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New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes
Journal of Radiation Research and Applied Sciences xxx (2018) 1e9
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New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes Saad EL-Din Hassan a, Salem S. Salem a, Amr Fouda a, *, Mohamed A. Awad b, Mamdouh S. El-Gamal a, Abdullah M. Abdo a a b
Department of Botany and Microbiology, Faculty of Science, Al-Azhar University, Nasr City, Cairo, Egypt Department of Zoology and Entomology, Faculty of Science, Al-Azhar University, Nasr City, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 April 2018 Received in revised form 18 May 2018 Accepted 20 May 2018 Available online xxx
The biosynthesis of nanoparticles by microorganisms especially endophytic species isolated from medicinal plant are the prime concern of researchers. In the present study, a novel, non-toxic, eco-friendly copper nanoparticles was biosynthesized by endophytic actinomycetes isolate Ca-1 and optimization processes have been endeavored. The endophytic actinomycete Streptomyces capillispiralis Ca-1, was isolated from healthy medicinal plant (Convolvulus arvensis) (L.) collected from Bahariya Oasis - Giza Governorate e Egypt. The isolate was identified by morphological, cultural and molecular identification techniques. The biosynthesis of CuNPs is confirmed by gradual change of biomass filtrate color from light blue into greenish brown color and characterized by an observation of a characteristic absorption peak by UV-Vis spectroscopy at 600 nm. Also, a spherical-monodispersed shaped CuNPs with particle size of 3.6 e59 nm were observed by Transmission Electron Microscopy (TEM). In addition, X-ray diffraction (XRD) exhibited pattern peaks corresponding to 110, 111, 200, 220, 311 and 222 planes, respectively that assigned to face centered cubic forms of metallic copper (JCPDS 04e0836). While FTIR results confirmed the occurrence of bioactive functional groups that are responsible for formation of CuNPs. Moreover, the biosynthesized CuNPs showed various biomedical applications against infectious microorganisms, biocontrol of phytopathogenic fungi and health nasty insects that represent the hopeful uses of copper nanoparticles to be applied as a unique approach to manage these healths threatening problems. © 2018 The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Keywords: Copper nanoparticles Endophytic actinomycetes Streptomyces capillispiralis Biomedical applications
1. Introduction Endophytes are bacteria, fungi or actinomycetes microorganisms that lodge within plant materials in a symbiotic association. A massive number of bioactive compounds produced by Endophytes not only useful for plants but also of economic importance to humans. Endophytic population was greatly influenced by climatic conditions and position where their host plant grows. (Fouda, Hamza, El-Din Hassan, Mohamed Eid, & Ewais, 2015). Endophytic actinomycetes are ubiquitous in most plants and colonize plants
* Corresponding author. E-mail addresses: [email protected], [email protected] (A. Fouda). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications.
tissues without exhibiting pathogenicity and found to be efficient producer of novel antibiotic and other useful compounds that applied in medicines & food industries. (Kumar and Jadeja, 2016); (Hassan, 2017). As an interdisciplinary approach in bio-chemical applications, nanotechnology is focusing on nanoparticles synthesis with amended antimicrobial and antioxidant properties against the worsening diseases and cancer (Duhan et al., 2017). Biosynthesis of nanoparticles using bio-green methods are advantageous over chemical and physical methods in synthesis process due to rapid, clean, simple, non-toxic, inexpensive and ecofriendly synthesis of nanoparticles (Phull et al., 2016); (Mohmed, Saad, FoudaElgamal, & Salem, 2017b) (Mohmed, Fouda, Elgamal, EL-Din HassanShaheen, & Salem, 2017a). Biosynthesis of nanoparticles have been studied from different biological communities such as plants, bacteria, actinomycetes, yeast and fungi which are
https://doi.org/10.1016/j.jrras.2018.05.003 1687-8507/© 2018 The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Hassan, S. E.-D., et al., New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes, Journal of Radiation Research and Applied Sciences (2018), https:// doi.org/10.1016/j.jrras.2018.05.003
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emerging as nano factories and have potential useful applications (Rasool & Hemalatha, 2017); (Foudaa, Saad, Elgamala, Mohmedb, & Salema, 2017) (Fouda et al., 2017). By Ended past few years, nanoparticles of noble metal paid extensive attention owing to their applications in diverse fields and improved physical, chemical and biological properties (Imada, Masuda, Kobayashi, Hamada-Sato, & Nakashima, 2010). By comparison to further noble metals such as Au, Pt and Ag, Cu is a lowcost material however it is also accountable to oxidation generally in nanoscale dimensions. Copper nanoparticles have various properties for example anti-microbial, optical, catalytic, antifungal, anti-insects and anti-cancerous (Dinda, HalderVazquezVazquez, Arturo Lopez-Quintela, & Mitra, 2015); (Fouda & Shaheen, 2017). In addition, it was found that, the copper nanoparticles have a significant antifungal activity against phytopathogenic fungi: Curvularia lunata MTCC No. 2030, Phoma destructiva DBT-66, Fusarium oxysporum MTCC No. 1755 and Alternaria alternata MTCC No. 6572 at low concentrations, which helps to use it in the future as one of the important applied factors in the agricultural field (Kanhed, BirlaGaikwadGadeSeabra, Rubilaret al, 2014). The present study aimed to biosynthesis and characterization of copper nanoparticles using endophytic actinomycetes isolated from medicinal plant. Also, evaluation the efficiency of biosynthesized nanoparticles in medical application as antimicrobial activity, bio-control for phytopathogenic fungi and larvicidal bioassay.
2. Materials and methods 2.1. Sample collection Healthy leaves of medicinal plant Convolvulus arvensis (L.), was collected from El-Bahariya Oasis, Giza Governorate, Egypt (28 210 3800 N - 28 540 5600 E). The collected sample was placed in a sterile bag and brought to the laboratory in an ice-box and directly subjected to selective isolation procedure. Plant picture is shown in plate (1).
2.2. Endophytic actinomycetes isolation In accordance to (Gangwar, Dogra, Gupta, & Kharwar, 2014), plant leaves were washed by running tap water to remove adhered epiphytes and soil debris. After drying under sterile conditions, tissue surfaces were sterilized by using 70% (v/v) ethanol for 5 min followed by sodium hypochlorite solution (2.5%) for 2 min. Surfacesterilized tissues were washed thrice in sterile distilled water. The last washing water was plated onto bacterial, fungal, and actinomycetes culture media of Nutrient agar, Czapek Dox agar, and Starch nitrate agar, respectively. The surface sterilization was confirmed by the absence of any microbial growth onto the previous cultural media. The sterilized leaves were cut into small pieces (0.5e1.0 cm) and placed on starch nitrate agar (SNA) medium supplemented with Nystatin (25 mg/ml) to suppress fungal growth and incubated at 30 ± 2 C for 14 days for actinomycetes isolation. The purified actinomycetes isolates were preserved in SNA slant for further study. 2.3. Extracellular biosynthesis of copper nanoparticles 2.3.1. Biomass preparation Three disks of freshly prepared culture of endophytic actinomycetes isolates were inoculated into 100 mL of starch nitrate broth (SNB) medium. The flasks were incubated at 30 ± 2 C for 4 days in rotary orbital shaker at 150 rpm. The biomass was harvested by passing through four layers of wool cloth. 2.3.2. Preparation of biomass filtrate The collected biomass was washed with sterilized distilled water to remove any media components and suspended in 100 ml distilled water for 72 h at 30 ± 2 C. The biomass filtrate obtained by passing it through Whatman filter paper No.1. Finally, filtrate is then collected and ready to use for further nanoparticles synthesis. 2.3.3. Preparation of copper nanoparticles using biomass filtrate The previously obtained biomass filtrate is being used for synthesis of copper nanoparticles as the following: 20 mM (CuSO4.5H2O) was added to 100 ml biomass filtrate and kept for 6 h at 35 C in dark condition. Biosynthesized cooper nanoparticles were primarily characterized by color change and UV-Vis spectroscopy (JENWAY 6305 Spectrophotometer) at a range of 300e700 nm to select most potent endophytic actinomycetes isolate. 2.4. Molecular identification of most potent endophytic actinomycetes isolate
Plate 1. Field picture of Convolvulus arvensis (L.).
A single colony was taken from the different endophytic actinomycetes isolates and washed in saline solution (NaCl 0.085%). DNA extraction was carried out using the (Thermo K0721) GeneJET Genomic DNA Purification Kit (Sigma, 168 Third Avenue Waltham, MA, USA) according to protocol supplied by the manufacturer. The DNA was used as a template for PCR amplification but with an additional purification step using GeneJET™ PCR Purification Kit (Thermo K0701). The 16S-rRNA gene of four selected isolates was amplified using Maxima Hot Start PCR Master Mix (Thermo K1051) (Sigma) in 20 ml reactions using the universal primer amplification, the primers used were 27 f (50 -AGAGTTTGATCCTGGCTCAG-30 ) and p1492r (50 -TACGGCTACCTTGTTACGACT). All amplification products were checked by electrophoresis on 1% agarose gels (Lane, 1991). The sequences obtained were compared with those deposited in the public databases and pairwise distances were calculated with the EzTaxon server (Kim et al., 2012). The sequences of the four isolates and those of their most closely related taxa retrieved from
Please cite this article in press as: Hassan, S. E.-D., et al., New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes, Journal of Radiation Research and Applied Sciences (2018), https:// doi.org/10.1016/j.jrras.2018.05.003
S.E.-D. Hassan et al. / Journal of Radiation Research and Applied Sciences xxx (2018) 1e9
GenBank were aligned using the CLUSTAL X program (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997) and checked for alignment inconsistencies. Phylogenetic distances were calculated with Kimura's two-parameter model (Kimura, 1980) and evolutionary trees were inferred using the neighbor-joining (Saitou & Nei, 1987). Phylogenetic analyses were performed with the software package MEGA7. 2.5. Characterization of biosynthesized copper nanoparticles The size and shape of CuNPs synthesized by endophytic actinomycetes isolate was determined through Transmission Electron Microscopy (TEM- JEOL 1010 Japan). The binding properties of copper nanoparticles using biomass filtrate were investigated by FTIR analysis (Agilent system Cary 630 FTIR model). X-ray diffraction (XRD) studies of nanoparticles were carried out using Shimadzu Scientific Instruments (SSI), Kyoto, Japan. X-Ray Diffraction patterns for CuNPs were obtained with the XRD- 6000 series, including stress analysis, residual austenite quantitation, crystallite size/lattice strain, crystallinity calculation, materials analysis via overlaid X-ray diffraction patterns Shimadzu apparatus using nickel-filter and Cu- Ka target, the estimation of the size of particles was performed by Scherrer's formula. 2.6. Antimicrobial activity for copper nanoparticles produced by most potent endophytic actinomycetes Antagonistic activity of biosynthesized CuNPs were assessed against prokaryotic and eukaryotic coded test organisms represented by Gram-positive bacteria (Staphylococcus aureus ATCC. 6538, Bacillus subtilis ATCC. 6633 & Bacillus dimenuta ATCC.19146), Gram-negative bacteria (Pseudomonas aeruginosa ATCC. 9022 & Escherichia coli ATCC. 8739), Unicellular Fungi (Candida albican ATCC.10231) and Multicellular Fungi (Aspergillus brasiliensis ATCC .16404) using agar well diffusion method. Mueller Hinton agar plates were seeded with overnight prokaryotic and unicellular fungi while multicellular fungi inoculated on Czapek Dox agar. One hindered microliter (100 ml) of Copper nanoparticles solution (20 mM) was added to each well (0.8 mm). To detect minimum inhibitory concentration (MIC), different concentration of CuNPs solution were made (5, 10, 15, 20 mM). The plates were incubated at 35 ± 2 C for 24 h (bacteria and unicellular fungi) and 28 ± 2 C for 72 h (multicellular fungi). The diameter of inhibition zones around wells were measured (mm) and the results were recorded. (Magaldi et al., 2004); (Valgas, SouzaSm^ ania, & Sm^ ania, 2007). All assays were performed in triplicate. 2.7. Bio-control for phytopathogenic fungi using CuNPs by Agar Plug Assay Phytopathogenic fungi represented by Alternaria spp., Aspergillus niger, Pythium spp. and Fusarium spp. were obtained from Plant pathology Department, Faculty of Agriculture, Zagazig University. Plugs (4 mm in diameter) of 3-day-old fungal cultures were transferred to plates amended with different concentrations of Copper nanoparticles (5, 10, 15 and 20 mM). Control for each fungus was prepared using culture medium without nanoparticles. All plates were incubated at 28 ± 2 C for 5 days. Radial growth of fungal colonies was recorded after 5 days. Growth inhibition percent was calculated using the radial growth of mycelium according to the following equation: Inhibition Percentage ¼ (Control Radial Growth -Treatment Radial Growth)/Control Radial Growth*100 (Mahdizadeh, Safaie, & Khelghatibana, 2015). All assays were performed in triplicate.
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2.8. Evaluation of larvicidal bioassay of biosynthesized nanoparticles against Culex pipiens and Musca domistica 2.8.1. Insects rearing Laboratory reared colony of Culex pipiens (Mosquito) and Musca domistica (House Fly) free from insecticides and pathogens obtained from animal house, Mosquitoes Research Department, Faculty of science, Al-Azhar University in Cairo was maintained starting from egg rafts. Then 3rd instar Larvae of that used in the tests were reared in a plastic cup containing dechlorinated water in case of mosquito (M. K. Ahmed et al., 2018), while house fly 3rd instar Larvae were reared in a plastic cup containing Brewer's dried grains, yeast extract and milk powder under the standard conditions of 28 ± 2 C temperature (Hogsette, 1992). 2.8.2. Larvicidal bioassay The assay was done on healthy late third instar larvae, three replicates for each test were used. Larvae of Culex pipiens were collected with a pasture pipette, placed on a filter paper to remove excess water then placed in cups containing 100 mL dechlorinated tap water. While larvae of Musca domistica were collected and placed in cup containing nutritional media and then different concentrations of the tested CuNPs were added then readjusted according to obtained results. Three controls cups containing distilled water free from CuNPs were used. All cups were covered with muslin cloth for protection. The observed mortality was recorded at (24, 48, 72 and 96 h) larvae were considered dead if there is no sign of any movement even after mild touch with a glass rod (Kumar, Chawla, Dhamodaram, & Balakrishnan, 2014), Percentage of mortality was corrected using of Abbott's formula: ([treatment Mortality%-control Mortality%]/100-control mortality %)*100 (Abbott, 1925). 2.9. Statistical analysis The means of three replications and standard error (SEr ±) were calculated for all the results obtained by Minitab® 18.1 program. 3. Results and discussions 3.1. Endophytic actinomycetes isolation and molecular identification Three endophytic actinomycetes isolates represented by Ca-1, Ca-2 and Ca-3 were isolated from Convolvulus arvensis leaves. These actinomycetes isolates were identified as Streptomyces sp. according to grown by cover slip culture methods which confirmed spiral shape of spore chain and pigment production as shown in Fig. 1. Similarly (Rasool & Hemalatha, 2017), synthesized CuNPs by endophytic actinomycetes. Biosynthesis of copper nanoparticles were optimized and exhibited optimal cultural conditions (pH, 9, Temp. 35 C, incubation period, 4 days, CuSO4.5H2O concentration, 20 mM) using three actinomycetes isolates. The biosynthesis of CuNPs were confirmed by gradual change of biomass filtrate color from light blue into greenish brown color (Fig. 2 A) Also, the absorption spectra were recorded over a range of 300e700 nm and showed the maximum absorbance peak at 600 nm for CuNPs biosynthesized by Ca-1 (Fig. 2 b). The color change occurs due to the excitation of surface plasmon character exhibited in nanoparticles corresponding to CuNPs. Some researchers observed that, the absorption spectra of copper nanoparticles ranged between 580 and 600 nm (Ponmurugan, Manjukarunambika, Elango, & Gnanamangai, 2016). While other noted that the maximum absorption wavelength for CuNPs were at 560 nm (Kuppusamy, IlavenilSrigopalram
Please cite this article in press as: Hassan, S. E.-D., et al., New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes, Journal of Radiation Research and Applied Sciences (2018), https:// doi.org/10.1016/j.jrras.2018.05.003
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Fig. 1. Cultural and microscopic characteristic of endophytic actinomycetes isolates, A-1: Actinomycetes isolate Ca-1 growing on SNA, A-2: microscopic slide for Ca-1 showing spiral shape (X ¼ 400), B-1: Actinomycetes isolate Ca-2 grown on SNA, B-2: microscopic slide for Ca-2 showing spiral shape (X ¼ 400), C-1: Actinomycetes isolate Ca-3 grown on SNA, C-2: microscopic slide for Ca-3 showing spiral shape (X ¼ 600).
Fig. 2. A, color change of biomass filtrate from light blue to greenish brown color, B: UV spectra for CuNPs showing maximum absorbance peak at 600 nm.
ManiamYusoff, Govindanet al, 2017). The exact position of the surface plasmon mainly depends on the particle size and stability of the corresponding CuNPs. (Díaz-Visurraga et al., 2012). Therefore, in our study, endophytic actinomycetes isolate Ca-1 was considered as most potent isolate due to color change and maximum absorbance peaks. Identification of most potent isolate Ca-1 was confirmed at molecular level based on DNA extraction followed by amplification and sequence analysis of 16S rDNA gene fragments that showed these endophytic actinomycetes isolate were identified as Streptomyces capillispiralis with similarity 91% to Streptomyces capillispiralis with GeneBank accession number NR 115408 (Fig. 3). The sequences was obtained from the present study deposited in GenBank under accession number of MH160824.
3.2. Characterization of biosynthesized copper nanoparticles Besides color change and UV-Vis spectroscopy, copper nanoparticles were also characterized by transmission electronic microscopy (TEM) to determine the shape and size of biosynthesized copper nanoparticles. As revealed in Fig (4), a spherical-monodispersed shaped CuNPs with particle size of 3.6e59 nm were observed. Also (Angajala, Pavan, & Subashini, 2014), founded that the CuNPs biosynthesis by Aegle marmelos correa showed diameter of 50e100 nm. In addition, XRD of CuNPs synthesized by Streptomyces capillispiralis Ca-1 exhibited pattern peaks corresponding to 110, 111, 200, 220, 311 and 222 planes, respectively that assigned to face
Please cite this article in press as: Hassan, S. E.-D., et al., New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes, Journal of Radiation Research and Applied Sciences (2018), https:// doi.org/10.1016/j.jrras.2018.05.003
S.E.-D. Hassan et al. / Journal of Radiation Research and Applied Sciences xxx (2018) 1e9
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Fig. 3. Phylogenetic analysis of 16S rRNA sequences of the actinomycetes isolate Ca-1 with the sequences from NCBI. The analysis was conducted with MEGA 6 using neighborjoining method.
to the binding of amide I band of protein with (N H) stretching (Rajeshkumar et al., 2013). Peaks at 1052.509 cm1 was characteristic of carboxylic (COO) residues (Dusane et al., 2011); (Golmoraj et al., 2011). A stretch at 857.198, 792.553 & 670.802 cm1indicated the Amide IV (OCN) stretch bending for protein. The stretch of (S S) band of protein was also found at 586.419 cm1could be attributed to the presence of a carbohydrate moiety (Jain, Mody, Mishra, & Jha, 2012). The result confirm that proteins play key role in formation of copper nanoparticles and act as capping and stabilizer agents in bio-synthesis of CuNPs as previously reported by (Ahmad et al., 2013); (Choudhary, Kumar, Kataria, Singh Cameotra, & Singh, 2016). 3.3. Antimicrobial activity and minimal inhibitory concentration (MIC)
Fig. 4. TEM image for CuNPs synthesized by Streptomyces capillispiralis Ca-1.
centered cubic forms of metallic copper (JCPDS 04e0836) as shown in Fig (5). Our result is in agreement with (Khalid, Shamaila, Zafar, & Shahzadi, 2015) revealed the face centered cubic crystal structure of copper nanoparticles. While FTIR results (Fig. 6) confirmed the occurrence of bioactive functional groups that are responsible for formation of CuNPs. Fig (6), showed that, strong peak at 3133.360 cm1 that show amide I in resonance with amide II over tone and the peak at (2230.062, 2107.958) indicated carboxylic acid (OH) stretch. On the other hand, Band at 1612.621 cm1correspond
The antimicrobial activities of biosynthesized CuNPs were judged on the basis of the zone of inhibition (mm); CuNPs produced by endophytic actinomycetes Streptomyces capillispiralis Ca1 exhibited maximum antimicrobial activities at maximum concentration (20 mM) against different pathogenic microorganisms as represented in Fig. (7). Hence, the maximum diameter of inhibition activities against different test microorganisms under investigation were recorded as the following: Bacillus dimenuta of 13.3 mm, Bacillus subtilis of 17.7 mm, Staphylococcus aureus of 16.3 mm, Escherichia coli of 14.3 mm, Pseudomonas aeruginosa of 18 mm, Candida albican of 18.7 mm and Aspergillus brasiliensis of 12 mm. As the antimicrobial effect was dose dependent, so that the MIC of biosynthesized CuPNs for each pathogenic test organism were determined. To accomplish this aim, different concentrations of CuNPs (5, 10, 15, 20 mM) were assayed. Henceforward, the biosynthesized CuNPs by Streptomyces capillispiralis Ca-1 exhibited MIC of 10 mM against Gram-positive bacteria, (Bacillus dimenuta, Bacillus subtilis & Staphylococcus aureus), Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) & unicellular fungi (Candida albican) while MIC of multicellular fungi (Aspergillus
Please cite this article in press as: Hassan, S. E.-D., et al., New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes, Journal of Radiation Research and Applied Sciences (2018), https:// doi.org/10.1016/j.jrras.2018.05.003
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Fig. 5. XRD pattern for CuNPs synthesized by endophytic actinomycetes Streptomyces capillispiralis Ca-1.
Fig. 6. FTIR spectra for CuNPs synthesized by endophytic actinomycetes Streptomyces capillispiralis Ca-1.
brasiliensis) was 20 mM. Copper nanparticles take action on abiotic surfaces besides the rapid death resistant of microbial strains and cause destruction of plasmid and genomic DNA, which has an implication in preventing the spread of infections and gene transfer (Bogdanovic et al., 2014) clearly demonstrated that CuNPs were able to reduce more than 98% of all tested strains at highest
CuNPs concentration after just 2 h. of contact. Also (Ramyadevi, Jeyasubramanian, Marikani, Rajakumar, & Rahuman, 2012), reported that maximum inhibition activity of copper nanoparticles were showed against bacteria (Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa), than the fungus like (Aspergillus flavus, A. niger) and it
Please cite this article in press as: Hassan, S. E.-D., et al., New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes, Journal of Radiation Research and Applied Sciences (2018), https:// doi.org/10.1016/j.jrras.2018.05.003
S.E.-D. Hassan et al. / Journal of Radiation Research and Applied Sciences xxx (2018) 1e9
Fig. 7. Antimicrobial activities for biosynthesized CuNPs against pathogenic test organisms at different concentration (5, 10, 15 and 20 mM).
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efficient control strategy of phytopathogenic fungi as presented in Fig. (8). Our results showed the promising antifungal activities at different concentrations (5, 10, 15 and 20 mM) of CuNPs by Agar Plug Assay. The maximum inhibition of 57.14, 63.81 & 58.05% against Alternaria spp., Aspergillus niger and Pythium spp. respectively were recorded at concentration 20 mM (maximum concentration) that inhibit more than 50% of various phytopathogenic fungal population while a maximum inhibition of 42.61% against Fusarium spp. was recorded. The possible mechanisms of action of metallic copper nanoparticles are based on the structure and function changes in the cells of fungi; furthermore, the nanoparticles affect structural DNA and disrupt its function, which leads to fungal microorganism's death (Cioffi et al., 2005). (Kanhed et al., 2014) reported that the synthesized CuNPs demonstrated the highest effectiveness against plant pathogenic fungi (Fusarium oxysporum, Phoma destructive, Curvularia lunata and Alternaria alternata) with diameter of inhibition activities of 24, 22, 21 and 18 mm respectively. Also (Johari, Ali, Reza Kalbassi, & Yu, 2014), elucidated that the best antifungal effects of nanoparticles ensued at 600 mg/L as the highest concentrations. In addition (Saharan et al., 2015), studies supports the efficiency of copper nanoparticles as potential an antifungal agent.
3.5. Larvicidal bioassay
Fig. 8. Bio-control of plant pathogenic fungi by CuNPs synthesized by Streptomyces capillispiralis Ca-1 at different concentrations (5, 10, 15, 20 mM).
also showed more zone of inhibition against E. coli of (26 mm) than C. albicans of (23 mm). 3.4. Bio-control for phytopathogens Effective management of fungal diseases for economically important cash crops is of vital need so that our study focusing on
To study the larvicidal bioassay against both Musca domistica (House Fly) and Culex pipiens (Mosquito) of biosynthesized CuNPs over time, mortality rate was measured after 24, 48, 72 and 96 h, respectively as presented in Fig. (9). Primarily, the maximum mortality against Musca domistica (House Fly) of 76.67, 90, 100 and 100% were observed at concentration of 10 mM that gradually increased by increasing time intervals after 24, 48, 72 and 96 h. respectively. Also, this concentration considered as IC90 concentration that inhibit more than 90% of the initial level after 48 h. While IC50 of CuNPs was recorded at concentration of 5 mM at end of time interval that decreased the viability of Musca domistica to 50% of the initial level. Our result also showed that, the maximum mortality against Culex pipiens (Mosquito) of 100% were observed at concentration of 2 mM and 1.5 mM at all-time intervals with exception the first 24 h. At concentration of 1.5 mM, a mortality percentage of 96.67% was recorded which considered as IC90 concentration. Whereas IC50 of CuNPs was recorded at concentration of 1 mM at different time interval that decreased the viability of Culex pipiens to 50% of the initial level. The possible mechanism behind the mosquito and Musca death is due to the penetration of metal nanoparticles orally or through breaching of the cuticle membrane,
Fig. 9. Larvicidal efficacy of CuNPs synthesized by Streptomyces capillispiralis Ca-1. A- Mosquito insect 3rd instar larvae B- Musca insect 3rd instar larvae.
Please cite this article in press as: Hassan, S. E.-D., et al., New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes, Journal of Radiation Research and Applied Sciences (2018), https:// doi.org/10.1016/j.jrras.2018.05.003
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thereby entering into the body cavity. In the body CUNPs binds to the sulfur containing proteins and phosphorous containing DNA leads to inhibition of protein and DNA synthesis. Therefore, the cellular membrane permeability might be decreased and disturbance in proton motive force causes morphological deformation and leads to death (Morsy, Rahem, & Allam, 2001). Likewise (Angajala et al., 2014), reported CuNPs of AmC (50e100 nm) possess better larvicidal efficacy with an LC50 of 582.73, 538.25 and 508.31 ppm against Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi. Moreover (Borase, Patil, Salunkhe, Salunke, & Patil, 2013), reported the photosynthesis of nanoparticles from leaves of E. tirucalli and tested against II nd and IV th instar larvae of A. aegypti (LC50 was 3.63 mg/L, LC90 was 11.21 mg/L; LC50 was 6.75 mg/L, LC90 was 15.96 mg/L) and A. stephensi (LC50 was 7.21 mg/L, LC90 was 17.45 mg/L; LC50 was 8.18 mg/L, LC90 was 15.76 mg/L). While (Rajkuberan et al., 2017) showed that a significant observation was noticed in LC50 and LC90 values at low concentrations causing 100% mortality. 4. Conclusion Our study suggested a green, rapid and eco-friendly synthetic approach to produce CuNPs at optimum reaction parameters that can easily achieved to obtain maximum concentration of copper nanoparticles with unique characteristics through the reduction of CuSO4.7H2O solution by biomass filtrate of endophytic Streptomyces capillispiralis. Besides, various biomedical applications of biosynthesized copper nanoparticles against infectious microorganisms, phytopathogenic fungi and health threatening insect were also investigated that represent the promising uses of copper nanoparticles to be applied as a novel approach to manage these heaths threatening problems. Declarations of interest None. References Abbott, W. S. (1925). A method of computing the effectiveness of an insecticide. Journal of Economic Entomology, 18, 265e267. https://doi.org/10.1093/jee/ 18.2.265a. Ahmad, Tokeer, Wani, Irshad A., Manzoor, Nikhat, Ahmed, Jahangeer, & Asiri, Abdullah M. (2013). Biosynthesis, structural characterization and antimicrobial activity of gold and silver nanoparticles. Colloids and Surfaces B: Biointerfaces, 107, 227e234. https://doi.org/10.1016/j.colsurfb.2013.02.004. Ahmed, M. K., Khatir, Abdulah, Sufyan H., & Mohammed, Nayla (2018). Evaluation of some insecticides against Culex pipiens, the dominant mosquito species in Abha city. International Journal of Horticulture, Agriculture and Food Science(IJHAF), 2, 4e17. https://doi.org/10.22161/ijreh.2.2.2. Angajala, Gangadhara, Pavan, Pasupala, & Subashini, R. (2014). One-step biofabrication of copper nanoparticles from Aegle marmelos correa aqueous leaf extract and evaluation of its anti-inflammatory and mosquito larvicidal efficacy. RSC Advances, 4, 51459e51470. https://doi.org/10.1039/C4RA10003D. Bogdanovi c, Una, Lazi c, Vesna, Vodnik, Vesna, Budimir, Milica, Markovi c, Zoran, & Dimitrijevi c, Suzana (2014). Copper nanoparticles with high antimicrobial activity. Materials Letters, 128, 75e78. https://doi.org/10.1016/j.matlet.2014.04.106. Borase, H. P., Patil, C. D., Salunkhe, R. B., Salunke, B. K., & Patil, S. V. (2013). Phytosynthesized silver nanoparticles: A potent biolarvicidal. Agent. Journal of Nanomedicine & Biotherapeutic Discovery. https://doi.org/10.4172/2155983X.1000111. Choudhary, Kumar, Manoj, Kataria, Jyoti, Singh Cameotra, Swaranjit, & Singh, Jagdish (2016). A facile biomimetic preparation of highly stabilized silver nanoparticles derived from seed extract of Vigna radiata and evaluation of their antibacterial activity. Applied Nanoscience, 6, 105e111. https://doi.org/10.1007/ s13204-015-0418-6. Cioffi, Nicola, Torsi, Luisa, Ditaranto, Nicoletta, Tantillo, Giuseppina, Ghibelli, Lina, Sabbatini, Luigia, et al. (2005). Copper nanoparticle/polymer composites with antifungal and bacteriostatic properties. Chemistry of Materials, 17, 5255e5262. https://doi.org/10.1021/cm0505244. Díaz-Visurraga, Judith, Daza, Carla, Pozo, Claudio, Becerra, Abraham, Plessing, Carlos von, & García, Apolinaria (2012). Study on antibacterial alginate-stabilized copper nanoparticles by FT-IR and 2D-IR correlation spectroscopy.
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Physicochemical characterization of biosurfactant and its potential to remove oil from soil and cotton cloth. Carbohydrate Polymers, 89, 1110e1116. Johari, Ali, Seyed, Reza Kalbassi, Mohammad, & Yu, Il Je (2014). Inhibitory effects of silver zeolite on in vitro growth of fish egg pathogen, Saprolegnia sp. Journal of Coastal Life Medicine, 2, 357e361. Kanhed, Prachi, Birla, Sonal, Gaikwad, Swapnil, Gade, Aniket, Seabra, Amedea B., Rubilar, Olga, et al. (2014). In vitro antifungal efficacy of copper nanoparticles against selected crop pathogenic fungi. Materials Letters, 115, 13e17. Khalid, Hina, Shamaila, S., Zafar, N., & Shahzadi, Shamaila (2015). Synthesis of copper nanoparticles by chemical reduction method. Science International, 27, 3085e3088. Kim, Ok-Sun, Cho, Yong-Joon, Lee, Kihyun, Yoon, Seok-Hwan, Kim, Mincheol, Na, Hyunsoo, et al. (2012). Introducing EzTaxon-e: A prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. 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New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes Saad EL-Din Hassan a, Salem S. Salem a, Amr Fouda a, *, Mohamed A. Awad b, Mamdouh S. El-Gamal a, Abdullah M. Abdo a a b
Department of Botany and Microbiology, Faculty of Science, Al-Azhar University, Nasr City, Cairo, Egypt Department of Zoology and Entomology, Faculty of Science, Al-Azhar University, Nasr City, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 April 2018 Received in revised form 18 May 2018 Accepted 20 May 2018 Available online xxx
The biosynthesis of nanoparticles by microorganisms especially endophytic species isolated from medicinal plant are the prime concern of researchers. In the present study, a novel, non-toxic, eco-friendly copper nanoparticles was biosynthesized by endophytic actinomycetes isolate Ca-1 and optimization processes have been endeavored. The endophytic actinomycete Streptomyces capillispiralis Ca-1, was isolated from healthy medicinal plant (Convolvulus arvensis) (L.) collected from Bahariya Oasis - Giza Governorate e Egypt. The isolate was identified by morphological, cultural and molecular identification techniques. The biosynthesis of CuNPs is confirmed by gradual change of biomass filtrate color from light blue into greenish brown color and characterized by an observation of a characteristic absorption peak by UV-Vis spectroscopy at 600 nm. Also, a spherical-monodispersed shaped CuNPs with particle size of 3.6 e59 nm were observed by Transmission Electron Microscopy (TEM). In addition, X-ray diffraction (XRD) exhibited pattern peaks corresponding to 110, 111, 200, 220, 311 and 222 planes, respectively that assigned to face centered cubic forms of metallic copper (JCPDS 04e0836). While FTIR results confirmed the occurrence of bioactive functional groups that are responsible for formation of CuNPs. Moreover, the biosynthesized CuNPs showed various biomedical applications against infectious microorganisms, biocontrol of phytopathogenic fungi and health nasty insects that represent the hopeful uses of copper nanoparticles to be applied as a unique approach to manage these healths threatening problems. © 2018 The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Keywords: Copper nanoparticles Endophytic actinomycetes Streptomyces capillispiralis Biomedical applications
1. Introduction Endophytes are bacteria, fungi or actinomycetes microorganisms that lodge within plant materials in a symbiotic association. A massive number of bioactive compounds produced by Endophytes not only useful for plants but also of economic importance to humans. Endophytic population was greatly influenced by climatic conditions and position where their host plant grows. (Fouda, Hamza, El-Din Hassan, Mohamed Eid, & Ewais, 2015). Endophytic actinomycetes are ubiquitous in most plants and colonize plants
* Corresponding author. E-mail addresses: [email protected], [email protected] (A. Fouda). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications.
tissues without exhibiting pathogenicity and found to be efficient producer of novel antibiotic and other useful compounds that applied in medicines & food industries. (Kumar and Jadeja, 2016); (Hassan, 2017). As an interdisciplinary approach in bio-chemical applications, nanotechnology is focusing on nanoparticles synthesis with amended antimicrobial and antioxidant properties against the worsening diseases and cancer (Duhan et al., 2017). Biosynthesis of nanoparticles using bio-green methods are advantageous over chemical and physical methods in synthesis process due to rapid, clean, simple, non-toxic, inexpensive and ecofriendly synthesis of nanoparticles (Phull et al., 2016); (Mohmed, Saad, FoudaElgamal, & Salem, 2017b) (Mohmed, Fouda, Elgamal, EL-Din HassanShaheen, & Salem, 2017a). Biosynthesis of nanoparticles have been studied from different biological communities such as plants, bacteria, actinomycetes, yeast and fungi which are
https://doi.org/10.1016/j.jrras.2018.05.003 1687-8507/© 2018 The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Hassan, S. E.-D., et al., New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes, Journal of Radiation Research and Applied Sciences (2018), https:// doi.org/10.1016/j.jrras.2018.05.003
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emerging as nano factories and have potential useful applications (Rasool & Hemalatha, 2017); (Foudaa, Saad, Elgamala, Mohmedb, & Salema, 2017) (Fouda et al., 2017). By Ended past few years, nanoparticles of noble metal paid extensive attention owing to their applications in diverse fields and improved physical, chemical and biological properties (Imada, Masuda, Kobayashi, Hamada-Sato, & Nakashima, 2010). By comparison to further noble metals such as Au, Pt and Ag, Cu is a lowcost material however it is also accountable to oxidation generally in nanoscale dimensions. Copper nanoparticles have various properties for example anti-microbial, optical, catalytic, antifungal, anti-insects and anti-cancerous (Dinda, HalderVazquezVazquez, Arturo Lopez-Quintela, & Mitra, 2015); (Fouda & Shaheen, 2017). In addition, it was found that, the copper nanoparticles have a significant antifungal activity against phytopathogenic fungi: Curvularia lunata MTCC No. 2030, Phoma destructiva DBT-66, Fusarium oxysporum MTCC No. 1755 and Alternaria alternata MTCC No. 6572 at low concentrations, which helps to use it in the future as one of the important applied factors in the agricultural field (Kanhed, BirlaGaikwadGadeSeabra, Rubilaret al, 2014). The present study aimed to biosynthesis and characterization of copper nanoparticles using endophytic actinomycetes isolated from medicinal plant. Also, evaluation the efficiency of biosynthesized nanoparticles in medical application as antimicrobial activity, bio-control for phytopathogenic fungi and larvicidal bioassay.
2. Materials and methods 2.1. Sample collection Healthy leaves of medicinal plant Convolvulus arvensis (L.), was collected from El-Bahariya Oasis, Giza Governorate, Egypt (28 210 3800 N - 28 540 5600 E). The collected sample was placed in a sterile bag and brought to the laboratory in an ice-box and directly subjected to selective isolation procedure. Plant picture is shown in plate (1).
2.2. Endophytic actinomycetes isolation In accordance to (Gangwar, Dogra, Gupta, & Kharwar, 2014), plant leaves were washed by running tap water to remove adhered epiphytes and soil debris. After drying under sterile conditions, tissue surfaces were sterilized by using 70% (v/v) ethanol for 5 min followed by sodium hypochlorite solution (2.5%) for 2 min. Surfacesterilized tissues were washed thrice in sterile distilled water. The last washing water was plated onto bacterial, fungal, and actinomycetes culture media of Nutrient agar, Czapek Dox agar, and Starch nitrate agar, respectively. The surface sterilization was confirmed by the absence of any microbial growth onto the previous cultural media. The sterilized leaves were cut into small pieces (0.5e1.0 cm) and placed on starch nitrate agar (SNA) medium supplemented with Nystatin (25 mg/ml) to suppress fungal growth and incubated at 30 ± 2 C for 14 days for actinomycetes isolation. The purified actinomycetes isolates were preserved in SNA slant for further study. 2.3. Extracellular biosynthesis of copper nanoparticles 2.3.1. Biomass preparation Three disks of freshly prepared culture of endophytic actinomycetes isolates were inoculated into 100 mL of starch nitrate broth (SNB) medium. The flasks were incubated at 30 ± 2 C for 4 days in rotary orbital shaker at 150 rpm. The biomass was harvested by passing through four layers of wool cloth. 2.3.2. Preparation of biomass filtrate The collected biomass was washed with sterilized distilled water to remove any media components and suspended in 100 ml distilled water for 72 h at 30 ± 2 C. The biomass filtrate obtained by passing it through Whatman filter paper No.1. Finally, filtrate is then collected and ready to use for further nanoparticles synthesis. 2.3.3. Preparation of copper nanoparticles using biomass filtrate The previously obtained biomass filtrate is being used for synthesis of copper nanoparticles as the following: 20 mM (CuSO4.5H2O) was added to 100 ml biomass filtrate and kept for 6 h at 35 C in dark condition. Biosynthesized cooper nanoparticles were primarily characterized by color change and UV-Vis spectroscopy (JENWAY 6305 Spectrophotometer) at a range of 300e700 nm to select most potent endophytic actinomycetes isolate. 2.4. Molecular identification of most potent endophytic actinomycetes isolate
Plate 1. Field picture of Convolvulus arvensis (L.).
A single colony was taken from the different endophytic actinomycetes isolates and washed in saline solution (NaCl 0.085%). DNA extraction was carried out using the (Thermo K0721) GeneJET Genomic DNA Purification Kit (Sigma, 168 Third Avenue Waltham, MA, USA) according to protocol supplied by the manufacturer. The DNA was used as a template for PCR amplification but with an additional purification step using GeneJET™ PCR Purification Kit (Thermo K0701). The 16S-rRNA gene of four selected isolates was amplified using Maxima Hot Start PCR Master Mix (Thermo K1051) (Sigma) in 20 ml reactions using the universal primer amplification, the primers used were 27 f (50 -AGAGTTTGATCCTGGCTCAG-30 ) and p1492r (50 -TACGGCTACCTTGTTACGACT). All amplification products were checked by electrophoresis on 1% agarose gels (Lane, 1991). The sequences obtained were compared with those deposited in the public databases and pairwise distances were calculated with the EzTaxon server (Kim et al., 2012). The sequences of the four isolates and those of their most closely related taxa retrieved from
Please cite this article in press as: Hassan, S. E.-D., et al., New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes, Journal of Radiation Research and Applied Sciences (2018), https:// doi.org/10.1016/j.jrras.2018.05.003
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GenBank were aligned using the CLUSTAL X program (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997) and checked for alignment inconsistencies. Phylogenetic distances were calculated with Kimura's two-parameter model (Kimura, 1980) and evolutionary trees were inferred using the neighbor-joining (Saitou & Nei, 1987). Phylogenetic analyses were performed with the software package MEGA7. 2.5. Characterization of biosynthesized copper nanoparticles The size and shape of CuNPs synthesized by endophytic actinomycetes isolate was determined through Transmission Electron Microscopy (TEM- JEOL 1010 Japan). The binding properties of copper nanoparticles using biomass filtrate were investigated by FTIR analysis (Agilent system Cary 630 FTIR model). X-ray diffraction (XRD) studies of nanoparticles were carried out using Shimadzu Scientific Instruments (SSI), Kyoto, Japan. X-Ray Diffraction patterns for CuNPs were obtained with the XRD- 6000 series, including stress analysis, residual austenite quantitation, crystallite size/lattice strain, crystallinity calculation, materials analysis via overlaid X-ray diffraction patterns Shimadzu apparatus using nickel-filter and Cu- Ka target, the estimation of the size of particles was performed by Scherrer's formula. 2.6. Antimicrobial activity for copper nanoparticles produced by most potent endophytic actinomycetes Antagonistic activity of biosynthesized CuNPs were assessed against prokaryotic and eukaryotic coded test organisms represented by Gram-positive bacteria (Staphylococcus aureus ATCC. 6538, Bacillus subtilis ATCC. 6633 & Bacillus dimenuta ATCC.19146), Gram-negative bacteria (Pseudomonas aeruginosa ATCC. 9022 & Escherichia coli ATCC. 8739), Unicellular Fungi (Candida albican ATCC.10231) and Multicellular Fungi (Aspergillus brasiliensis ATCC .16404) using agar well diffusion method. Mueller Hinton agar plates were seeded with overnight prokaryotic and unicellular fungi while multicellular fungi inoculated on Czapek Dox agar. One hindered microliter (100 ml) of Copper nanoparticles solution (20 mM) was added to each well (0.8 mm). To detect minimum inhibitory concentration (MIC), different concentration of CuNPs solution were made (5, 10, 15, 20 mM). The plates were incubated at 35 ± 2 C for 24 h (bacteria and unicellular fungi) and 28 ± 2 C for 72 h (multicellular fungi). The diameter of inhibition zones around wells were measured (mm) and the results were recorded. (Magaldi et al., 2004); (Valgas, SouzaSm^ ania, & Sm^ ania, 2007). All assays were performed in triplicate. 2.7. Bio-control for phytopathogenic fungi using CuNPs by Agar Plug Assay Phytopathogenic fungi represented by Alternaria spp., Aspergillus niger, Pythium spp. and Fusarium spp. were obtained from Plant pathology Department, Faculty of Agriculture, Zagazig University. Plugs (4 mm in diameter) of 3-day-old fungal cultures were transferred to plates amended with different concentrations of Copper nanoparticles (5, 10, 15 and 20 mM). Control for each fungus was prepared using culture medium without nanoparticles. All plates were incubated at 28 ± 2 C for 5 days. Radial growth of fungal colonies was recorded after 5 days. Growth inhibition percent was calculated using the radial growth of mycelium according to the following equation: Inhibition Percentage ¼ (Control Radial Growth -Treatment Radial Growth)/Control Radial Growth*100 (Mahdizadeh, Safaie, & Khelghatibana, 2015). All assays were performed in triplicate.
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2.8. Evaluation of larvicidal bioassay of biosynthesized nanoparticles against Culex pipiens and Musca domistica 2.8.1. Insects rearing Laboratory reared colony of Culex pipiens (Mosquito) and Musca domistica (House Fly) free from insecticides and pathogens obtained from animal house, Mosquitoes Research Department, Faculty of science, Al-Azhar University in Cairo was maintained starting from egg rafts. Then 3rd instar Larvae of that used in the tests were reared in a plastic cup containing dechlorinated water in case of mosquito (M. K. Ahmed et al., 2018), while house fly 3rd instar Larvae were reared in a plastic cup containing Brewer's dried grains, yeast extract and milk powder under the standard conditions of 28 ± 2 C temperature (Hogsette, 1992). 2.8.2. Larvicidal bioassay The assay was done on healthy late third instar larvae, three replicates for each test were used. Larvae of Culex pipiens were collected with a pasture pipette, placed on a filter paper to remove excess water then placed in cups containing 100 mL dechlorinated tap water. While larvae of Musca domistica were collected and placed in cup containing nutritional media and then different concentrations of the tested CuNPs were added then readjusted according to obtained results. Three controls cups containing distilled water free from CuNPs were used. All cups were covered with muslin cloth for protection. The observed mortality was recorded at (24, 48, 72 and 96 h) larvae were considered dead if there is no sign of any movement even after mild touch with a glass rod (Kumar, Chawla, Dhamodaram, & Balakrishnan, 2014), Percentage of mortality was corrected using of Abbott's formula: ([treatment Mortality%-control Mortality%]/100-control mortality %)*100 (Abbott, 1925). 2.9. Statistical analysis The means of three replications and standard error (SEr ±) were calculated for all the results obtained by Minitab® 18.1 program. 3. Results and discussions 3.1. Endophytic actinomycetes isolation and molecular identification Three endophytic actinomycetes isolates represented by Ca-1, Ca-2 and Ca-3 were isolated from Convolvulus arvensis leaves. These actinomycetes isolates were identified as Streptomyces sp. according to grown by cover slip culture methods which confirmed spiral shape of spore chain and pigment production as shown in Fig. 1. Similarly (Rasool & Hemalatha, 2017), synthesized CuNPs by endophytic actinomycetes. Biosynthesis of copper nanoparticles were optimized and exhibited optimal cultural conditions (pH, 9, Temp. 35 C, incubation period, 4 days, CuSO4.5H2O concentration, 20 mM) using three actinomycetes isolates. The biosynthesis of CuNPs were confirmed by gradual change of biomass filtrate color from light blue into greenish brown color (Fig. 2 A) Also, the absorption spectra were recorded over a range of 300e700 nm and showed the maximum absorbance peak at 600 nm for CuNPs biosynthesized by Ca-1 (Fig. 2 b). The color change occurs due to the excitation of surface plasmon character exhibited in nanoparticles corresponding to CuNPs. Some researchers observed that, the absorption spectra of copper nanoparticles ranged between 580 and 600 nm (Ponmurugan, Manjukarunambika, Elango, & Gnanamangai, 2016). While other noted that the maximum absorption wavelength for CuNPs were at 560 nm (Kuppusamy, IlavenilSrigopalram
Please cite this article in press as: Hassan, S. E.-D., et al., New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes, Journal of Radiation Research and Applied Sciences (2018), https:// doi.org/10.1016/j.jrras.2018.05.003
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Fig. 1. Cultural and microscopic characteristic of endophytic actinomycetes isolates, A-1: Actinomycetes isolate Ca-1 growing on SNA, A-2: microscopic slide for Ca-1 showing spiral shape (X ¼ 400), B-1: Actinomycetes isolate Ca-2 grown on SNA, B-2: microscopic slide for Ca-2 showing spiral shape (X ¼ 400), C-1: Actinomycetes isolate Ca-3 grown on SNA, C-2: microscopic slide for Ca-3 showing spiral shape (X ¼ 600).
Fig. 2. A, color change of biomass filtrate from light blue to greenish brown color, B: UV spectra for CuNPs showing maximum absorbance peak at 600 nm.
ManiamYusoff, Govindanet al, 2017). The exact position of the surface plasmon mainly depends on the particle size and stability of the corresponding CuNPs. (Díaz-Visurraga et al., 2012). Therefore, in our study, endophytic actinomycetes isolate Ca-1 was considered as most potent isolate due to color change and maximum absorbance peaks. Identification of most potent isolate Ca-1 was confirmed at molecular level based on DNA extraction followed by amplification and sequence analysis of 16S rDNA gene fragments that showed these endophytic actinomycetes isolate were identified as Streptomyces capillispiralis with similarity 91% to Streptomyces capillispiralis with GeneBank accession number NR 115408 (Fig. 3). The sequences was obtained from the present study deposited in GenBank under accession number of MH160824.
3.2. Characterization of biosynthesized copper nanoparticles Besides color change and UV-Vis spectroscopy, copper nanoparticles were also characterized by transmission electronic microscopy (TEM) to determine the shape and size of biosynthesized copper nanoparticles. As revealed in Fig (4), a spherical-monodispersed shaped CuNPs with particle size of 3.6e59 nm were observed. Also (Angajala, Pavan, & Subashini, 2014), founded that the CuNPs biosynthesis by Aegle marmelos correa showed diameter of 50e100 nm. In addition, XRD of CuNPs synthesized by Streptomyces capillispiralis Ca-1 exhibited pattern peaks corresponding to 110, 111, 200, 220, 311 and 222 planes, respectively that assigned to face
Please cite this article in press as: Hassan, S. E.-D., et al., New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes, Journal of Radiation Research and Applied Sciences (2018), https:// doi.org/10.1016/j.jrras.2018.05.003
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Fig. 3. Phylogenetic analysis of 16S rRNA sequences of the actinomycetes isolate Ca-1 with the sequences from NCBI. The analysis was conducted with MEGA 6 using neighborjoining method.
to the binding of amide I band of protein with (N H) stretching (Rajeshkumar et al., 2013). Peaks at 1052.509 cm1 was characteristic of carboxylic (COO) residues (Dusane et al., 2011); (Golmoraj et al., 2011). A stretch at 857.198, 792.553 & 670.802 cm1indicated the Amide IV (OCN) stretch bending for protein. The stretch of (S S) band of protein was also found at 586.419 cm1could be attributed to the presence of a carbohydrate moiety (Jain, Mody, Mishra, & Jha, 2012). The result confirm that proteins play key role in formation of copper nanoparticles and act as capping and stabilizer agents in bio-synthesis of CuNPs as previously reported by (Ahmad et al., 2013); (Choudhary, Kumar, Kataria, Singh Cameotra, & Singh, 2016). 3.3. Antimicrobial activity and minimal inhibitory concentration (MIC)
Fig. 4. TEM image for CuNPs synthesized by Streptomyces capillispiralis Ca-1.
centered cubic forms of metallic copper (JCPDS 04e0836) as shown in Fig (5). Our result is in agreement with (Khalid, Shamaila, Zafar, & Shahzadi, 2015) revealed the face centered cubic crystal structure of copper nanoparticles. While FTIR results (Fig. 6) confirmed the occurrence of bioactive functional groups that are responsible for formation of CuNPs. Fig (6), showed that, strong peak at 3133.360 cm1 that show amide I in resonance with amide II over tone and the peak at (2230.062, 2107.958) indicated carboxylic acid (OH) stretch. On the other hand, Band at 1612.621 cm1correspond
The antimicrobial activities of biosynthesized CuNPs were judged on the basis of the zone of inhibition (mm); CuNPs produced by endophytic actinomycetes Streptomyces capillispiralis Ca1 exhibited maximum antimicrobial activities at maximum concentration (20 mM) against different pathogenic microorganisms as represented in Fig. (7). Hence, the maximum diameter of inhibition activities against different test microorganisms under investigation were recorded as the following: Bacillus dimenuta of 13.3 mm, Bacillus subtilis of 17.7 mm, Staphylococcus aureus of 16.3 mm, Escherichia coli of 14.3 mm, Pseudomonas aeruginosa of 18 mm, Candida albican of 18.7 mm and Aspergillus brasiliensis of 12 mm. As the antimicrobial effect was dose dependent, so that the MIC of biosynthesized CuPNs for each pathogenic test organism were determined. To accomplish this aim, different concentrations of CuNPs (5, 10, 15, 20 mM) were assayed. Henceforward, the biosynthesized CuNPs by Streptomyces capillispiralis Ca-1 exhibited MIC of 10 mM against Gram-positive bacteria, (Bacillus dimenuta, Bacillus subtilis & Staphylococcus aureus), Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) & unicellular fungi (Candida albican) while MIC of multicellular fungi (Aspergillus
Please cite this article in press as: Hassan, S. E.-D., et al., New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes, Journal of Radiation Research and Applied Sciences (2018), https:// doi.org/10.1016/j.jrras.2018.05.003
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Fig. 5. XRD pattern for CuNPs synthesized by endophytic actinomycetes Streptomyces capillispiralis Ca-1.
Fig. 6. FTIR spectra for CuNPs synthesized by endophytic actinomycetes Streptomyces capillispiralis Ca-1.
brasiliensis) was 20 mM. Copper nanparticles take action on abiotic surfaces besides the rapid death resistant of microbial strains and cause destruction of plasmid and genomic DNA, which has an implication in preventing the spread of infections and gene transfer (Bogdanovic et al., 2014) clearly demonstrated that CuNPs were able to reduce more than 98% of all tested strains at highest
CuNPs concentration after just 2 h. of contact. Also (Ramyadevi, Jeyasubramanian, Marikani, Rajakumar, & Rahuman, 2012), reported that maximum inhibition activity of copper nanoparticles were showed against bacteria (Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa), than the fungus like (Aspergillus flavus, A. niger) and it
Please cite this article in press as: Hassan, S. E.-D., et al., New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes, Journal of Radiation Research and Applied Sciences (2018), https:// doi.org/10.1016/j.jrras.2018.05.003
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Fig. 7. Antimicrobial activities for biosynthesized CuNPs against pathogenic test organisms at different concentration (5, 10, 15 and 20 mM).
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efficient control strategy of phytopathogenic fungi as presented in Fig. (8). Our results showed the promising antifungal activities at different concentrations (5, 10, 15 and 20 mM) of CuNPs by Agar Plug Assay. The maximum inhibition of 57.14, 63.81 & 58.05% against Alternaria spp., Aspergillus niger and Pythium spp. respectively were recorded at concentration 20 mM (maximum concentration) that inhibit more than 50% of various phytopathogenic fungal population while a maximum inhibition of 42.61% against Fusarium spp. was recorded. The possible mechanisms of action of metallic copper nanoparticles are based on the structure and function changes in the cells of fungi; furthermore, the nanoparticles affect structural DNA and disrupt its function, which leads to fungal microorganism's death (Cioffi et al., 2005). (Kanhed et al., 2014) reported that the synthesized CuNPs demonstrated the highest effectiveness against plant pathogenic fungi (Fusarium oxysporum, Phoma destructive, Curvularia lunata and Alternaria alternata) with diameter of inhibition activities of 24, 22, 21 and 18 mm respectively. Also (Johari, Ali, Reza Kalbassi, & Yu, 2014), elucidated that the best antifungal effects of nanoparticles ensued at 600 mg/L as the highest concentrations. In addition (Saharan et al., 2015), studies supports the efficiency of copper nanoparticles as potential an antifungal agent.
3.5. Larvicidal bioassay
Fig. 8. Bio-control of plant pathogenic fungi by CuNPs synthesized by Streptomyces capillispiralis Ca-1 at different concentrations (5, 10, 15, 20 mM).
also showed more zone of inhibition against E. coli of (26 mm) than C. albicans of (23 mm). 3.4. Bio-control for phytopathogens Effective management of fungal diseases for economically important cash crops is of vital need so that our study focusing on
To study the larvicidal bioassay against both Musca domistica (House Fly) and Culex pipiens (Mosquito) of biosynthesized CuNPs over time, mortality rate was measured after 24, 48, 72 and 96 h, respectively as presented in Fig. (9). Primarily, the maximum mortality against Musca domistica (House Fly) of 76.67, 90, 100 and 100% were observed at concentration of 10 mM that gradually increased by increasing time intervals after 24, 48, 72 and 96 h. respectively. Also, this concentration considered as IC90 concentration that inhibit more than 90% of the initial level after 48 h. While IC50 of CuNPs was recorded at concentration of 5 mM at end of time interval that decreased the viability of Musca domistica to 50% of the initial level. Our result also showed that, the maximum mortality against Culex pipiens (Mosquito) of 100% were observed at concentration of 2 mM and 1.5 mM at all-time intervals with exception the first 24 h. At concentration of 1.5 mM, a mortality percentage of 96.67% was recorded which considered as IC90 concentration. Whereas IC50 of CuNPs was recorded at concentration of 1 mM at different time interval that decreased the viability of Culex pipiens to 50% of the initial level. The possible mechanism behind the mosquito and Musca death is due to the penetration of metal nanoparticles orally or through breaching of the cuticle membrane,
Fig. 9. Larvicidal efficacy of CuNPs synthesized by Streptomyces capillispiralis Ca-1. A- Mosquito insect 3rd instar larvae B- Musca insect 3rd instar larvae.
Please cite this article in press as: Hassan, S. E.-D., et al., New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes, Journal of Radiation Research and Applied Sciences (2018), https:// doi.org/10.1016/j.jrras.2018.05.003
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thereby entering into the body cavity. In the body CUNPs binds to the sulfur containing proteins and phosphorous containing DNA leads to inhibition of protein and DNA synthesis. Therefore, the cellular membrane permeability might be decreased and disturbance in proton motive force causes morphological deformation and leads to death (Morsy, Rahem, & Allam, 2001). Likewise (Angajala et al., 2014), reported CuNPs of AmC (50e100 nm) possess better larvicidal efficacy with an LC50 of 582.73, 538.25 and 508.31 ppm against Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi. Moreover (Borase, Patil, Salunkhe, Salunke, & Patil, 2013), reported the photosynthesis of nanoparticles from leaves of E. tirucalli and tested against II nd and IV th instar larvae of A. aegypti (LC50 was 3.63 mg/L, LC90 was 11.21 mg/L; LC50 was 6.75 mg/L, LC90 was 15.96 mg/L) and A. stephensi (LC50 was 7.21 mg/L, LC90 was 17.45 mg/L; LC50 was 8.18 mg/L, LC90 was 15.76 mg/L). While (Rajkuberan et al., 2017) showed that a significant observation was noticed in LC50 and LC90 values at low concentrations causing 100% mortality. 4. Conclusion Our study suggested a green, rapid and eco-friendly synthetic approach to produce CuNPs at optimum reaction parameters that can easily achieved to obtain maximum concentration of copper nanoparticles with unique characteristics through the reduction of CuSO4.7H2O solution by biomass filtrate of endophytic Streptomyces capillispiralis. Besides, various biomedical applications of biosynthesized copper nanoparticles against infectious microorganisms, phytopathogenic fungi and health threatening insect were also investigated that represent the promising uses of copper nanoparticles to be applied as a novel approach to manage these heaths threatening problems. Declarations of interest None. References Abbott, W. S. (1925). A method of computing the effectiveness of an insecticide. Journal of Economic Entomology, 18, 265e267. https://doi.org/10.1093/jee/ 18.2.265a. Ahmad, Tokeer, Wani, Irshad A., Manzoor, Nikhat, Ahmed, Jahangeer, & Asiri, Abdullah M. (2013). Biosynthesis, structural characterization and antimicrobial activity of gold and silver nanoparticles. Colloids and Surfaces B: Biointerfaces, 107, 227e234. https://doi.org/10.1016/j.colsurfb.2013.02.004. Ahmed, M. K., Khatir, Abdulah, Sufyan H., & Mohammed, Nayla (2018). Evaluation of some insecticides against Culex pipiens, the dominant mosquito species in Abha city. International Journal of Horticulture, Agriculture and Food Science(IJHAF), 2, 4e17. https://doi.org/10.22161/ijreh.2.2.2. Angajala, Gangadhara, Pavan, Pasupala, & Subashini, R. (2014). One-step biofabrication of copper nanoparticles from Aegle marmelos correa aqueous leaf extract and evaluation of its anti-inflammatory and mosquito larvicidal efficacy. RSC Advances, 4, 51459e51470. https://doi.org/10.1039/C4RA10003D. Bogdanovi c, Una, Lazi c, Vesna, Vodnik, Vesna, Budimir, Milica, Markovi c, Zoran, & Dimitrijevi c, Suzana (2014). Copper nanoparticles with high antimicrobial activity. Materials Letters, 128, 75e78. https://doi.org/10.1016/j.matlet.2014.04.106. Borase, H. P., Patil, C. D., Salunkhe, R. B., Salunke, B. K., & Patil, S. V. (2013). Phytosynthesized silver nanoparticles: A potent biolarvicidal. Agent. Journal of Nanomedicine & Biotherapeutic Discovery. https://doi.org/10.4172/2155983X.1000111. Choudhary, Kumar, Manoj, Kataria, Jyoti, Singh Cameotra, Swaranjit, & Singh, Jagdish (2016). A facile biomimetic preparation of highly stabilized silver nanoparticles derived from seed extract of Vigna radiata and evaluation of their antibacterial activity. Applied Nanoscience, 6, 105e111. https://doi.org/10.1007/ s13204-015-0418-6. Cioffi, Nicola, Torsi, Luisa, Ditaranto, Nicoletta, Tantillo, Giuseppina, Ghibelli, Lina, Sabbatini, Luigia, et al. (2005). Copper nanoparticle/polymer composites with antifungal and bacteriostatic properties. Chemistry of Materials, 17, 5255e5262. https://doi.org/10.1021/cm0505244. Díaz-Visurraga, Judith, Daza, Carla, Pozo, Claudio, Becerra, Abraham, Plessing, Carlos von, & García, Apolinaria (2012). Study on antibacterial alginate-stabilized copper nanoparticles by FT-IR and 2D-IR correlation spectroscopy.
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Please cite this article in press as: Hassan, S. E.-D., et al., New approach for antimicrobial activity and bio-control of various pathogens by biosynthesized copper nanoparticles using endophytic actinomycetes, Journal of Radiation Research and Applied Sciences (2018), https:// doi.org/10.1016/j.jrras.2018.05.003