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Current Nanoscience, 2014, 10, 000-000
Application of Biosynthesized Silver Nanoparticles in Agricultural and Marine Pest Control M. Yokesh Babu*1, V. Janaki Devi1, C.M. Ramakritinan2, R. Umarani3, Nayimabanu Taredahalli4 and A.K.Kumaraguru1 1
Department of Marine and Coastal Studies, Madurai Kamaraj University, Madurai-625021, Tamilnadu, India; Department of Marine & Coastal Studies, Madurai Kamaraj University, Madurai-625021. Tamilnadu-India; 3 Department of Zoology, Devanga Arts College, Aruppukottai, Madurai-626101. Tamilnadu-India; 4Department of Entomology, University of Agricultural Sciences, Gandhi Krishi Vignana Kendra, Bangalore 560065 2
Abstract: The present study is focused on the applications of biosynthesized silver nanoparticles in various fields. Silver nanoparticles are synthesized in vitro using marine bacteria Shewanella algae bangaramma in the laboratory. The silver nanoparticles are characterized by using UV–Vis spectrum, TEM, FTIR, EDAX, XRD and AFM analysis. The synthesized silver nanoparticles are spherical, crystalline and 5-30 nm in diameter. They were found to have both larvicidal and bactericidal activities. There is no mortality in the control. The maximum LC 50 and LC 90 values with 95% confidential limit (4.529 mg/ml (2.478 - 5.911), 9.580 mg/ml (7.528-14.541)) were observed with III- instar larvae of Lepidiota mansueta (Burmeister). In exposed groups the mortality of the larvae was significantly increased in all concentrations (p < 0.0001). The order of bactericidal activity against marine fouling bacteria is found to be Pseudomonas spp. < Vibrio cholera < Roseobacter spp. < Alteromanas spp. To date, this is the first report on the marine bacteria mediated synthesis of silver nanoparticles in culture medium which has effective larvicidal and antifouling activities.
Keywords: Antifouling, biosynthesis, larvicidal, Lepidiota mansueta, Shewanella algae, Silver nanoparticles. INTRODUCTION Pests are a key antagonist against agricultural production systems. White grubs are common insect pests of sugarcane throughout the world . Lepidiota mansueta Burmeister, is a major sugarcane white grub, which causes heavy damage to sugarcane by root invasion . The III- instar larvae of the grub damages the underground part of the stem and root in sugarcane. This leads to the reduction of growth, and ultimately death . In India, these grubs damage the potato and colocasia plants (http://www.ainpwhitegrubs.com) also. Recently, scientists along with farmers have been trying to control the grubs using conventional insecticides through the soil medium . This same practice has been in operation in the western part of Uttar Pradesh as a collaborative programme to control the white grubs by spraying the aggregated pheromone along with chlorpyriphos/imidacloprid. However, these methods have been found to be less effective against the larvae of Lepidiota mansueta and these insecticides in themselves cause considerable damage to the ecosystem. Continuous usage of these organic insecticides increases the toxic load in the soil and this has accumulated in higher organisms throughout the food web, leading to proteotoxicity and genotoxicity in successive generations . Hence, search for an alternative controlling strategy against the grubs is the need of the hour .
*Address correspondence to this author at the Department of Marine and Coastal Studies, Madurai Kamaraj University, Madurai-625021, Tamilnadu, India; Tel: +91-7845577145; E-mail: [email protected]
Similarly in aquatic ecosystems, marine pests (fouling bacteria) are a critical issue. The mode of entry of marine pests is the ballast water, fouling on vessels, niche areas and pipelines (www.marinepests.gov). It significantly causes heavy damages to the marine vessels and economic sphere is severely affected. Bacteria are the primary fouler in the marine environment . TBT and copper containing antifouling paints are continuously used against the fouling organisms. These chemicals affect non-targeted organisms by manifested as synergistic. In this concern, an optional study for antifouling products is necessary to protect the non targeted organisms and to avoid bio-fouling. Hence alternative methods of controlling the above pests need to be developed considering urgently. Recently, nanotechnology has become one of the novel and almost emerging approach for controlling the bacteria, fungi and insects . With the above backdrop information, this work is mainly focused on to control these pests using biosynthesized silver nanoparticles. MATERIAL AND METHODS Chemicals All analytical reagents and media components were purchased from Hi-media (Mumbai, India) and silver nitrate was procured from Merck (India) Isolation and Characterization of Bacteria The brown seaweed Gracilaria corticata was collected from Pudhumadam, Southeast coast of India. It was identi© 2014 Bentham Science Publishers
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fied at MKU, Pudhumadam, India. The sample was washed thrice with sterile filtered seawater and crushed with mortar and pestle in aseptic conditions. 1 ml of crushed seaweed suspension was inoculated on Zobell Marine agar 2216 and incubated at 28°C for 24hrs. Subsequently, the metal reducing bacteria were isolated based on screening techniques. Without loss of viability, the isolated bacteria can tolerate up to 5mM silver nitrate with the precipitation of brown silver nanoparticles. The bacterium was identified based on morphological, physiological and biochemical characterization according to the methods of Bergey’s manual of determinative bacteriology  followed by ribosomal DNA sequencing. The amplification of DNA was carried out by colony PCR with FP 5’ GGC GTG CTT AAC ACA TGC AAG TCG 3’, RP 5’ GCG GCT GGC ACG TAG TTA G 3’ primers using standard protocols. The amplified PCR products were sequenced by ABI prism DNA sequencer with BigDye terminator. Synthesis of Silver Nanoparticles The characterized isolate was inoculated in Zobell marine broth 2216 and incubated at 28 °C for 24 hrs and the cell growth was measured at 660 nm. At the time of 1.0 OD the 3.5 mM silver nitrate was added in the bacterial culture and incubated at 28 °C under agitation (150 rpm) for 24 hrs in dark condition. As negative controls, bacterial cultures (without silver nitrate) and Zobell marine broth 2216 with silver nitrate (without bacterial cells) were run simultaneously. The bio-reduction of the silver ions in the culture was monitored and the nanoparticle synthesis was confirmed by ultraviolet-visible (UV-vis) spectrophotometer in the range of 300–600 nm at a resolution of 1 nm. The stability of the silver nanoparticles was checked up to three months. Recovery of Silver Nanoparticles Ultrasonic disruption of control and test bacterial cells was carried out with ultrasonic processor (250w Syclon ultrasonic cell disruptors, USA) over three cycles of 15 sec periods, with an interval of 45 s. The sample was centrifuged at 20,000 rpm for 30 min, after centrifugation the silver nanoparticles were lyophilized. Characterization of Silver Nanoparticles The lyophilized sample was subjected to FTIR Spectroscopy (SHIMAD) analysis. Two milligrams of the sample were mixed with 200 mg KBr (FT-IR grade) and pressed into a pellet. The pellet was placed into the sample holder, followed by the FT-IR spectrum was recorded in the range 500 -5000 cm–1 in FT-IR spectroscopy at a resolution of 4 cm-1. TEM studies were carried out using Jeol 2100 microscope operating at 120 kV accelerating voltage. Samples were prepared by placing a drop of silver nanoparticles solution (lyophilized sample dissolved in deionized water) on carbon-coated copper TEM grid. The grid was allowed to dry at room temperature for 3 hrs then the particle size and structure was analyzed under TEM.
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The silver nanoparticles and other elemental materials were present in the experimental solution which was confirmed by EDAX. The X-ray powder diffraction (XRD) patterns were recorded using a Philips PW1710 diffractometer with the Cu KR radiation (ì) 1.5405A° at a scanning rate of 0.02 degrees per second in 2 ranging from 30° to 80°. For XRD analysis the samples were prepared on a glass substrate. In the AFM analysis, the slides were scanned with APE Research-model no: A100SGS microscope. Lyophilized sample was resuspended in milliQ water, 0.1 ml was placed on the cover slip to make a thin film, and allowed to dry for 30 min before being scanned under AFM. The AFM characterization was carried out in ambient temperature. Larvicidal Activity of Silver Nanoparticles Lepidiota mansueta, late III- instar larvae were collected from Jorhat (Assam). During preliminary screening with the laboratory trial, the larvae of Lepidiota mansueta were reared in the Department of Entomology UAS, GKVK, Bangalore. Artificial diets have been fed for larval feeding . The acute toxicity test was conducted to determine the LC50 and LC90 values of synthesized silver nanoparticle. Five test concentrations (5.0, 10.0, 15.0, 20.0, 25.0 mg/ml) were prepared (dissolved in deionized water) and mixed individually with the artificial diet. Larvae were fed this diet throughout the experimental period. Two replicates with 20 larvae were used in each test concentration, and the control group was maintained in the same conditions. The experiments were repeated thrice. After exposure, mortality was observed according to Abbott (1925)  and the observations were recorded at every 24 hrs for 15 days. Furthermore, the dead larvae were collected from the exposed groups and crushed with milliQ water, after sonication and centrifugation the accumulation of silver nanoparticles was confirmed by TEM (same procedure mentioned above). Statistical Analysis The determination of LC50 and LC90 values was performed using EPA Probit analysis version 1.5. A one-way analysis of variance (ANOVA) with Dunnett’s test was used to calculate significant differences between control and experimental groups, P < 0.05 as significant. Linear regression models were used to determine the relationship between exposure concentration and mortality rate. The analyses were performed using GraphPad prism, version 6 and the results are reported as mean ± SD. Antifouling Activities of Silver Nanoparticles The wood panel (24 cm X 24 cm) was incubated for 24 hours in marine water (3m depth), then the fouling bacteria were isolated from the wood panel and identified as Vibrio cholera, Pseudomonas spp. Roseobacter spp. and Alteromanas spp. (Bergey’s manual of determinative bacteriology). These bacteria were individually spread on Zobell marine agar 2216 plates, biosynthesized silver nanoparticle discs were placed on the medium with the concentration of 20
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g/discs, and control disc was maintained with sterile milliQ water; and incubated the plates at 28 °C for 24 hrs. The experiment was repeated thrice and the zone of inhibition was measured. TEM image was taken only for highly responded Alteromonas spp. to study the mechanism of antibacterial activity of silver nanoparticles. RESULTS AND DISCUSSION Biosynthesis of Silver Nanoparticles Previous studies demonstrate that the silver nanoparticles have been synthesized using marine bacteria , fungi , cyanobacteria  and algae . In this study we have identified a new marine bacterial strain reducing silver nitrate Shewanella algae strain bangaramma, using physiological, biochemical characters and 16s rDNA sequencing (NCBI Gene bank JQ029718). Shewanella spp. is a Gram-negative, metal-reducing bacterium. It utilizes numerous metals (Fe (III-), Mn (IV), Cr (VI), U (VI), and Au (III-)) as terminal electron acceptors [15, 16]. Wang et al.  reported that, the Shewanella spp. has the potential to reduce Ag + to Ag0 within the cell; these deposits were easily recovered by reductive precipitation. In this study, after 30 minutes exposure and incubation of bacterial culture plates with silver nitrate the medium turn to light brown in color; it indicates that the bacteria have initiated the silver nitrate reduction, and after 12 hrs, it turns to dark brown in color. It showed that the reaction medium behaves with time kinetics. Normally, the bacteria synthesize silver nanoparticles in 24 hrs reaction time. According to Sachin et al.  the marine bacteria Idiomarina spp. synthesizes silver nanoparticles in 42 hrs, where as Shewanella algae strain bangaramma initiated the nanoparticle synthesis within 30 mins and the stability of silver nanoparticles was retained up to three months.
Fig. (1a). Absorption spectrum of silver nanoparticles synthesized using Shewanella algae bangaramma.
Fig. (1b). FTIR spectrum of silver nanoparticles synthesized by Shewanella algae bangaramma.
Konishi et al.  reported, different strains of Shewanella algae isolated from diverse habitats, was able to reduce the gold and platinum in anaerobic condition with H2 gas. Takashi and coworkers  observed that the cell extract of Shewanella algae was synthesized the gold nanoparticles at room temperature. In the case of Shewanella algae bangaramma, silver nanoparticle synthesis was rapid, commercial and trouble-free than the previous studies. UV Spectrophotometry The silver nanoparticles absorbed radiation in the visible region of the electromagnetic spectrum (380- 450 nm) owing to excitation of surface plasmon vibrations which was responsible for color changes in the medium. In UV–vis spectrum, a strong, broad peaks were observed at 432 nm and 425 nm for 30 min and 12 hrs respectively (Fig. 1a), this significant observation indicates that the reduction of the silver ions. The same peak (425 nm) was retained from 12 hrs to three months of incubation periods. The broadening and slight shifting of the surface plasmon resonance was probably owing to the dampening of the surface plasmon resonance caused by the change in the refractive index of the surrounding medium and the modification of the size and shape of the silver nanoparticles in the medium [20, 21]. The
Fig. (1c). Different X-ray emission spectrum of silver nanoparticles synthesized by Shewanella algae bangaramma.
peak intensity was slowly elevated in reaction time, but no change was observed in peak position that suggests the nucleation of silver nanoparticles initiated merely reaction time.
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tides/ proteins produced by bacteria were involved in the reduction and moreover helps to stabilized the silver nanoparticles . Energy Dispersive X-ray EDX analysis shows the optical absorption peak at 3 keV (Fig. 1c), which is typical for metallic silver nanocrystallites  whereas other signals from Cl, Mg, K and Na atoms were also recorded in the spectrum, because of X-ray emission from other cellular materials. X-ray Diffraction Fig. (1d). X-ray diffraction pattern of the silver nanoparticles synthesized by Shewanella algae bangaramma.
X-ray Diffraction (XRD) pattern shows intense Bragg’s reflections that had been indexed on the basis of metal nano structure. The broadening of Bragg’s peaks indicated the formation of nanocrystals. A few intense characteristic peaks of silver at 2 values of 38.0, 45.45, 66.119, and 75.53 deg corresponding to (111), (200), (220), and (311) planes and exhibit that the synthesized silver nanoparticles were crystalline in nature (Fig. 1d). The diffraction peaks were found to be broad in XRD spectrum, around their bases indicated that the silver particles are in nanosizes, and based on Scherer’s equation Phkl = k/ 1/2 cos, the size of the nanoparticles was found to be ~ 50 nm), Acetobacter xylinum (> 50 nm) , Klebsiella pneumonia, E. coli (< 50 nm) , Corynebacterium spp. SH09 (15 nm) , Aeromonas spp. SH10 (6.4 nm)  and Morganella spp. (20-25 nm) . Larvicidal Activity of Silver Nanoparticles The mortality was observed at different doses such as 5.0, 10.0, 15.0, 20.0, 25.0 mg/ml. The calculated LC50 and LC90 values (with 95 % confidence limits) of silver nanoparticles against late III- instar larvae of Lepidiota mansueta on
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ent mortality was also noticed in all doses (p < 0.005). After initial exposure, no significant mortality was observed in all test concentrations at 24 hrs. In increased exposure time, the mortality was also increased significantly. Based on the statistical analysis, the larvicidal activity of synthesized silver nanoparticles was time and dose dependent.
Fig. (3). % of mortality ( ± SD) of Lepidiota mansueta III instar larvae after exposure to synthesized silver nanoparticles.
Fig. (2). Transmission electron microscopy image of Shewanella algae synthesized silver nanoparticles.
15th day was 4.529 mg/ml (2.478-5.911 mg/ml) and 9.580 mg/ml (7.528 -14.541 mg/ml) respectively (Table 1). No significant mortality was observed in control during the experimental period. Significant dose dependent mortality was observed in all exposed groups based on statistical analysis. The variation in control and between the exposed groups was p < 0.0001 (Fig. 3 and Table 2). Considerable time dependTable 1.
In TEM image, > 15 nm sized nanoparticles were majorly presented in exposed larvae (Fig. 4). This image confirmed that the accumulation of silver nanoparticles in Lepidiota mansueta III- instar larvae is more than 15 nm in size which is responsible for larval death. Stadler et al.  successfully applied nano alumina against two-grain pests Sitophilus oryzae and Rhyzopertha dominica. Mohammad rouhani and his coworkers  reported that physically synthesized pure silver nanoparticles are toxic against Aphis nerii (424.67 mg/ml) than combined with zinc nanoparticles (539.46 mg/ml). Abduz Zahir and Jakubowski  reported that the plant mediated synthesis of silver nanoparticles showed the LD50 value 44.69 ± 5.80 mg/kg in 14 days incubation against Sitophilus oryzae and its efficacy was higher than other chemical pesticides. The larvicidal activity of biosynthesized silver nanoparticles was studied on earlier mos-
Mean value (± SE) of Lepidiota mansueta larvae responded to Shewanella algae bangaramma synthesized silver nanoparticles
Silver nanoparticles Concentration
No. of exposed
Number of response (mean ± SE) 1st day
0 ± 0.00
0 ± 0.00
0 ± 0.00
0 ± 0.00
0 ± 0.00
0 ± 0.00
0 ± 0.00
0 ± 0.00
0 ± 0.00
0 ± 0.00
LC 50 value ( lower-upper with 95% confidence limits)
24.962 (21.125- 37.438)
LC 90 value ( lower-upper with 95% confidence limits)
46.128 (32.841- 140.871)
(n=20/experiment (10+10(duplicate)) the experiment repeated thrice, as a result total number of exposed 60).
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Regression equations, coefficients of determination and summary of regression ANOVA for larvicidal activity of synthesized silver nanoparticles against Lepidiota mansueta III- instar larvae exposed to various concentrations 95% confidence intervals
Y = 0.7687*X - 1.499
0.5643 to 0.9731
Y = 1.139*X - 2.192
0.8611 to 1.416
Y = 1.389*X - 1.213
1.272 to 1.506
Y = 1.398*X - 0.4034
1.298 to 1.499
Y = 1.364*X + 1.013
1.199 to 1.528
Concentrations of synthesized silver nano particles
ANOVA P - value
Fig. (4). Transmission electron microscopy image of silver nanoparticles from exposed Lepidiota mansueta.
quito larvae C. quinquefasciatus (LC50 = 27.49 and 4.56 mg/l; LC90 = 70.38 and 13.14 mg/l) and A. subpictus (LC50 = 27.85 and 5.14 mg/l; LC90 = 71.45 and 25.68 mg/l) ; Mimosa pudica synthesized silver nanoparticles against A. subpictus (LC50 = 0.69 mg/l); C. quinquefasciatus (LC50 = 1.10 mg/l) ; Nelumbo nucifera synthesized silver nanoparticles against the larvae of A. subpictus (LC50 = 0.69 mg/l; LC90 = 2.15 mg/l) and the larvae of C. quinquefasciatus (LC50 = 1.10 mg/l; LC90= 3.59 mg/l) . Nano silver synthesized by filamentous fungus Cochliobolus lunatus was tested against second, third, and fourth instar larvae of A. aegypti (LC50 1.29, 1.48, and 1.58; LC90 3.08, 3.33, and 3.41 mg/l) and A. stephensi (LC50 1.17, 1.30, and 1.41; LC90 2.99, 3.13, and 3.29 mg/l) . In contrast with Mohammad Rouhani et al. , the present study revealed most of the larvae were dead at low concentrations and the remaining larvae were not able to pupate properly. These interactions have potentially disrupted the vital cell processes like enzyme function, and gene translation . As proved in an earlier study’ the toxicity of silver NPs was generated free oxygen radicals in larvae by inhibiting the copper utilizing detoxification enzymes. These oxidative stresses were subsequently results in DNA damage and ultimately induce apoptosis that leads to cell death [42, 43].
Antifouling Activities of Silver Nanoparticles The Gram negative bacteria are the opening fouler and it attached to the surfaces of solid materials when exposed to sea water . Metal nanoparticles based paints are one of the most promising alternatives to shun these problems. In this study the application of silver nanoparticles as an antibacterial agent against fouling bacteria were investigated using disc diffusion method and the mechanism was studied by TEM [44, 45]. Based on the disc diffusion method, the clear lysis zone was formed by silver nanoparticles against Vibrio cholera, Pseudomonas spp. Roseobacter spp. and Alteromanas spp. The zone of inhibition was 13.5 ± 0.17 mm, 13.3 ± 0.22 mm, 12.8 ± 0.33 mm, and 18.2 ± 0.28 mm respectively. The study approves the Pal et al.  statement; when compared to other shapes the spherical nanoparticle inhibits the bacterial replication at low concentrations. TEM images of silver nanoparticles exposed Alteromanas spp. for different time periods are shown in Fig. (5). In this image, the control bacterial cells retained the normal structure (Fig. 5a) in 24 hrs, but the exposed bacterial cells
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Fig. (5). Transmission electron microscopy image of bactericidal activity of silver nanoparticles on marine fouling bacteria Alteromonas sp. a) Control bacterial cell b) Silver nanoparticle bound on bacterial cell after exposure, c) Silver nanoparticles forms pits and gap formation in bacterial cell wall d) dead bacterial cell.
were bounded with silver nanoparticles after immediate exposure (Fig. 5b). Subsequently the bacterial cell was ruptured (Fig. 5c) and finally all the cell materials were oozing out in 24 hrs time period (Fig. 5d). The TEM image confirms the incorporation of silver nanoparticles into the cell; it resolved the contraction of cell by forming pits with irregular shapes in the outer membrane and changes in membrane permeability, followed by progressive release of membrane proteins [46-48]. This observation is a clear explanation of the antibacterial mode of action of these silver nanoparticles.
CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS The source for writing this manuscript was provided by Dr. B.A. AMMA and Dr. A.P. SAKTHI, Melmaruvathur Adhiparasakthi College, Melmaruvathur, Tamilnadu, India.
The highly stable silver nanoparticles synthesized by seaweed associated marine bacteria Shewanella algae bangaramma have the ability to control biofouling in marine ecosystem. Therefore it is recommended that it can be used in antifouling paints. Moreover its larvicidal activity can be used in controlling white grub. Organic chemical impacts in the ecosystems can also be controlled. Silver nanoparticles have been described as ‘oligodynamic’ because of its ability to exert a bactericidal effect at low concentrations. This nano level has nothing to do with the other flora and fauna of the ecosystem. From this study it is concluded that the silver nano particle synthesized is economical, efficient and ecofriendly and it is strongly recommended as a hopeful challenger for agricultural and marine pests.
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Received: June 20, 2013
Revised: August 6, 2013
Accepted: September 9, 2013
  
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