Introduction
Plant-infectious diseases caused by pinewood nematodes
(PWN) have become alarming over the years and causing significant damage to
pinewood despite efforts to contain them. Ethnobotanical studies have revealed
the causative agent of pine wilt disease to be B. xylophilus.
Preliminary investigation reported B. xylophilus to be native to
North-America and later introduced to Japan and subsequently spread into China,
Korea and into Europe (Portugal and Spain) (Proença
et al. 2010). Prior to the inception of PWNs, research studies
have revealed the association of possible toxin producing bacteria. These
bacteria enter a symbiotic relationship with the PWN as a way of extending
their lifespan in the pinewood. The activity of the so-called symbiotic
bacteria within the pinewood lead to plant damage by destroying the absorption
and transportation systems of plants which subsequently leads to wilting and
eventual death (Alves et al. 2018; Su et
al. 2020). PWNs
convey varying bacterial diversity depending on the country and location (Vicente et al. 2011). Nonetheless, it
has been hypothesized that differences in bacterial community structure in
different forest ecosystems are the cause of differences in bacterial species
associated with the pine wood nematode (PWN) in certain areas which tend to
justify the pathophysiology of pine wilt diseases (PWD). With PWD native to
America, Cedrus deodara, for example, has been reported to be
predominant in the United States (Wingfield et
al. 1982) and Japan (Mamiya 1983),
while Pseudomonas seems to be the
abundant in China (Proença et al. 2010;
Zhao et al. 2011).
The Potency of already existing control measures for
associated symbiotic bacteria of PWDs remains unspecified as reports indicate
that these measures tend not to have the full potential to control PWNs and in some cases causes environmental perturbation (Sabry 2019). For instance, the major treatment
explored for treating PWDs which involve the spraying of insecticides has been
reported to be inadequate to discontinue the spread of the damages of the
symbiotic bacteria (Shinya et al. 2013).
The issue therefore makes it obligatory to seek new antimicrobials and/or new
effective means of treating infectious diseases triggered by bacteria
associated with pine wilt disease (PWD).
The surging advancement of nanotechnology in agriculture
has gained substantial consideration worldwide since it can be applied to any
system of agriculture through potential and well-ordered release and targeted
supply of agrochemicals towards PWN (Jo et al.
2009; Servin et al. 2015; Sabry 2019). For instance, developing a
pesticide incorporated with AuNPs have been reported to be lethal to nematodes
without any negative impact on plants (Tsyusko et
al. 2012; Thakur and Shirkot 2017). This has spurred interest in
studying the antibacterial action of NPs against PWNs (Vallet-Regí et al. 2019). Several research investigations
have been focused on the drug delivery and disease site targeting efficiency of
NPs since their introduction, which is relevant to their usage as a control
option to nematode infections. Nonetheless, the efficacy and activities of NPs
in agriculture lies with their synthesis approach. Over the period, researchers
has extensively explored the conventional chemical
synthesis approach of obtaining NPs (Xu et
al. 2007; Luty-Błocho et al. 2011; Ojea-Jiménez et al.
2011; Ansari et al. 2020), however, the chemical synthesis
approach has been reported to be toxic especially in an in vivo experiment (Goodman et
al. 2004; Chen et al. 2009; Lasagna-Reeves et al. 2010).
It is therefore necessary to resort to a safer and simpler approach of NPs
synthesis that involves using plants and their parts. In our recent work, we
successfully synthesized a novel AuNPs using only leaf extract of Parkia biglobosa and investigated its potential to inhibit the growth of
some selected clinical isolates (Shine et al.
2020). Also, Akintelu et al.
(2021) used Garcinia kola pulp extract to synthesize gold
nanoparticles and explored it antibacterial potency. It is against this
background, Parkia biglobosa,
a popular medicinal plant in the northern Ghana is used both as reducing and
stabilizing agents to obtain gold nanoparticles from the reduction of
chloroauric gold (HAuCl4) from Au3+ to Au0.
The as-synthesized AuNPs were explored for their antimicrobial potential
against isolated symbiotic bacteria (Staphylococcus
Aureus, Pseudomonas Syringae, Bacillus Anthracis and Escherichia Coli) from pine wilt nematode, B. xylophilus. The biosynthesized AuNPs has the
potential to shorten the lifespan of the B.
xylophilus associated symbiotic bacteria by inhibiting their growth to
curtail a further damage to pinewood.
Materials and Methods
HAuCl4
was obtained from Sigma Aldrich, Pine branches were collected from the
suburbs of Zhenjiang (Jiangsu, China). Parkia
biglobosa leaf and stem bark were
also obtained from the university for development studies, Tamale, Ghana.
Source, isolation, and
culture of nematode (Bursaphelenchus xylophilus)
The isolation of the
nematodes used in this experiment followed the protocol by (Han et al. 2003; Zhang et al. 2014).
Naturally, infected (symptomatic) black pine (Pinus thunbergii) tissues were cut into small chips of
about 1×1 cm pieces using a saw and cutter. The chips were weighed with an
electronic balance. Total chips of 162 g were divided into groups of units with
each at 20 g. Using the Baermann funnel method as described by (Zhang et
al. 2014), with some modification, about 20 g of the chips were put on a
cheesecloth placed inside the Baermann funnel. Warm water at about 25°C was
used to soak the chips in the funnel and ice water bottles were put beneath the
end of the funnel tube to maintain ample cold temperatures. The setup was put
into an incubator and left for 6 h. Nematode suspension was collected after 6
h. 2 mL of the solution was taken to observe the presence and population of
nematode under a high-power microscope (Caikon XTL, China). The nematode was
identified as B. xylophilus cultured with the fungus Botrytis cinerea
on a low nitrogen potato dextrose agar (PDA) plate. Subsequently, the plates
were further incubated until the fungal mycelia were entirely consumed by the
nematodes and then separated from the culture medium by using a Baermann funnel
to obtain an aqueous suspension of nematodes for a subsequent test.
Isolation and culture of
bacteria strains from nematodes body
In this study, Strains of bacteria were isolated from the body of B. Xylophilus by raw liquid and washed liquid treatments. In the raw liquid
treatment, nematode suspension containing about 1200 nematode population was
ground (with a push and pull, up to 40 oscillations using a mortar and pestle)
under aseptic conditions. The suspension was diluted to different gradients (10-3,
10-4, 10-5) and cultured on a Petri dish containing beef
extract medium (NA) at a concentration of (0.005 g/mL, 1.99 g/mL and 8.0 g/mL), with (4) plates
as technical replicates. The same procedure was repeated for PDA medium at a concentration of (0.02 g/mL, 0.2 g/mL, 0.07 g/mL and
0.04 g/mL). The coated plates were incubated at 20–24°C to observe bacteria
growth.
In the washing liquid treatment procedure, a nematode solution
containing about 2000 nematode population was taken and shook vigorously in
low-temperature cold water for 200 times and then centrifuged at 8000 rpm after
which the supernatant was removed and washed with sterile water 2–3 times. The
mixture was grounded under aseptic conditions and then diluted to various concentrations
(10-2, 10-3 and 10-4). These were coated on
the beef extract and PDA medium as described above and incubated at 20–24°C to
observe bacteria growth. Bacteria growth was
observed 4–5 days after inoculation (DAI). Several bacterial colonies were
marked from the various plate containing different treatments. Using bacterial
characteristics such as morphology, shape, color, colonies of the bacteria
strains were isolated into the freshly prepared medium. Pure bacteria colonies
were isolated with beef extract medium in a slanted and liquid tube and
preserved for subsequent use.
Gram staining and Molecular
Identification isolates
The microscope was used to
identify isolated bacteria. Gram stain was done to classify the bacteria into
gram-positive and gram-negative. Bacteria single colonies representing each bacteria obtained from the culture plates were further
identified by molecular method following the protocol (Kai et al. 2019) by using 16S rRNA gene sequence. The
samples were sequenced by Nanjing Huizhi Zhiyuan Biotechnology Co., Ltd.,
China.
Extraction and Phytochemical screening
The samples were defatted and
8 g each of the leaves and stem powder was measured into 25 mL beaker each. 24 mL
of hexane was added to the samples. The solution was stirred continuously for
90 min by use of a magnetic stirrer. The stirring was stopped and the sample
left for 30 min before decanting. The sample was then filtered under vacuum and
subjected to extraction and phytochemical screening. The screening techniques
were carried as reported by (Akinyemi et al.
2005).
Test for flavonoids
1 mL of aqueous extract was
added to 1 mL of 10% lead acetate solution. The formation of a yellow
precipitate was taken as a positive test for flavonoids.
Test for Alkaloids
3 mL of aqueous extract was
stirred with 3 mL of 1% HCl on a steam bath. Mayer’s and Wagner’s reagents were
then added to the mixture. Turbidity of the resulting precipitate was taken as
evidence for the presence of alkaloids.
Test for tannins
About 2 mL of the aqueous
extract was stirred with 2 mL of distilled water and few drops of FeCl3
solution added. The formation of a green precipitate was an indication for the
presence of tannins.
Test for saponins
5 mL of aqueous extract was
shaken vigorously with 5 mL of distilled water in a test tube and warmed to 100°C.
The formation of stable foam was taken as an indication for the presence of
saponins.
Test for terpenoids
2 mL of the organic extract
was dissolved in 2 mL of chloroform and evaporated to dryness. 2 mL of
concentrated sulphuric acid was then added and heated for about 2 min. A
greyish colour indicates the presence of terpenoids.
Tests for steroids
2 mL of ethanol extract was
dissolved in 2 mL of chloroform and 2 mL concentrated sulphuric acid were
added. Formation of a red colour in the lower chloroform layer was used as an
indicator for the presence of steroids.
Tests for carbohydrates
To 3 mL of the aqueous
extract was added about 1 mL of iodine solution. A purple colouration at the
interphase indicates the presence of carbohydrate.
Tests for glycosides
Salkowski’s test
2 mL of each extract was
dissolved in 2 mL of chloroform. 2 mL of sulphuric acid was added carefully and
shaken gently. A reddish brown colour indicates the presence of a steroidal
ring (that is a glycone portion of glycoside).
Tests for anthraquinones
Borntrager’s test
3 mL of aqueous extract was
shaken with 3 mL of benzene, filtered and 5 mL of 10% ammonia solution was
added to the filtrate. The mixture was shaken and the presence of a pink, red
or violet colour in the ammoniacal (lower) phase indicates the presence of free
anthraquinones.
Test for proteins
Buiret’s test
A 2 mL of aqueous extract was
added to the buiret’s reagent. The color changed to purple showing presence of
protein.
Biosynthesis
Measure 5 g of powdered leaf
and stem into two different beakers and add 25 mL of deionized water to each
sample. Stir to obtain a homogenous mixture and was left for 24 h. The samples
were filtered using Whatman filter paper. 9 mL of 1MHauCl4 was measured
into two beakers and 1 mL each of the leaf and stem was added to the
HauCl4. A pronounced colour change was observed for both leaf and
stem where the leaf mediated AuNPs exhibited a dark purple colour while that of
the stem was ruby red.
Characterization
After colour change was
observed Ultraviolet-visible spectrophotometer (SpectraMax i3, Silicon Valley
USA) was used to determine the plasmon resonance. A 96 well costar clear plate
was used for the analysis of the AuNPs, and water was used as the blank.
Transmission Electron microscopy (TEM) was used to analyze the shape, sizes,
and dispersion of the gold transmission electron microscope (TEM). It was
performed TEM was performed on a field emission transmission electron
microscope (Tenai G2 F30 S-TWIN, FEI) at an acceleration voltage of 300 kV.
FT-IR was also used to analyze the functional group of the NPs. Further
analysis was done by XRD to ascertain the crystallinity of the gold nanoparticles.
The purified purified freeze-dried AuNPs were blended with KBr to obtain
pellet and were subjected to FTIR spectroscopy. The FT-IR spectra were measured
at a resolution of 4 cm-1 in the transmission mode (4000 cm-1–400
cm-1) Fourier transform infrared spectroscopy (FTIR) on a Bruker
spectrometer (Tensor 27).
Antibacterial study
0.9 g of nutrient broth was
measured into a 100 mL beaker and 50 mL of water added. The culture media was
then measured 2 mL each into a glass tube and autoclaved for 15 min at 121oC.
The media was afterward inoculated with the isolated bacteria for re-culturing.
The inoculum was then incubated for 18hrs and shook at 230 ppm. 9.5 g of
Mueller Hinton agar was measured into 250 mL of water, stirred, and autoclaved
at 15 min with punched paper disks and the Petri dishes. After sterilizing, the
agar was distributed to the Petri dishes and allowed to solidify. Whiles
awaiting the solidification of the agar, the paper discs of diameter 6 mm were
impregnated with 5 uL/mL of the gold nanoparticles. A cotton swab was
used to smear the culture on the agar and incubated for 24 h at 37oC.
The inhibition zone was evaluated using a straight rule.
Statistical analysis
The data was analysed using
Genstat 11th edition in a One-way analysis of variance (ANOVA) and
the results presented as mean of three replicates. Means separation was done
using bonferroni tests at 5% significant level.
Results
The microscope was used to identify isolated bacteria. Gram stain was
done to classify the bacteria into gram-positive and gram-negative. The pink colour indicating gram-negative while purple represents
gram-positive bacteria and their morphology were used to specify them. Staphylococcus
aureus is as represented in Fig. 3A and 3B is a Gram-positive round-shaped
bacteria that is a member of the Firmicutes, usually part of the microbiota of
the body in the forms of germs found on the skin or in even a healthy
individual’s nose causing skin, bone, joint and respiratory infections (Curran and Al-Salihi 1980; Cole et al. 2001).
Pseudomonas syringae on the other hand is a rod-shaped, Gram-negative bacterium with polar
flagella as exhibited in Fig. 3C and 3D. It is a host-specific pathogen whose
interactions in plants can take two strikingly courses. Thus, in virulent
interactions with susceptible plants, the bacteria multiply for several days
before producing visible symptoms such as necrotic lesions on leaves and fruits
whiles the avirulent interactions with resistant plants the bacteria trigger a
rapid localized, defense-associated, programmed death cell known as the
hypersensitive response (Collmer et al.
2002).
Bacillus anthracis is a Gram-positive, endospore-forming rod-shaped bacterium with a
width of 1.0–1.2 and µm and a length of 3–5 µm as shown in Fig. 3E and 3F. Bacillus anthracis is the anthrax
agent, a prevalent animal and sometimes human disease and the only mandatory
pathogen in the genus Bacillus (Spencer 2003). This disease can be known as a
zoonosis, because by transmitted from infected animals to humans has long been
considered a potential biological weapon (Spencer
2007).
E. coli as shown in Fig. 3G and 3H is a Gram-negative, facultatively anaerobic,
rod-shaped, coliform bacterium of genus Escherichia
that is commonly found in the lower intestine of warm-blooded organisms (Singleton 2004; Tenaillon et al. 2010).
After the gram staining to obtain the morphotype of the strains, molecular
identification was carried out using 16RS sequencing (Supporting information).
The present study focused
mainly on the qualitative chemical evaluation of the phytochemical constituents
of extract from the stem and leaves of Parkia biglobosa. The study revealed
the presence of alkaloids, tannins, cardiac glycosides and flavonoids,
terpenoids, saponins and glycosides in the stem whiles the leaves exhibited
cardiac glycosides, flavonoids, saponins, alkaloids, proteins, and amino acids
and steroids (Table 1). The phytochemicals present have been shown to possess
medicinal activity as well as physiological activity in previous studies by (Udobi and Onaolapo 2009; Builders et al. 2012).
These results show that Parkia biglobosa stem and leaves can be a
potential source of useful compounds that can be used to synthesize
nanoparticles for new antimicrobial drugs. The presence of these phytochemicals
justifies the traditional medicinal uses of this plant by the local people in
West and East Africa.
%20517-526_files/image002.png)
Fig. 1 a) A plate showing Staphylococcus aureus
colonies of agar b) A plate
exhibiting gram-stain of Staphylococcus
aureus, c) A plate showing Pseudomonas syringae colonies on agar d) A plate showing gram stain of Pseudomonas syringae,
e) A plate showing Bacillus anthracis colonies on agar, f) A plate showing gram stain Bacillus
anthracis
%20517-526_files/image004.png)
Fig. 2: Uv-vis absorption spectrum of a) Chloroauric gold, b)
leaf-mediated gold nanoparticle, c)
stem-mediated gold nanoparticles
Table 1: Phytochemical analysis of ethanolic leaf and stem
extract
|
Phytochemical Test |
Stem Leaves |
|
+ + |
|
|
+ + |
|
|
Terpenoids |
+ - |
|
Anthraquinones |
- - |
|
+ + |
|
|
+ + |
|
|
Alkaloids |
+ + |
|
Carbohydrates |
- - |
|
Tannins |
+ - |
|
Proteins and amino acids |
- + |
|
Steroids |
- + |
Inference (+) = Present (-) = Absent
The initial colour of the plant extracts was brown whiles the gold salt
was yellow however after adding the plant extracts to the gold salt, a colour
change was optically observed. The colour of the leaf mediated-AuNPs was dark
purple while that of the stem mediated-AuNPs was observed to be ruby red.
Thereafter, the UV-vis was used to determine the plasmon resonance and the
absorbance with the range of wavelength 230–1000 nm with 5
min intervals for 30 min. It was revealed in Fig. 1 and
2A and B that the plasmon resonance was recorded at 530 nm for both the L-AuNPs
and S-AuNPs. The absorbance for L-AuNPs started from 2 min whiles that of stem
started from 3 min and progressively rise as the time increases till it
attained stability.
The leaf mediated AuNPs also
had an optical colour change from dark brown to dark purple and were supported
with the UV-vis registering absorbance from 2 min and completed at around 30 min.
The TEM exhibited the shape of the Leaf nanoparticle to be spherical,
triangular, and irregular, and rod-shaped as
demonstrated in Fig. 3A, B and C.
Conversely, the complete
synthesis of the stem mediated AuNPs was observed at 30 min as indicated in Fig.
2B accompanied by a colour change from dark brown to ruby red. As shown in Fig. 3D, E and F, the TEM revealed truncated,
pentagonal, nanorod, spherical, triangular shape with sizes 3–28 nm.
From Fig. 4A, the FT-IR of the
leaf revealed bands at 615 Cm-1, 777 Cm-1 1060 Cm-1
1143 Cm-1 1394 Cm-1 1630 Cm-1 2038 Cm-1,
2860 Cm-1 2937 Cm-1, 3303 Cm-1 3487 Cm-1. The band revealed at 615 Cm-1
and 3303 Cm-1 could be a C-H stretch vibrations while that of 777 Cm-1
can be attributed to aromatic C-H bend, 1143 Cm-1
C-O stretch, 1394 CH3 C-H. An intense band located at 1630 Cm-1 was
attributed to benzene ring stretch however the band at 2038 Cm-1 was
not assigned. The band at 2860 Cm-1 and 2937 Cm-1 correspond
to aliphatic CH stretch 3487 Cm-1 can also
be likened to O-H stretching and N-H stretching vibrations, respectively.
On the other hand, as
exhibited in Fig. 4B, the stem also revealed bands at 613 Cm-1 C-H
stretch vibrations, 793 Cm-1 aromatic CH vibrations which could be
alkanes. 1080 Cm-1 C-C stretching vibrations, 1279 Cm-1
C-O stretching vibrations, 1614 Cm-1 Aromatic C=C stretching, 2085 Cm-1
C=conjugated 2922 Cm-1 asymmetrical stretching CH2, 3311
Cm-1 and 3465 Cm-1 could be assigned OH stretch (alcohols
Phenols). From the FT-IR results of the S-AuNPs, we
hypothesized phenolic compounds and flavonoids to be responsible for the
reduction process and stabilization of the AuNPs. This finding is supported
by experiments carried out by (Gopinath et
al. 2014; Markus et al. 2017; Chahardoli et al. 2018).
%20517-526_files/image006.png)
Fig. 3: a) Transmission electron
micrograph of the Leaf-
meditated AuNPs on a scale
of 20 nm, b)
Transmission electron micrograph of the Leaf- meditated AuNPs on a scale of and
100 nm, c) Transmission
electron distribution graph of Leaf- meditated AuNPs, d) Transmission electron micrograph of the
stem mediated AuNPs 20 nm, e)
Transmission electron micrograph of the stem mediated
AuNPs on the scale of 100 nm, f)
Transmission electron distribution graph stem mediated AuNPs
%20517-526_files/image008.png)
Fig. 4 a) Fourier Transmission Infrared of the leaf mediated
nanoparticles, b) Fourier
Transmission Infrared of the stem mediated nanoparticles
Table 2: Antibacterial screening of Parkia biglobosa stem and leaf mediated AuNPs against the
bacteria isolates (Data were expressed as mean of 3 replicates)
|
Bacteria strain |
Inhibition Zone
(mm) |
||
|
|
G-S |
L-AuNPs |
S-AuNPs |
|
B. A |
0.00 |
40.00 |
42.50 |
|
E.C |
0.00 |
37.50 |
46.00 |
|
P. S |
0.00 |
51.00 |
50.50 |
|
S. A |
0.00 |
62.50 |
66.00 |
Inference B.A=Bacillus
anthracis E.C. Escherichia coli, P.S Pseudomonas syringae, S.A Staphylococcus aureus
%20517-526_files/image010.png)
Fig. 5: X-ray powder diffraction graph of the stem- AuNPs and leaf-AuNPs
The FT-IR results show the
presence of phenols which could be the reduction agent for the AuNPs (Sadeghi et al. 2015; Markus et al.
2017; Chahardoli et al. 2018).
To further probe into the crystal structure of the
AuNPs synthesized, X-ray diffraction (XRD) experiment was performed which
revealed that, both PS-AuNPs and PL-AuNPs displayed four pronounced diffraction
peaks corresponding to the (111), (200), (220) and (311) planes of Au, at a
2θ of approximately 38.184°, 44.392°, 64.578° and 77.547°, respectively as
shown in Fig. 5 and 6 above. In totality, the planes and peaks observed with
the gold nanoparticles are, respectively, the typical peaks for the (111),
(200), (220) and (311) planes of Au (JCPDS: 04–0784) NPs, indicating
the successful synthesis of a reduction of the chloroauric gold to obtain gold nanoparticles
with a cubic structure, Fm-3m (225). The intensity of the (111) peak
is much stronger than those of other peaks, suggesting that the Au (111) plane
is the predominant crystal facet in the synthesized Au nanoparticles.
As represented in Table 2 above the AuNPs tested against the Pseudomonas
syringae recorded an inhibition of 50.50 mm for the PS-AuNPs and 51 mm for
the PL-AuNPs. Moreover, the zone of inhibition of Escherichia coli was
evidenced for PS-AuNPs at 46 mm whiles that of PL-AuNPs was recorded at 37.5 mm.
Notwithstanding a significant inhibition of PS-AuNPs at 62 mm and PL-AuNPs had
an inhibition zone at 66 mm for Staphylococcus aureus. The gold
nanoparticles colloids also had an inhibition on the growth of Bacillus
anthracis where PS-AuNPs inhibited to a diameter of 42.5 mm whiles PL-AuNPs
recorded 40 mm. Lastly, the G.S however did not have any inhibition on the
isolates.
Discussion
Pseudomonas syringae, Escherichia coli, Staphylococcus aureus and Bacillus
anthracis were identified. Pseudomonas syringae is a gram-negative
bacteria pathogen that infects over 50 species of pathovars leading to canker
and leaf spots in plants. Escherichia coli is a causative agent of
gastrointestinal infections (Cabral 2010),
normally found in contaminated water and food. Bacillus anthracis on the
other hand is a gram-positive bacterium classified as zoonotic because it
infects both humans and animals. It causes anthrax which may be fatal in some
cases. Similarly, Staphylococcus aureus is a gram-positive bacterium
associated with skin and respiratory infections.
%20517-526_files/image012.png)
Fig. 6: Plates with the zone of inhibition of both the
stem-mediated AuNPs (PS-AuNPs) and Leaf-mediated AuNPs (PL-AuNPs) against; a) Pseudomonas syringae
(P.S), b) Escherichia coli.
(E.C), c) Staphylococcus aureus
(S.A), d) Bacillus anthracis
The disk diffusion method was used to evaluate the antibacterial
efficacy of the bacteria isolates. Our findings recorded for both stem and leaf
mediated synthesized AuNPs at 50% each for Pseudomonas syringae supports
works done by (Mishra et al. 2014).
Moreover, our susceptibility reports of the AuNPs on the inhibition of E.
coli are supported by (Ali et al.
2011). The results of the biosynthesized gold nanoparticle inhibiting
the growth of the Bacillus anthracis are, however, in contrast to a
report by (Abbai et al. 2016) who
stated that AuNPs did not have any inhibition efficacy on Bacillus anthracis
in their study conducted.
It was realized that the effectiveness of the AuNPs on the gram-positive
bacteria compared to gram-negative bacteria is significant. This is because the
cell wall of gram-positive bacteria is single-layered which allows easy entry
of the nanoparticles, whiles the cell wall of the gram-negative bacteria is
wavy and double-layered. Although the mechanism of AuNPs activities in the
inhibition of the growth bacteria is not well documented, gold nanoparticles has
been reported to create holes in the cell wall, allowing
cell contents to leak, resulting in cell death (Mishra
et al. 2014). The potency of metal nanoparticles against
bacterial pathogens can also be due to the presence of antibacterial
macromolecules, such as proteins and enzymes which are adsorbed on the surface
of synthesized nanoparticles and which increase the antimicrobial activity of
nanoparticles (Balakumaran et al. 2016).
As a potential mechanism, AuNPs can bind to the important characteristics of
the bacterial outer membrane and thus lead to structural changes, degradation
and ultimately cell death. This is because they interact with the DNA in the
bacterial cells and interfere with the transcription of the strain’s DNA,
resulting in stunted growth and cell damage (Bauer
et al. 2004; Rai et al. 2010; Srivastava and Mukhopadhyay 2015).
The present results in the treatment of E. coli and S. aureus
with biosynthesized AuNPs showed a higher inhibition as compared to the reports
of researchers using various plant extracts such as Prunus armeniaca, Curcuma
pseudomontana, Carica papaya, Solanum nigrum, Areca
catechu (Muniyappan and Nagarajan 2014; Muthuvel
et al. 2014; Rajan et al. 2015; Srivastava and Mukhopadhyay 2015;
Muthukumar et al. 2016). This could be due to the different
bacteria strains, the different structure and composition of cell walls and the
smaller sizes of the P. biglobosa mediated AuNPs produced. Factors such
as the phytochemical composition of the plant extracts, properties of the
nanoparticles, and particle shape and surface can also be involved in the
site-specific reaction of bacterial strains. The ineffectiveness of the G.S on
the isolates could be attributed to the large particle size of the gold salt
which did not allow penetration through the cell wall of the bacterial strains.
Conclusion
A biosynthesized nanoparticle was obtained by using
the leaf and stem extracts of Parkia biglobosa to reduce chloroauric gold to obtain their nanoparticles form. The Parkia
biglobosa mediated AuNPs were evaluated by UV-Vis, FTIR, TEM, and XRD which
confirmed the successful synthesis of AuNPs. The as-synthesized AuNPs
were able to inhibit the growth of the symbiotic bacteria isolated from the
Pine Wilt Nematode with the highest inhibitory activity from the greatest to
the lowest in the order staphylococcus aureus < Pseudomonas syringae < Bacillus
anthracis < Escherichia coli. This method of producing NPs by
employing plants and plant components has proven to be a viable option for
obtaining toxic-free NPs safe for biomedical and pharmaceutical application.
With the extensive research into NPs for antimicrobial properties, their use in
pesticide production will demonstrate a new generation way of an improved
agricultural practices.
Acknowledgement
The authors immensely
acknowledge the financial supports from Jiangsu special Approved Professor
Program Sujiaoshi ([2015]17).
Author Contributions
Joan Shine Davids: Conceptualization, Data curation, Formal
analysis, Investigation, Methodology, Writing original draft; Micheal Ackah:
Resources, visualization; Emmanuel Okoampah: Writing review and editing; Sandra
Senyo Fometu: Writing review and editing; Zhang Jianping: Supervision,
analysis; Wu Guohua: Funding Acquisition, Project administration, supervision,
Validation.
Conflict of Intrest
Authors declare no conflicts of
interests among institutions.
Data Availability
This work does not involve animals hence.
Ethics Approval
Not applicable to this article.
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