Lowercase letters represent data recorded in the spring of 2010

1978)

b
B
a
A

b
B
a
A

e Uppercase letters represent data recorded in the autumn of 2010. Means followed by the same letter do not differ signicantly (P  0.05) according to Fisher's protected LSD test.
f Least signicant difference value (LSD) value at

a 0.05.

same solvent to overcome the limitation of spectral interpretation.


The spectra obtained at 80  C were interpreted as trans-protopinium after comparison with the literature (Tousek et al., 2005).
The two spectra obtained at 25 and 80  C were compared. The
major peaks in the alkaloid at 25  C were interpreted as trans-

protopinium and the minor peaks as cis-protopinium by comparing


the data with the literature (Tousek et al., 2005). The transformed
and stable states of the two isomers were obtained by measuring
the spectra at 80  C in the same solvent, as described earlier.
In the present study with cis- and trans-protopinium, we

I. Naz et al. / Crop Protection 81 (2016) 138e144

143

Table 3
Bare root dip effect of different concentrations of cis- and trans-protopinium against Meloidogyne incognita on tomato cv. Moneymaker and plant growth parameters under
naturally infested eld conditions of Dargaia.
Cis- and transprotopiniu m (mg
mL1)

GIb

Females g1 of
root

Rf (Pf/
Pi)

Plant height
(cm)

Fresh shoot weight Dry shoot weight Fresh root weight Root length
(g)
(g)
(g)
(cm)

Yield (kg/
plot)

0 (Control)c

5.0
ad
5.0
Ae
3.6 b
3.0 B
2.3 c
2.2
BC
1.5 d
1.7C
0.79
0.92

148.0 a

1.50 a

46.6 c

47.3 c

28.6 b

75.3 a

27.7 d

17.3 d

105.0 A

1.9 A

52.3C

49.3C

33.3 D

94.6 A

33.0 C

18.3 B

85.3
78.7
70.6
55.3

b
B
b
C

0.61 b
0.58 B
0.41 b
0.42C

50.3
62.0
54.7
65.3

50.6
61.3
59.3
66.0

29.3
41.0
32.3
46.6

53.0
57.0
44.3
46.6

34.3
44.0
42.5
50.3

19.3
19.2
21.6
21.6

38.3 c
30.0 D
20.07
14.9

0.23 b
0.20 D
0.51
0.13

63.0 a
76.6 A
6.2
6.9

100
200

300
f

LSD (P < 0.05)

bc
B
b
B

c
B
b
B

73.3 a
82.0 A
4.4
8.3

b
C
b
B

38.0 a
51.3 A
4.1
4.6

b
B
bc
BC

35.6 c
43.0C
11.6
12.5

c
B
b
B

52.9 a
65.0 A
5.9
6.7

c
B
b
A

23.5 a
23.6 A
1.3
2.2

Data are means of 10 replicated plants per treatment using the combination of two experiments.
Galling index: 0 no gall on roots; 1 1e2; 2 3e10; 3 11e30; 4 31e100; 5 >100 galls per root (Taylor and Sasser, 1978).
The treatment with 0 mg mL1 was H2O (control) mixed with DMSO (1%, v/v).
d
Lowercase letters represent data recorded in the spring of 2010.
e
Uppercase letters represent data recorded in the autumn of 2010. Means followed by the same letter do not differ signicantly (P  0.05) according to Fisher's protected
LSD test.
f
Least signicant difference value (LSD) value at a 0.05.
b
c

observed that the AUCPHI and AUCPM were directly related to


concentration and incubation time. Both the AUCPHI and the
AUCPM tended to be 100% over 120 h of incubation at the highest
concentrations. Naz et al. (2013b) recently reported a similar dosedependent effect of two nematicidal phytochemicals (i.e., nonacosane-10-ol (alcohol) and 23a-homostigmast-5-en-3b-ol (sterol)) against M. incognita. Both compounds caused 100% larval
mortality and 95% hatch inhibition over 120 h of incubation. Previously, as many as 33 alkaloids including isoquinoline alkaloids
(such as protopine, cryptopine, sinactine, stylopine, and many
others), protoberberine, and benzylisoquinoline were extracted
from members of the Fumariaceae family (Sasu et al., 2002;
Maqbool et al., 2004). Most of these alkaloids and their derivatives have been evaluated for their effect on other microbes
such as bacteria, fungi, and viruses (Orhan et al., 2007). Reports on
the in vitro nematicidal activity of pure alkaloids are scarce, but the
in vivo anti-nematodal activity of alkaloids of F. parviora has not
been studied yet. Al-Shaibani et al. (2009) revealed that the protopine alkaloid of F. parviora had a strong effect on helminths of
cattle. A unique nitrogen-containing 10-membered cyclic ketone of
protopine was suggested to possess various pharmacological activities, including antiarrhythmic, antithrombotic, and antiplatelet
aggregation effects (Song et al., 2000). Protopine has been shown to
inhibit the activity of acetylcholine esterase and cause nerve
disruption (Kim et al., 1999). As both acetylcholine and dopamine
serve as neurotransmitters in M. incognita (Stewart et al., 2001), the
isoquinoline alkaloid in the present study might have disrupted
these enzymes. Acetylcholine is involved in the movement of
larvae, egg laying, control of wave initiation during movement,
pharyngeal pumping, and male mating behavior (Garcia et al.,
2001). The toxic compound may also have disrupted the cell
membrane of the nematode larvae upon exposure.
Several alkaloids are used either medicinally or as lead structures for novel synthetic drugs (Fester, 2010). Increasing concentrations of cis- and trans-protopinium were found to be more
potent than only distilled water. A single application of the compound as a root drench in M. incognita-inoculated tomato resulted
in an increase in plant parameters and decrease in nematode
reproduction after 2 months. Current studies corroborate our previous ndings on the nematicidal potential of the root and stem
bark extracts of F. parviora against M. incognita (Naz et al., 2013a),

with methanolic root extracts signicantly reducing the GI, females, eggs, and Rf at a concentration of 3000 ppm. Thoden et al.
(2007) reported similar results, with pyrrolizidine alkaloids of
Chromolaena odorata suppressing M. incognita juveniles on lettuce
seedlings at a concentration of 7 ppm, whereas juveniles were
completely inhibited at concentrations as high as 70 and 350 ppm.
Other alkaloids have shown good potency against plant-parasitic
nematodes. Chitwood (2002) showed that monocrotaline (a pyrrolizidine alkaloid) from Crotalaria spectabilis inhibited the movement of M. incognita at a concentration of 10 mg ml1. Similar results
were observed in our eld trials, where bare-root dip treatment of
tomato with different concentrations of cis- and trans-protopinium
reduced the GI, number of females, and the nematode reproduction
factor in the spring and autumn of 2010. Other researchers have
obtained similar results, with bare-root dip treatment of tomato
with castor leaf extracts effectively suppressing root-knot nematodes (Akhtar and Alam, 1990). The effect of aqueous castor leaf
extracts on M. incognita could be enhanced further in combination
with Paecilomyces lilacinus spores (Rao et al., 1999).
At all studied concentrations (100e300 mg mL1), cis- and transprotopinium caused the elongation of the stem and accumulation
of green and dry weights, in addition to greater parameters of plant
growth than in the control in the greenhouse trials. The fresh and
dry shoot weights were enhanced when cis- and trans-protopinium
was applied closer to the rhizosphere of the tomato root as
compared with the untreated water controls, mainly due to the
parasitic control of the nematode. At low concentrations, the
compound completely protected tomato plants from M. incognita
infection in the season greenhouse trials. The concentrations used
did not cause any phytotoxicity, as plant height (57.6 and 61.0 cm)
and root length (22.6 and 28.0 cm) were enhanced at a higher
concentration of 300 mg mL1 in the spring and autumn of 2010,
respectively. Similar results were observed in our eld trials,
wherein the bare-root dip treatment of tomato with different levels
of cis- and trans-protopinium increased plant growth parameters
with an attendant yield increase over untreated controls. The
alkaloid can boost the plant resistance/defense system. Plant
resistance or defense reactions against plant pathogens induced by
phytochemicals (Chitwood, 2002) have been reported. Although
the mode of action of plant extracts and alkaloids is not well understood, some alkaloids (e.g., pyrethrins) cause paralysis before

144

I. Naz et al. / Crop Protection 81 (2016) 138e144

death. Pyrethrins are considered to possess neurotoxic effects


(Chitwood, 2002). To date, the nematicidal effects of isoquinoline
alkaloids of F. parviora have not been established, although its
crude extracts successfully controlled the population of M. incognita on tomato (Naz et al., 2013a).
Botanicals are economical and eco-friendly nematicides and
nematostatics. Cis- and trans-protopinium were found to suppress
M. incognita eggs and J2s in the laboratory, although this compound
showed the most favorable results in the greenhouse and eld
settings with an attendant yield increase in the eld. We propose
that a second treatment 2 weeks after the rst treatment could
improve the results of trans-protopinium. Cis- and trans-protopinium can be considered a potential tool to manage this widespread pest playing an important role in IPM and IDM. F. parviora
plants are common and abundant in the wheat elds of Pakistan,
particularly in the plain areas. These ndings are signicant for the
management of root-knot nematodes damaging tomato crops in
the Khyber Pakhtunkhwa province, thus eliminating the use of
expensive and toxic synthetic nematicides. Further, these phytochemicals are benecial due to their lower cost compared with
chemicals and the easy availability of the plant.
Acknowledgments
The funds provided by the Higher Education Commission of
Pakistan (HEC) through indigenous scholarships (Batch-VI) are
highly appreciated.
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