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Research
, Volume: 16( 1) DOI: DOI: 10.37532/0974-7494.2022.16(1)

Biosynthesis, Characterization, Nematicidal Efficacy of Silver Nanoparticles Synthesized using Solanum nigrum Fruit against Root Knot Nematode Meloidogyne incognita

*Correspondence:
Saranya Thiruvenkataswamy
Department of Zoology,
Bharathiar University,
Tamil Nadu,
India
E-mail: [email protected]

Received: 04-November-2019, Manuscript No. tsnsnt-21-4205; Editor assigned: 08-November-2019, PreQC No. tsnsnt- 21-4205; Reviewed: 22-November-2019, QC No. tsnsnt-21-4205; Revised: 28-June-2022, QI No. tsnsnt-21-4205; Manuscript No. tsnsnt-21-4205; Published: 26-July-2022; DOI: 10.37532/0974-7494.2022.16(1).140

Citation: Thiruvenkataswamy S, Paulpandi S, and Narayanasamy M, et al. Biosynthesis, Characterization, Nematicidal Efficacy of Silver Nanoparticles Synthesized using Solanum nigrum Fruit against Root Knot Nematode Meloidogyne incognita. Nano Tech Nano Sci Ind J. 2022;16(1):140.

Abstract

The present study to synthesis Sliver nanoparticles by using fruit extract of European black nightshade (Solanum nigrum) and to test their characterized along with their potentiality to control the Meloidogyne incognita at different concentration. The synthesized AgNPs were characterized by UV-Vis Spectroscopy, Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction analysis (XRD). The synthesized AgNPs result of UV-Spectroscopy and the strong broad peak located at 443 nm. In SEM analysis, the synthesized silver nanoparticles was clearly seen that in a spherical shape. The size of AgNPs range is 30 nm. In FTIR analysis, IR bands of synthesised AgNPs observed functional groups are methyl, methylene and methoxy groups, secondary amines and vinyl groups. In XRD analysis, the average size of AgNPs particles is 29 nm and are crystalline in nature. In nematicidal activity of AgNPs on M. incognita at different concentrations the percent mortality caused by 2.5 μg/ml (100%) of AgNPs was higher than the percent mortality caused by 0.5 μg/ml (48%) of AgNPs.

Keywords

Silver nanoparticle; Solanum nigrum fruit; Root knot nematode; Meloidogyne incognita

Introduction

Plant-parasitic nematodes are recognized as major agricultural pathogens and are known to attack plants and cause crop losses throughout the world. These parasitic worms attack crop root systems, they siphon crucial growth nutrients reducing crop yields. The surviving plants also are more vulnerable to secondary infections, drought, and other stresses. The vast majority of plant parasitic nematode damage is caused by sedentary endoparasitic forms, in particular, the root-knot (Meloidogyne spp.), soybean cyst (Heterodera glycines), and potato cyst (Globodera spp.) nematodes, which impact a wide range of crops, such as soybeans, potatoes, bananas, cotton, corn, strawberries, and tomatoes [1]. Several phytoparasitic nematode species cause histological damages to roots, including the formation of visible galls (e.g. by root-knot nematodes), which are useful characters for their diagnostic in the field. Root-Knot Nematode (RKN) is one of the most destructive pathogen of a wide variety of crops, and causes annual crop loss of approximately US $100 billion globally [2]. They exist in soil in areas with hot climates or short winters. About 2000 plants worldwide are susceptible to infection by root-knot nematodes and they cause approximately 5% of global crop loss. Root-knot nematode larvae infect plant roots, causing the development of root-knot galls that drain the plant's photosynthate and nutrients. Infection of young plants may be lethal, while infection of mature plants causes decreased yield.

Root-knot nematodes (Meloidogyne spp.) are one of the three most economically damaging genera of plant-parasitic nematodes on horticultural and field crops. Root-knot nematodes are distributed worldwide, and are obligate parasites of the roots of thousands of plant species, including monocotyledonous and dicotyledonous, herbaceous and woody plants. The genus includes more than 90 species, with some species having several races. Four Meloidogyne species (M. javanica, M. arenaria, M. incognita, and M. hapla) are major pests worldwide, with another seven being important on a local basis. Meloidogyne occurs in 23 of 43 crops listed as having plant-parasitic nematodes of major importance, ranging from field crops, through pasture and grasses, to horticultural, ornamental and vegetable crops. If root-knot nematodes become established in deep-rooted, perennial crops, control is difficult and options are limited. Researchers all over the world are engaged in standardizing the root-knot nematode management strategies by following non-chemical and eco-friendly alternatives such as sanitation, soil management, organic amendments, fertilization, biological control and heat-based methods to stabilize vegetable production [3]. In recent years, yield losses have been strongly associated with Root-Knot Nematode (RKN) of Meloidogyne spp. and weeds, the invasion of which are high when crops are grown under intensive regimens [4].

In past decades, synthetic nematicides have been the only weapon used against these parasites, but their use is now a days somewhat reduced, and many products have actually been phased-out from the market because their indiscriminate use affected no target organisms with consequential problems for the environment [5,6]. The development of an alternative ecofriendly tool to control crop-damaging nematodes represents an important challenge. Soil solarization is the most diffused nonchemical method to control nematodes, but this is effective only up to a depth of 20 cm in the soil, and when the temperature increases, those nematodes still surviving in the deeper soil layers can migrate upward and reinfest the previously disinfested area. The practice of intercropping could be another way to delay or inhibit nematodes’ reinfestation, although it is not always suitable when intensive cropping is involved [7]. Nanotechnology is an important branch which includes development of biologically made experimental process for the synthesis of nanoparticles. Recently, nanotechnology has become more and more important in the biomedical and pharmaceutical areas as possible and antimicrobial due to existence of infectious diseases and the appearance of antibiotic- resistant strains [8]. The field of Nano biotechnology, has received considerable attention in the usage of various biological things [9]. Nanoparticles can be synthesized by few methods: Chemical and physical and another one important method is biosynthesis. Biosynthesis of nanoparticles is a developing area in the field of nanotechnology which is eco-friendly [10]. In the field of nanotechnology, biosynthesis of nanoparticles is the important area which has economically and eco-friendly tremendous benefits compared to the chemical and physical methods of synthesis [11].

Among the all noble metal nanoparticles, silver nanoparticle are an arch product from the field of nanotechnology which has gained boundless interests because of their unique properties such as chemical stability, good conductivity, catalytic and most important antibacterial, anti-viral, antifungal in addition to anti-inflammatory activities which can be incorporated into composite fibers, cryogenic superconducting materials, cosmetic products, food industry and electronic components [12]. The use of plants as the production assembly of silver nanoparticles has drawn attention, because of its rapid, ecofriendly, non-pathogenic, economical protocol and providing a single step technique for the biosynthetic processes. The reduction and stabilization of silver ions by combination of biomolecules such as proteins, amino acids, enzymes, polysaccharides, alkaloids, tannins, phenolics, saponins, terpinoids and vitamins which are already established in the plant extracts having medicinal values and are environmental benign, yet chemically complex structures [13]. A lot of literature has been reported to till date on biological syntheses of silver nanoparticles using microorganisms including bacteria, fungi and plants because of their antioxidant or reducing properties typically responsible for the reduction of metal compounds in their respective nanoparticles. Although; among the various biological methods of silver nanoparticle synthesis, microbe mediated synthesis is not of industrial feasibility due to the requirements of highly aseptic conditions and their maintenance. Therefore, the use of plant extracts for this purpose is potentially advantageous over microorganisms due to the ease of improvement, the less biohazard and elaborate process of maintaining cell cultures [14].

European black nightshade (Solanum nigrum) or locally just black nightshade, duscle, garden nightshade, garden huckleberry, hound's berry, petty morel, wonder berry, small-fruited black nightshade, or popolo is a species in the Solanum genus. Black nightshade is a common herb or short-lived perennial shrub. It seen in many wooded areas as well as disturbed habitats. It reaches a height of 30 cm to 120 cm (12 to 47 inches), leaves 4.0 cm to 7.5 cm (1.6 to 3.0 inches) long and 2 cm to 5 cm (1 to 2 inches) wide; ovate to heart shaped. The flowers have petals greenish to whitish, recurred when aged and surround prominent bright yellow anthers. The berry is mostly 6 mm to 8 mm (0.24 to 0.31 inches) in diam., dull black or purple-black in color. In India, another strain is see with berries that turn red when ripe. Parts of this plant can be toxic to livestock and humans [15]. Nonetheless, ripe berries and cooked leaves of edible strains are used as food in some locales, and plant parts are used as a traditional medicine. Many of the other "black nightshade" species as refer as "Solanum nigrum". Sometimes S. nigrum is puzzled for the more toxic deadly nightshade, Atropa belladonna, in a different Solanaceae genus altogether.

A comparison of the fruit shows that the black nightshade berries grow in bunches, the deadly nightshade berries grow individually. S. nigrum, a widely used plant in Asian culture medicine, has been shown to possess numerous activities such as antitumorigenic, antioxidant, anti-inflammatory, hepatoprotective, diuretic, and antipyretic [16].

Materials and Methods

Fruit material and chemical

The fresh fruits were collected from the S. nigrum plant in the nearest filed. Silver nitrate and deionized water were used throughout the experiment. The silver nitrate was purchased from Sigma Aldrich.

Preparation of S. nigrum fruit extract

S. nigrum fruits were washed with deionized water to remove the dust particles. About five gram fruits were boiled in 100 ml deionized water for 10 mins at 100°C. Then the boiled solution was allowed cool at room temperature and then the solution was filtered through Whatman No. 1 filter paper. Filtered solution was stored at 4°C for further use.

Synthesis of silver nanoparticles

To prepare 20 mM stock solution of silver nitrate (Sigma Aldrich), 169.87 mg of AgNO3 was dissolved in 50 ml deionized water. For preparing biogenic silver nanoparticles, 50 ml fruit extract was added drop wise to 50 ml silver nitrate solution (conc. 20 mM) so that the final concentration of the mixture remained 10 mM. The mixture was kept at room temperature and the color change was obtained after 90 mins. The brown colour indicate silver ions occurred rapidly. The nanoparticles were purified by centrifuged at 4500 rpm for 15 mins, wash with deionized water. The centrifuged method for do again and again to get the purity of the nanoparticles. At the end, the precipitate deposited at the bottom of tube was collected carefully and dried with the help of hot air oven.

Characterization of AgNPs

Bioreduction of Ag+ in aqueous solution was monitored by sampling of aliquots (0.2 mL) of the suspension, then diluting the samples with 2 mL de-ionized water. UV-Vis spectroscopy analyses of silver nanoparticles produced were carried out as a function of bioreduction time at room temperature on Ultraviolet-Visible spectroscopy. The presence of green-synthesized silver nanoparticles was confirmed by sampling the reaction mixture at regular intervals, and the absorption maxima was scanned by UV-Vis, at the wavelength of 200 nm-800 nm in a UV-3600 Shimadzu spectrophotometer at 1 nm resolution. Furthermore, the dried AgNPs was used for Scanning Electron Microscopy (SEM), Energy-Dispersive Spectroscopy (EDS), Fourier Transform Infrared (FTIR) analyses and X-Ray Diffraction (XRD).

Multiplication of M. incognita

The nematodes, M. incognita used in the present studies were collected from a single egg mass and multiplied on a tomato plant. For multiplication, 3 weeks old tomato seedlings were transferred in earthen pots containing 2 kg of sterilized soil. The soil was prepared with Red soil, Farmyard manure and sand in the ratio of 2:1:1. The pot was inoculated with 1000 M. incognita individuals.

Extraction of M. incognita egg mass and recovery of second stage juveniles

The tomato roots were washed in tap water and cut into 2 to 3 cm small pieces. The roots were taken in a sterile bottle and then treated with 0.5% sodium hypochlorite solution to digest the gelatinous matrix and vigorously shaken for 5-10 minutes. The roots were then washed well in running tap water to remove the excess of sodium hypochlorite. The washed roots were filtered through 100 mesh, 200 mesh and 500 mesh sieves accordingly and eggs on the 500 mesh sieve were collected. The eggs were allowed to hatch using modified Baermann funnel method that comprises concave mesh roofed with filter paper in a petridish with water.

Mortality bioassay

To see the effects of green synthesized silver nanoparticles on the mortality of M. incognita J2s, 100 μl of freshly hatched juvenile suspension which contains 20 nematodes were added to the wells of 96 well cell culture plate and 100 μl of different concentrations of AgNPs (0.5 μg/ml, 0.1 μg/ml, 1.5 μl/ml, 2 μg/ml and 2.5 μg/ml) were added.

The wells containing distilled water served as control. There were 5 replications for each treatment. The mortality rate was recorded after 12 hours, 24 hours and 48 hours. Nematodes are considered alive if they moved or appeared as a winding shape and are considered dead if they do not move when probed with a fine needle.

Data collection

The percent mortality was calculated and corrected by Abbott’s formula as follows,

Mortality (%)=(t-c)/(100-c) × 100

Where t:Percent mortality in the treatment; c:Percent mortality in the control.

Results and Discussion

UV-Visible absorption spectroscopy studies

The UV-Visible spectra analysis was carried out in the present study. Reduction of AgNO3 was visually evident from the color change (brownish-yellow) of the reaction mixture after 90 mins. The intensity of the brown color increased in direct proportion to the incubation period. This may be due to the excitation of the Surface Plasmon Resonance (SPR) effect and the reduction of AgNO3. The silver nanoparticles obtained were characterized by UV-visible spectroscopy and the characteristic absorption peaks at 443 nm (Figure 1) in the spectrum confirmed the formation of silver nanoparticles.

Spectra

Figure 1: UV-Spectra image of AgNPs synthesized using Solanum nigrum fruit extract fruit extract. Note: EquationAgNPs after 90 mins incubation,Equation 30 mins incubation, Equation60 mins incubation.

Characterization of silver nanoparticles by SEM analysis

SEM images (Figure 2) recorded at high magnifications of the AgNPs synthesized by treating AgNO3 solution with S. nigrum fruit extract. The results show AgNPs were predominantly uniform size, and the shape of the particles was predicted. The percentage of resulting nanoparticles were spherical and in the size of 30 nm.

SEM

Figure 2: SEM image of AgNPs.

Fourier transform infrared spectroscopy (FTIR) studies

The characterization of the plant extract and the resulting silver nanoparticles was analyzed in FTIR Figure 3. The absorbance bands analysis in bioreduction are observed in the region of 500 cm-1-4000 cm-1 are 1024.02 cm-1, 1383.68 cm-1, 1629.55 cm-1, 2921.63 cm-1, 3449.30 cm-1. These peaks could be assigned to the C-H, C=O, -OH Stretch, Secondary amine NH Stretch, C-H bend, and C-O groups respectively.

FTIR

Figure 3: FTIR image of AgNPs.

X-ray diffraction (XRD) studies

A XRD profile of biosynthesized silver nanoparticles is shown in Figure 4. The diffraction peaks at 2θ=38.20º, 44.40º, 64.60º and 77.60º corresponding lattice plane values were indexed as (1 1 1), (2 0 0), (2 2 0) and (3 1 1) reflections planes of a Faced Centre Cubic (FCC) originating from the silver substrate lattice of silver were obtained. According to Debye-Scherrrer’s equation the size of the synthesized AgNPs is about 29 nm.

XRD

Figure 4: XRD image of AgNPs.

Nematicidal activity of AgNPs

Figure 5 shows the percentage mortality of M. incognita treated with AgNPS. Maximum mortality was caused at ‘2.5 µg/ml’ and minimum at ‘0.5 µg/ml’ concentrations. Similarly, time also had significant effects on juvenile mortality It was confirmed that the mortality rate of nematodes was directly proportional to the time and concentration of the AgNPs. From this plot, which reveals the dose and time response relationship at 12, 24, and 48 of exposure, it is evident that the mortality of M. incognita J2s was not an immediate incident but increased gradually overtime.

Characterization of silver nanoparticles

Nanoparticles characterization is significant to understand and manage the nanoparticles synthesis and applications. Characterization is achieved by using different variety of techniques such as UV-vis spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), powder X-Ray Diffractometry (XRD) and Scanning Electron Microscopy (SEM), [17-21]. These techniques are used for determination of different parameters such as particle range, character, crystallinity, and surface area. Silver is known as very good antimicrobial agent against various pathogen used for several decades. In the recent year, researchers paid more attention on the green synthesis and applications of AgNPs using various plant extracts. The nanoparticle obtained by the bio-reduction of silver ions using S. nigrum fruit extract is due to active reducing ingredients in the extracellular filtrate which turns the extract to dark brown color. UV-vis absorption spectroscopy technique can be used to investigate protein-ligand complex formation and explore the structural changes in proteins [22]. It is a method (indirect) to examine the bioreduction of AgNPs from aqueous AgNO3 solution. The broad spectrum for AgNPs was observed at 443 nm due to arising excitation of Surface Plasmon Resonance (SPR) [23].

FESEM technique was applied to determine the nano size and shape of metallic silver particles produced. The FESEM image of silver nanoparticles synthesized by fruit extract was assembled on the surface due to interactions such as hydrogen bonding and electrostatic interactions between the bio-organic capping molecules bound to the AgNPs. According to the scanning electron micrograph, the morphology of the silver nanoparticles is in aggregated form from 25 nm to 30 nm. It is well-known that when liquids that contain fine particles were evaporated on a flat surface, the particles accumulate along the outer edge and form typical structures. The FESEM analysis was performed after 10 days following the completion of experiments and the AgNPs did not agglomerate. Results strongly confirmed that S. nigrum leaf extracts might act both as reducing and stabilizing agent in production of AgNPs described that the smaller size makes particles penetrate the cell easily [24]. It is noteworthy to mention that the geometrically triangular nanoparticles are having very sharp vertexes and edges that would be more powerful in damaging the target cells.

FTIR spectroscopy was used to characterize and identify biomolecules that were bound specifically on the synthesized AgNPs. The spectra of synthesized nanoparticles showed the distinct peak in the range of 1024.02 cm-1, 1383.68 cm-1, 1629.55 cm-1, 2921.63 cm-1, 3449.30 cm-1. Major peaks were observed at 2921 cm-1 that could be assigned to the C-H stretching vibrations of methyl, methylene and methoxy groups. The mechanism of adsorption and capping of AgNPs by S. nigrum can be explained through coordination of carbonyl bond (3449 cm-1), therefore electron transfer from C=O to Ag-NP (Qiu et al.) [6]. 3449 cm-1 - OH Stretch, 1629 cm-1 Secondary amine NH Stretch, 1383 cm-1 -Vinyl C-H bend, and 1024 cm-1 might be contributed by the C-O groups of the polysaccharides in the aqueous extract of plant. Further it is suggested that the asymmetric -CH3-bending modes of methyl groups of protein may be responsible for the reduction of silver nitrate ions. The XRD pattern clearly shows that the silver nanoparticles synthesized by the green method are crystalline in nature. Apart from these peaks responsible for silver nanoparticles, the recorded XRD pattern shows additional un-assigned peaks. This may be due to the formation of the crystalline bio-organic compounds that are present in the S. nigrum fruit extract (Figure 5).

Meloidogyne

Figure 5: Percent mortality of Meloidogyne incognita treated with AgNPS % mortality at various concentrations followed by the same letter in superscript are not significantly different at P<0.05% according to Duncan’s multiple range. Note: Equation12 h Equation24 h Equation48 h

Thus, the XRD pattern proves to be strong evidence in favour of the UV-visual spectra for the presence of silver nanocrystals. The XRD patterns displayed here are consistent with earlier reports. Apart from these, other unidentified peaks at 2θ=27.63º, 31.96º and 46.08º arises, possibly due to organic impurities present in the sample. These unidentified peaks in the XRD are also apparent in other AgNPs works also.

Nematicidal activity of AgNPs

The AgNPs synthesized by S. nigrum fruit extracts showed high nematicidal activity against the M. incognita J2s [25,26]. High mortality was obtained by 2.5 µg/ml and low mortality was obtained by 0.5 µg/ml of AgNPs reported that the silica nanoparticles induce premature aging phenotype in C. elegans reported that the intracellular uptake of silver nanoparticles prevents nematodes to lay eggs and this leads to the embryos to develop internally in C. elegans reported that the silver nanoparticles induce dose- response effects on the nematode C. elegans found that the application of silver nanoparticles in root knot nematode significantly reduce the nematode population [27,28]. Nematicidal effect of AgNPs against root-knot nematodes likely applies to other genera of plant-parasitic nematodes and also to plant-pathogenic fungi, because its mode of action is not specific but associated with disrupting multiple cellular mechanisms including membrane permeability, ATP synthesis, and response to oxidative stress in both eukaryotic and prokaryotic cells. For this reason, AgNPs is a broad-spectrum antimicrobial agent capable of affecting plantpathogenic bacteria and fungi.

Conclusion

In the present study, experiments have been conducted to investigate the synthesis of silver nanoparticles using S. nigrum fruit extract and the synthesized nanoparticles have been characterized along with their potentiality to control the M. incognita at different concentrations. Following are the outcome of the present study. Synthesis of silver nanoparticles was carried out by adding different concentration of S. nigrum fruit extract to get optimal size of nanoparticles. These nanoparticles was characterized by using UV-Spectroscopy, SEM, FTIR and XRD to find the nanoparticle size, shape, functional groups and cyrstallinity. The synthesized silver nanoparticles was analyzed by UV-Spectroscopy and the strong broad peak located at 443 nm. In SEM analysis, the synthesized silver nanoparticles was clearly seen that in a spherical shape. The size of AgNPs range is 30 nm. In FTIR analysis, IR bands of synthesised AgNPs observed functional groups are methyl, methylene and methoxy groups, secondary amines and vinyl groups. The different peaks of the silver nanoparticles observed in XRD analysis, the average size of particles is 29 nm and are crystalline in nature. Significant result was obtained in nematicidal activity of AgNPs on M. incognita at different concentrations. Percent mortality caused by 2.5 µg/ml (100%) of AgNPs was higher than the percent mortality caused by 0.5 µg/ml (48%) of AgNPs.

Disclosure

The authors report no conflict of interest in this work. There is no funding agency for this study.

References

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