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Transactions on Engineering and Computing Sciences - Vol. 12, No. 1
Publication Date: February 25, 2024
DOI:10.14738/tecs.121.16310.
Timi, D., Maino, M., & Gopalakrishnan, S. (2024). Nematicidal Assessment of Plant Mediated Green Synthesized Silver
Nanoparticles Under Laboratory Conditions. Transactions on Engineering and Computing Sciences, 12(1). 214-225.
Services for Science and Education – United Kingdom
Nematicidal Assessment of Plant Mediated Green Synthesized
Silver Nanoparticles Under Laboratory Conditions
David Timi
Department of Applied Sciences,
PNG University of Technology, PMB, Lae, PNG
Macquin Maino
Department of Agriculture,
PNG University of Technology, PMB, Lae, PNG
Subramaniyam Gopalakrishnan
Department of Applied Sciences,
PNG University of Technology, PMB, Lae, PNG
ABSTRACT
Plant mediated biosynthesis of silver nanoparticles (AgNPs) and the bioactivity
under laboratory condition on plant parasitic nematode, Meloidogyne incognita, is
discussed. In the present study, aqueous extracts of the aerial parts of a medicinal
weed, Euphorbia geniculata was utilized to construct AgNPs. The synthesized AgNPs
was characterized by Ultraviolet-visible (UV-Vis.), Fourier Transform-infrared (FT- IR), X-ray Diffraction (XRD) spectrometer and Scanning Electron Microscopy (SEM)
analysis. The species identification of M. incognita was accomplished via the DNA- PCR technique. The green biosynthesized AgNPs demonstrated comparable
nematicidal activity with the reference chlorpyrifos against Meloidogyne incognita
identified through DNA assay in PCR.
INTRODUCTION
Nematodes are multi-cellular organisms and they are considered to be the most numerous
organisms on earth.1 These mostly microscopic worm-like creatures, range in sizes from 0.3
mm to over 8 m such as Placentonema gigantissimum that inhabits the placenta of a sperm
whale.2 They are so numerous that a handful of soil may contain thousands of them. Their
habitat ranges from the ocean floor to the mountain tops.2Many of the nematodes are parasitic
and feed on plants, bacteria, fungi, protozoa, other nematodes, animals and humans such as
Wuchereria bancrofti that causes elephantiasis throughout the tropical region. Others are
involved in the decomposition of organic matters and are referred to as free-living.2, 3
The knowledge of plant parasitic nematodes of Meloidogyne species has been well
documented.3 Knowing and managing plant parasitic nematodes is important in agriculture as
these organisms are annually responsible for 15% crop loss that is about 78 billion US$ in
monitory value globally.3, 4 Meloidogyne are microscopic soil-borne nematode species and are
known world-wide as the most plant parasitic pest that limits agricultural crop productivity.5, 6
Among the root-knot Meloidogyne species, there are four major ones that affect agricultural
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Timi, D., Maino, M., & Gopalakrishnan, S. (2024). Nematicidal Assessment of Plant Mediated Green Synthesized Silver Nanoparticles Under
Laboratory Conditions. Transactions on Engineering and Computing Sciences, 12(1). 214-225.
URL: http://dx.doi.org/10.14738/tecs.121.16310
crops. In the order of their dominance and severity of damage to crops, they include M.
incognita, M. javanica, M. arenaria and M. hapla.
7 Meloidogyne hapla mainly inhabits the cooler
higher altitude soil environment while the other three species predominantly inhibit the
warmer tropical lowlands.6 Together they account for 95% of the root-knot nematodes in
arable lands.8 Meloidogyne incognita is the dominant of the Moloidogyne species worldwide and
is found frequently in tropical and subtropical climates.9 It feeds on a wide range of agricultural
crops such as tomato, sweet potato and carrot to name a few, as well as other forest plants in
general.7 According to Lamberti and Taylor (1979), it accounts for about 64% of nematode
population in tropical regions and about 70 - 80% in subtropical climates.10
From a number of methods used to identify nematode species, two commonly used at present
are firstly, the identification based on perennial pattern morphology and secondly, the method
based on PCR technique. The latter is the recent advancement that has enabled exact
identification of the four major species of the root-knot nematodes and is based on their genetic
marker.7, 11
Several methods are employed in the control and management of nematode populations and
some of these include chemical, cultural, biological, sanitation, solarisation and botanical
control.
7, 9 Among these methods, the chemical control method is the most common approach
but it has its serious health and environmental issues. The tactic of botanical control is less
practiced but safe, environment friendly, and the materials utilized are cheap and are readily
available naturally. These biological materials are a rich source of biomolecules which can be
utilized to construct metal nanoparticles such as silver nanoparticles that can be used as an
alternative nematicide which is the basis of this research.
Technology of nano science is a rapidly emerging area of research that has application in all
areas of sciences and engineering disciplines including agriculture.12 Nanotechnology is based
on the manipulation of atoms and molecules at nanoscale level (1-100 nm) because of the
unique physical, chemical and biological properties demonstrated by certain nanomaterials.13
Silver nanoparticle (AgNPs) is one such nanomaterial that has drawn attention for its
application in agricultural research primarily due to its outstanding chemical and biological
properties.14 Among metal nanoparticles, silver is more biocompatible and has excellent
antimicrobial properties due to its high surface-to-volume ratio, hence, its application as an
antimicrobial and insecticidal agent.14, 15
In recent past, there have been numerous literature reports of promising nematicidal activities
demonstrated by green synthesized AgNPs.16, 17, 18 In the present investigation, aqueous
extracts of a medicinal herb, Euphorbia geniculata (Euphorbiaceae), (Figure 1) was utilized to
fabricate silver nanoparticles and assayed against Meloidogyne incognita to examine the
nematicidal property of these nanoparticles under laboratory conditionals.
Euphorbia geniculata is an annual weed that grows up to 60 cm high in moist tropical and
subtropical regions to an altitude of up to over 1000 m on a wide variety of soils and it is
considered a pest that affects varieties of agricultural crops.19 There has been no documented
report of the ethno-botanical use of this plant. However, the medicinal use of a related species,
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Euphorbia hirta is well documented.19 No specific chemical information of E. geniculata is also
found, but, triterpenoids, flavanoids, alkaloids, glycosides, tannins, steroids saponins, lignins,
phytosterols, polyphenols and benzoic acid were found to occur in other related species
including E. Hirta.
20 This study is a continuation of the biological assessment of AgNPs
fabricated via aqueous extract of E. geniculata.
21
MATERIALS AND METHODS
Preparation of Silver Nitrate Solution
Using an analytical balance, 9.5 g of silver nitrate was weighed and transferred into a 500 ml
volumetric flask, dissolved and made up to volume with distilled water giving a concentration
of 0.1177 moles L-1. From this, 2.5 ml was transferred to 100 ml volumetric flask with a 5 ml
pipette and diluted to mark with distilled water, giving a final concentration of 0.003 moles L-1.
Sampling and Preparation of the Plant Sample
The aerial parts of fresh disease-free plant (Figure 1) were collected and 10 g of these were
weighed and washed thoroughly with tap water and then rinsed with distilled water.
Figure 1: Euphorbia geniculate
Aqueous Extract of the Plant Sample
A 10 g of the plant material were chopped to smaller pieces with a kitchen knife and placed into
250 ml volumetric flask containing 200 ml of distilled water and heated on a bunsen burner to
the first sign of boiling, then removed and cooled to room temperature. The solution was then
filtered through a Whatman No. 1 filter paper and the filtrates were used to synthesize silver
nanoparticles.
Synthesis of Silver Nanoparticles
Preparation of AgNPs:
A measuring cylinder was used to obtain 50 ml of aqueous plant extracts and transferred into
250 ml volumetric flasks and to these, 100 ml of 3.0 mM of silver nitrate were added, shaken
and placed directly under the sun. A colour change from pale yellow to dark brown indicated
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Timi, D., Maino, M., & Gopalakrishnan, S. (2024). Nematicidal Assessment of Plant Mediated Green Synthesized Silver Nanoparticles Under
Laboratory Conditions. Transactions on Engineering and Computing Sciences, 12(1). 214-225.
URL: http://dx.doi.org/10.14738/tecs.121.16310
the formation of silver nanoparticles, followed by the agglomeration of the particles. After 2 h,
the mixture was removed and placed in a room for agglomerated colloidal AgNPs to settle over- night before centrifugation at 2500 rpm. The supernatants were discarded and the AgNPs were
washed twice with about 50 ml of distilled water by centrifugation per wash. The supernatants
were discarded and the remaining AgNPs were dried and stored at 4oC in a small sample bottle.
About 250 mg of these nanomaterials were sent to India for spectral analysis.
Characterization:
Characterization of the synthesized AgNPs included UV-Visible spectroscopy, Fourier
Transform Infra-red spectroscopy (FT-IR), X-ray Diffraction spectroscopy (XRD) and Scanning
Electron Microscopy (SEM). The UV-vis. analysis of the phytosynthesized AgNPs was carried
out using a Varian Cary 50 Bio UV-visible spectrophotometer. The absorption maxima (λmax) of
the coloured solution of AgNPs in a cuvette (1 cm path length) was taken from a wavelength
range set at 200 – 700 nm and slit width at 1 nm. FT-IR analysis was carried out in the range of
450 to 4,000 cm−1. XRD pattern was recorded using Cu Kα radiation (λ = 1.54060 Å) with nickel
monochromator in the range of 2θ from 10° to 70°. The morphology and size range of the
synthesized AgNPs were examined by SEM.
Molecular Identification of Meloidogyne incognita
Experimental Location:
Molecular identification of Meloidogyne incognita and laboratory tests were done at the Unitech
Biotechnology Centre (UBC) in the Department of Agriculture of PNG University of Technology,
(Unitech) Taraka Campus Lae, Morobe Province.
Bio-trapping of Meloidogyne incognita:
Beefsteak, a highly susceptible tomato variety was used to bio-trap M. incognita. Seeds were
initially germinated in tray boxes using unsterilized soil for three weeks. The seedlings were
then transplanted into 15 cm diameter polystyrene pots containing soil naturally infested with
Meloidogyne spp. These were then raised in a glasshouse for about two months before they
were harvested and brought to the laboratory for extraction of embedded females and isolation
of egg masses.
Identification of Meloidogyne incognita:
The nematode species of M. incognita was identified using DNA-based PCR technique. Bi- directional oligo DNA primers (Table 1) were used to amplify the sequence characterized
amplified region (SCAR) according to procedure described by Zijlstra et al. (2000).11 DNA from
nematodes were isolated according to procedure described by Powers and Harris (1993).22
Female Meloidogyne spp. entrapped inside the infected root system of beefsteak tomato were
milled using mortar & pestle and DNA was extracted according to Powers and Harris (1993).22
PCR amplification was carried out in a total reaction volume of 25 μL containing 5 μL of DNA
template (lysate), 2.5 μL 10x Kapa Taq buffer with dye and Mg2+ (Geneworks), 0.5 μL 10 mM
dNTPs, 0.6 μL 10 μM Fine/Rinc primers, 0.5 μL 5 U/μL of Taq DNA polymerase (Geneworks),
and nuclease-free water was added to final reaction volume. The PCR thermocycler was
programmed with an initial denaturation at 94oC for 2 min followed by 35 cycles at 94oC for 30
s and 72oC for 1 min. The reaction was terminated with 1 cycle at 72oC for 5 min.
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Pre-stained PCR products (DNA) were loaded into the well of 1% agarose gel with a molecular
marker (Biorad). Loaded products were submerged in 1x TBE buffer containing 0.5 μg ml-1
ethidium bromide in mini gel tank and fractionated at 100v for 50 min. DNA bands specific to
M. incognita were finally identified under a UV spectrophotometer using a UV box (Vilber
Lourmat) and were recorded.
Table 1: Nucleotide sequences of Meloidogyne species-specific primers and expected
sizes of their typical amplified DNA bands.131
Primer Sequence Size of DNA
Primer Name (5’-3’) bps Specificity
Finc(1) ctctgcccAATGAGCTGTCC 1200 M. incognita
Rinc ctctgcccTCACATTAAG
Mi-F
(2) TAGGCAGTAGGTTGTCGGG 1350 M. incognita
Mi-R CAGATATCTCTGCATTGGTGC
Laboratory Assay of Biosynthesized AgNPs against M. incognita
Rearing M. incognita on Beefsteak Tomato:
Seeds of beefsteak tomato were germinated and raised on heat-sterilized soil tray before being
transplanted after two weeks into 15 cm diameter polystyrene pots containing sterilized soil.
These seedlings were allowed to grow for one week before eggs and juveniles (J2s) from
identified M. incognita were used to inoculate the seedling and were allowed to regenerate on
roots of the host plant for subsequent studies.
Isolation of Eggs for Laboratory Susceptibility Test:
Eggs and J2s were harvested from roots of the infected beefsteak reared in the glasshouse.
Infected tomato pots were fully submerged into a bucket of water and the soil was carefully
washed off from the roots. To obtained mature eggs, the egg masses on infected roots were
dipped into 0.05% sodium hypochlorite solution and gently swirled for 5 min. This freed the
eggs from the roots and were separated by filtration using a 38 μm Endacott sieve and then
washed into a sample vial to be used for testing and also to incubate to produce J2s. Those not
for immediate use were stored in a refrigerator at 15oC.
Experimental Design and Treatments:
Experiments in the laboratory were arranged in small glass petri dishes on benches in
replicates of three. There were nine sets of experiments which included blank (distilled water
as positive control), commercial nematicide (chlorpyrifos, 1% w/v) as positive control,
undiluted AgNPs, and 10x, 100x, 1000x, 2500x, 5000x and 10,000x dilution of AgNPs. For
simplicity, each set of treatments were tested separately and simultaneously for both eggs and
J2s.
Bioassay of AgNPs Against Eggs and J2s:
Eggs – Ten eggs extracted from roots of beefsteak tomato were transferred into nine different
petri-discs (small) using sterile pipette tips and to these different treatments (distilled water,
chlorpyrifos, and different dilutions of AgNPs) were added. The mortality of eggs was observed
and recorded at 24 h intervals.
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Timi, D., Maino, M., & Gopalakrishnan, S. (2024). Nematicidal Assessment of Plant Mediated Green Synthesized Silver Nanoparticles Under
Laboratory Conditions. Transactions on Engineering and Computing Sciences, 12(1). 214-225.
URL: http://dx.doi.org/10.14738/tecs.121.16310
Juveniles (J2s) – Some egg suspensions extracted from roots of beefsteak tomato were
transferred to a small petri-dish and incubated at room temperature overnight to allow them
to hatch into J2s. From these, ten J2s were transferred into nine different small petri-dish and
different treatments (distilled water, chlorpyrifos, and the different dilutions of AgNPs were
added. The mortality of the J2s were then monitored and recorded at a 24 h interval.
RESULTS
Colour Change
The formation of silver nanoparticles was evident from the change of colour of the reaction
mixture from a yellow to a dark brown solution within 20 min (Figure 2).
Figure 2: Formation of AgNPs indicated by the change of colour from yellow to dark brown
solution.
Characterization
The Uv-vis spectrum (Figure 3) of the AgNPs generated from the aqueous extract of Euphorbia
geniculata exhibits an absorption peak at 450 nm that is characteristic of the Uv-vis signal of
the surface plasmon resonance of silver nanoparticles. FT-IR frequencies of the bio-molecules
involved in the reduction of silver ion (Figure 4) shows peaks at 3410 (N-H), 2925 (aliphatic C- H), 2851(aliphatic C-H), 1449 (C-H bending) and 1031 cm-1.
23 The XRD pattern of the AgNPs
(Figure 5) shows three distinct diffraction patterns 2θ peak values at around 38o, 44o and 64o
that are characteristic of the planes (111), (200) and (220) for the face-centered cubic silver.24
Image from the scanning electron microscopy (SEM) (Figure 6) shows the morphology of the
AgNPs to be mainly spherical and their size distribution ranges from 13 to 45 nm.
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Figure 3: Uv-vis. Spectrum of AgNPs synthesized From Extract of E. geniculata
Figure 4: FT-IR Spectrum of AgNPs Synthesized From E. geniculata
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0
0.0
5
1 0
1 5
2 0
2 5
3 0
3 5
4 0
4 5
5 0
5 5
6 0
6 5
7 0
7 5
8 0
8 5
9 0
9 5
100.0
cm-1
%T
1
3410
2925
2851
1449 1031
513
502
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Timi, D., Maino, M., & Gopalakrishnan, S. (2024). Nematicidal Assessment of Plant Mediated Green Synthesized Silver Nanoparticles Under
Laboratory Conditions. Transactions on Engineering and Computing Sciences, 12(1). 214-225.
URL: http://dx.doi.org/10.14738/tecs.121.16310
Figure 5: XRD Pattern of AgNPs Synthesized from Extract of E. Geniculata
Figure 6: SEM Image of AgNPs Synthesized from Extract of E. geniculata
Identification of Nematode Species
Molecular Identification of M. incognita:
The nematode species M. incognita was identified using DNA-based technique. Oligo DNA
primers used in PCR amplification process yielded expected DNA products for M. incognita
(Figure 7). Lanes 3 to 6 on 1% agarose gel showed the expected DNA bands of 1200 bps using
SCAR primers characteristic of M. incognita.
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Figure 7: PCR products. Lane 1 is Biorad DNA marker; Lane 2 is negative control; Lanes 3 – 6 are
positive amplifications (1200 bps) of M. incognita (Zijlstra et al., 2000)11
Laboratory Test on Mortality of J2s
The AgNPs tested against infective J2 showed promising toxic activity and the results are
presented in Table 2. Tests revealed that undiluted AgNPs as well as dilutions at 10, 100, 1000
and 2500 caused 100% mortality in 24 h, showing the same outcome as the commercial
nematicide, chlorpyrifos. No mortality was recorded from J2s treated with water alone. At that
time interval, 50% mortality was recorded at higher dilution of 5000, while no deaths were
observed at 10,000 dilutions of AgNPs (Table 2). The trend of mortality at the highest dilutions
was similar to that observed from treatments with water alone. The undiluted AgNPs were kept
as aqueous suspensions in ~30 ml of distilled water.
Table 2: Mortality of J2 M. incognita exposed to AgNPs synthesized from E. geniculata
Treatments Average mortality of J2 from population of 10 juveniles as % of control
Dilutions (1/x) n 24 48 72 96
Control (H2O) 10 0 0 0 50
Chlorpyrifos (1%) 10 100 100 100 100
Undiluted 10 100 100 100 100
10 10 100 100 100 100
100 10 100 100 100 100
1000 10 100 100 100 100
2500 10 100 100 100 100
5000 10 50 60 70 90
10,000 10 0 0 0 60
The undiluted AgNPs were kept as aqueous suspensions in ~30 ml of distilled water.
Laboratory Tests on Hatchability of Eggs
The effect of silver nanoparticles on the hatchability of the eggs of M. incognita was also
examined and the results are presented in Table 3. The tests showed that AgNPs effectively
suppressed the eggs from hatching during the 96-h observation period similar to the positive
control, chlorpyrifos (1%). For the eggs treated with negative control (water), there was normal
1 2 3 4 5 6
1.2 kbps
1 kb
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Timi, D., Maino, M., & Gopalakrishnan, S. (2024). Nematicidal Assessment of Plant Mediated Green Synthesized Silver Nanoparticles Under
Laboratory Conditions. Transactions on Engineering and Computing Sciences, 12(1). 214-225.
URL: http://dx.doi.org/10.14738/tecs.121.16310
life process that led to the hatchability of eggs from 48 h onward. Exposure of the eggs at 5000x
and 10000x dilution of the AgNPs did not seem to affect the hatchability of eggs as the J2 M.
incognita were observed to leave the eggs from 48 h onwards (Table 3)
Table 3: Egg hatchability of M. incognita exposed to AgNPs synthesized from E.
geniculata
Treatments Egg hatchability as % of control
Dilution (1/x) n 24 48 72 96
Control (H2O) 10 0 40 70 100
Chlorpyrifos (1%) 10 0 0 0 0
Undiluted 10 0 0 0 0
10 10 0 0 0 0
100 10 0 0 0 0
1000 10 0 0 0 0
2500 10 0 0 0 0
5000 10 0 20 70 70
10,000 10 0 30 70 90
DISCUSSION & CONCLUSION
In biological assays, correct identification of target organism is an important aspect. This
process facilitates tailored experimentation, data collection, analysis and interpretation. This
study showed that M. incognita can be identified among other Meloidogyne species using
species-specific DNA primers in PCR detection technique. Species-specific DNA product of
approximately 1.2 kbps was amplified in PCR reactions and detected in agarose gel under UV
trans-illumination. PCR technique in the use of single J2 nematodes proved that the technique
is reliable in diagnosis and differentiation of the four major Meloidogyne spp. that affect crops.
Population of M. incognita identified through this process were used in subsequent laboratory
experiments.
Meloidogyne incognita is a significant harmful pest for most agricultural crops, reducing growth
and yield of susceptible plants. The results show that infective juveniles of M. incognita exposed
to AgNPs experienced high lethal effect and further, AgNPs showed strong suppressive effects
on the hatchability of the eggs. The effect on both the J2 and the eggs was comparable to the
commercial nematicide chlorpyrifos even at 2500x dilution of the nanomaterial. At 5000x
dilution, the effect of the nanoparticles was negligible. From the egg hatchability tests, it was
interesting to observe that chlorpyrifos at 1% concentration was rupturing the eggs unlike the
nanoparticles, which only suppressed the eggs from hatching without rupturing them.
This research finding is seen to be consistent with previous research studies on laboratory
assessment of silver nanoparticles when applied against Meloidogynes spp.
25, 26 It further
demonstrated the excellent biocidal properties of AgNPs phytosynthesized from species of
Euphorbiaceae.21, 27 Additionally, it further suggests that AgNPs of plant mediated biosynthesis
can be utilized for biological applications in the control of agricultural pests.21, 26, 27 The overall
result under the laboratory conditions was encouraging and this led to further greenhouse and
field assessment of the AgNPs against M. incognita.
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ACKNOWLEDGEMENT
The authors are grateful to the Research Committee of PNG University of Technology for the
funding assistance. From the Biotech Centre of the Department of Agriculture, PNG University
of Technology, Prof. Tom Okpul is acknowledged for the use of the facility to conduct DNA
analysis in PCR and Mrs Totave Kamen provided technical assistance.
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Laboratory Conditions. Transactions on Engineering and Computing Sciences, 12(1). 214-225.
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