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Synthesis of chitosan based nanoparticles and their in vitro evaluation against phytopathogenic fungi
International Journal of Biological Macromolecules 62 (2013) 677–683
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Synthesis of chitosan based nanoparticles and their in vitro evaluation against phytopathogenic fungi Vinod Saharan a,∗ , Akanksha Mehrotra a , Rajesh Khatik a , Pokhar Rawal b , S.S. Sharma b , Ajay Pal c a Department of Molecular Biology and Biotechnology, Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur, Rajasthan, India b Department of Plant Pathology, Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur, Rajasthan, India c Department of Biochemistry, College of Basic Sciences and Humanities, CCS Haryana Agricultural University, Hisar, Haryana, India
a r t i c l e
i n f o
Article history: Received 8 July 2013 Received in revised form 24 September 2013 Accepted 11 October 2013 Available online 16 October 2013 Keywords: Antifungal activity Chitosan Plant fungi Saponin Nanoparticle
a b s t r a c t The main aim of present study was to prepare chitosan, chitosan-saponin and Cu-chitosan nanoparticles to evaluate their in vitro antifungal activities. Various nanoparticles were prepared using ionic gelation method by interaction of chitosan, sodium tripolyphosphate, saponin and Cu ions. Their particle size, polydispersity index, zeta potential and structures were confirmed by DLS, FTIR, TEM and SEM. The antifungal properties of nanoparticles against phytopathogenic fungi namely Alternaria alternata, Macrophomina phaseolina and Rhizoctonia solani were investigated at various concentrations ranging from 0.001 to 0.1%. Among the various formulations of nanoparticles, Cu-chitosan nanoparticles were found most effective at 0.1% concentration and showed 89.5, 63.0 and 60.1% growth inhibition of A. alternata, M. phaseolina and R. solani, respectively in in vitro model. At the same concentration, Cu-chitosan nanoparticles also showed maximum of 87.4% inhibition rate of spore germination of A. alternata. Chitosan nanoparticles showed the maximum growth inhibitory effects (87.6%) on in vitro mycelial growth of M. phaseolina at 0.1% concentration. From our study it is evident that chitosan based nanoparticles particularly chitosan and Cu-chitosan nanoparticles have tremendous potential for further field screening towards crop protection. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The advancement in nanotechnology has markedly extended the application of nanomaterials in plants. From past 5 to 8 years, nanomaterials have also been utilized in agriculture fields mostly in crop protection [1,2]. Plant pathogenic fungi cause plant and seed diseases to the most economically important crops and result in quantitative and qualitative losses in agriculture [3]. Due to excessive use of chemical fungicides, the environmental hazards to human, flora and fauna have raised major concerns over the years. Moreover, the uncontrolled use of chemical agents can cause development of resistance in phytopathogenic fungi against fungicides. More surface area, activation of novel reactive groups and unique physico-chemical properties enable nanoparticles more effective against microbes at very low dose [4,5]. Among the nanoparticles, use of metal-based nanoparticles were initiated widely in crop protection [6–13]. But the possible environmental toxicity due to unpredicted nature of metal nanoparticles have raised serious
∗ Corresponding author. Tel.: +91 9461180586; fax: +91 294 2420447. E-mail address: [email protected] (V. Saharan). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.012
questions of their application in crops [12–19,5]. These issues have ignited considerable interest among scientists and researchers in search for bio-based nanomaterials for crop protection [19,20]. Chitosan based nanoparticles are preferably used worldwide for various applications owing to their biodegradability, high permeability, non-toxicity to human and cost effectiveness [20]. It has been shown that chitosan has broad antifungal activities [21–24]. However, the insolubility of bulk chitosan in aqueous media limits its wide spectrum application as an antifungal agent. Moreover, it has relatively low antifungal activity in bulk form [20]. Therefore, various strategies have been employed to enhance its antifungal activity [23,25,26]. The chelating property of chitosan towards various organic and inorganic compounds makes it a suitable biopolymer for improvement in stability, solubility and biocidal activity [20]. Saponins, the complex glycosidic compounds, are known for their fungistatic activities [27] and their self assemble property in aqueous media have been successfully exploited in chitosan-saponin nanoformulation against cancer cells [28]. Copper (Cu) compounds are well known for their antifungal nature and have been used with chitosan for antibacterial [29] and antifungal activities [30]. Mainly, the biocidal studies of chitosan nanoparticles have been carried out on bacteria and fungi and a meagre
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study has been reported on pathogenic plant fungi [29,31,32]. In present investigation, chitosan based nanoparticles viz. chitosan, chitosan-saponin and Cu-chitosan nanoparticles were prepared by blending of saponins and Cu which were further examined against phytopathogenic fungi viz. Alternaria alternata, Macrophomina phaseolina and Rhizoctonia solani. 2. Materials and methods 2.1. Materials Chitosan (80% N-deacetylation) and saponin were obtained from HiMedia, Mumbai, India. Sodium tripolyphosphate was supplied by SRL, Mumbai, India. PVDF (Poly vinylidene difluoride) syringe filters (pore size 0.22 m) were purchased from HiMedia, Mumbai, India. Three fungal species, Alternaria alternate, Macrophomina phaseolina and Rhiszoctonia solani were obtained from Department of Plant Pathology, Rajasthan College of Agriculture, Maharan Pratap University of Agriculture, Udaipur. 2.2. Preparation of nanoparticles 2.2.1. Preparation of chitosan nanoparticles Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with TPP anions [33]. For scale-up synthesis of nanoparticles, the given method was modified. Chitosan was dissolved at 0.1% level (w/v) in 1% (v/v) acetic acid followed by overnight stirring on magnetic stirrer (Remi Laboratory Instruments, Mumbai, India) at 200 rpm and filtered through a PVDF syringe filter (pore size 0.22 m). TPP was dissolved at 1% level (w/v) in ultrapure water and filtered through PVDF membrane syringe filter (pore size 0.22 m). The cross-linking of chitosan with TPP at equal volume was performed by Pediatric set (Romsons, Agra, India) under magnetic stirrer at 700 rpm. The resulting formulation was subjected to centrifugation for 10 min at 10,000 rpm and the pellet was re-suspended in ultrapure water followed by ultra-sonication (with 3.0 mm probe, Sonicator, Lark Innovative, Chennai, India) at 80% pulser ration for 100 s at 4 ◦ C. Centrifugation followed by ultra-sonication was repeated three times and the precipitated nanoformulation was lyophilized and stored at 4 ◦ C for further analysis. 2.2.2. Preparation of chitosan-saponin nanoparticles Chitosan-saponin nanoparticles were prepared by ionic crosslinking using TPP, as described earlier with certain modifications [34]. Saponin was dissolved at 0.5% level (w/v) in ethanol and stirred mix with chitosan solution [0.1% (w/v) in 1% (v/v) acetic acid] in a volumetric ratio of 10:1. TPP [1% (w/v) in aqueous] was mixed with chitosan-saponin solution using Pediatric set (Romsons, Agra, India) under magnetic stirrer at 700 rpm. Further purification of nanoparticles was done as described in chitosan nanoparticles preparation. 2.2.3. Preparation of Cu-chitosan nanoparticles Cu-chitosan nanoparticles were prepared as described earlier by Jaiswal [35]. The procedure as used for preparation of chitosan nanoparticles was used with extra addition of 0.01% CuSO4 (HiMedia, Mumbai, India) solution into the formulation before the finishing of cross-linking reaction. The amount of entrapped Cu ions into the chitosan nanoparticles was determined by double beam Atomic Absorption Spectrophotometry (AAS4141 model, Electronics Corporation of India Ltd, India). After centrifugation steps, as discussed previously, the left-over supernatant was subjected to AAS analysis to quantify free copper [36].
2.3. Characterization of nanoparticles 2.3.1. Dynamic light scattering (DLS) measurements DLS was used for the measurement of average particle size, polydispersity index (PDI) and zeta potential of nanoparticles on high performance particle Zetasizer HPPS-5001 (Malvern, UK). Each sample was analyzed in triplicate at 25 ◦ C at a scattering angle of 90◦ [37]. Pure water was used as a reference for dispersing medium. The results are given as the average particle size obtained from the analysis of three different batches, each of them measured three times.
2.3.2. Fourier transform infrared (FTIR) analysis To confirm the synthesis of various nanoparticles, FTIR analysis was done. For FTIR each sample was prepared in potassium bromide (KBr) as a pellet under 1:99 ratio of sample and KBr, and it was recorded by ABB FTLA 2000-100 (Quebec, Canada) at a resolution limit of 16 cm–1 [29].
2.3.3. Transmission electron microscopy (TEM) observation In TEM, the various nanoparticles were diluted in ultrapure water and mixed with 2% uranyl acetate solution; a drop of the mixture was deposited onto a standard copper grid covered by a holey carbon film, and dried at ambient temperature before observation. After evaporation of the liquid, the samples were fixed on the carbon film of the grid, and visualized under transmission electron microscopy model H-7650 (Hitachi, Japan) with 40–120 kV accelerating voltage [38].
2.3.4. Scanning electron microscopy (SEM) observation Scanning electron microscope was used to study the surface morphology of nanoparticles. The samples were dried by critical point drying (CPD, Emitech) and mounted on aluminium stubs and then coated with gold using a Sputter coater model E-1010 (Emitech). The samples were examined using scanning electron microscope model S 2700 (Hitachi, Japan) with 15 kV accelerating voltage [28].
2.4. Antifungal activities Poison food technique was used to measure the antifungal activity [39]. Different concentrations (0.001, 0.005, 0.01, 0.02, 0.06 and 0.1%, w/v) of various nanoparticles in aqueous solution were used in antifungal activity test against three fungus species viz. A. alternate, M. phaseolina and R. solani. Potato dextrose agar medium (HiMedia, Mumbai, India) was prepared and poured in Petri dishes (90 mm × 15 mm, HiMedia, Mumbai, India) with above mentioned percentages of various nanoparticles, separately. Mycelial bit from peripheral end of uniform size (diameter, 5.0 mm) was taken from 7 days old culture of test pathogens and placed in the centre of test Petri dishes. All the Petri dishes were incubated at 28 ± 1 ◦ C for 7 days and the observation of radial mycelial growth was recorded when control Petri dish cover full growth (90 mm). All the treatments consisted of three replications and experiment was repeated twice. The inoculated plates were compared with control (without nanoparticles) to calculate the % inhibition rate of mycelia of the pathogen by using the formula given by Vincent [40] %Inhibition rate =
Mc − Mt × 100 Mc
where Mc is the mycelial growth in control, Mt is the mycelial growth in treatment.
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Fig. 1. The size distribution by intensity in aqueous solution. (a) Chitosan NPs, (b) Chitosan-saponin NPs, and (c) Cu-chitosan NPs.
2.5. Spore germination method The antifungal activities of nanoparticles (0.001, 0.005, 0.01, 0.02, 0.06 and 0.1%, w/v) on spore germination of A. alternata were tested. Spore suspension (1.0 × 103 spores/ml) of A. alternata was prepared aseptically from 7 days old pure culture; 50 l of spore suspension and 50 l of nanoparticle at above mentioned concentrations in aqueous were taken on glass slides (HiMedia, Mumbai, India) in 10 replicates. All the treatments were maintained at 28 ± 1 ◦ C for 8 h and the observations were made under microscope to calculate the % inhibition rate by counting the number of spore germinated compared to control. %Inhibition rate
=
Gc − Gt × 100 Gc
where Gc is the germination in control and Gt is the germination in treatment. 2.6. Statistical analysis Statistical analysis for the data was performed with JMP software version 8 [41] using Turkey–Kramer HSD test for determining significant differences among treatment at p = 0.05 level. Each
experiment was repeated twice and each treatment consists of three replicates.
3. Results and discussion 3.1. Particle size, polydispersity index and zeta potential The size distribution profile, shown in Fig. 1a–c, represents mean diameter of chitosan, 192.2 ± 2.5 nm (ranges from 150.1 to 390.2 nm); chitosan-saponin, 373.9 ± 4.1 nm (ranges from 200.8 to 990.6 nm) and Cu-chitosan nanoparticles, 196.4 ± 2.2 nm (range from 180.0 to 487.9 nm). Subsequently, chitosan, chitosan-saponin and Cu-chitosan nanoparticles showed 0.6, 1.0 and 0.5 PDI values, respectively. The PDI data indicate that chitosan and Cu-chitosan nanoparticles showed narrow size distribution compared to chitoan-saponin nanoparticles. Zeta potential of chitosan (+45.33 mV) and Cu-chitosan (+88 mV) was higher than that of chitosan-saponin nanoparticles (+31 mV). Zeta potential is an important parameter for stability of nanoparticles in aqueous media, therefore chitosan and Cu-chitosan nanoparticles with higher zeta potential showed stability in aqueous solution in present study [29,32].
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Fig. 2. FTIR spectra: (a) chitosan, (b) chitosan NPs, (c) chitosan-saponin NPs, and (d) Cu-chitosan NPs.
3.2. FTIR study FTIR analysis was performed to confirm the interaction of chitosan, TPP, saponin and Cu. Bare chitosan was characterized with some specific peaks located at 1643, 903 and 3456 cm–1 which relate to amide ( CONH2 ), anhydro glucosidic ring and primary amine ( NH2 ), respectively (Fig. 2a). In the case of chitosan nanoparticles, the observed peaks 1643 and 3456 cm–1 get shifted from higher wave number region to lower wave number region as 1636 and 3410 cm−1 (Fig. 2b). The reduction in stretching frequency could be attributed to the TPP interaction with ammonium group of chitosan and more hydrogen bonding in chitosan–TPP complex [33]. The saponin interaction with chitosan was confirmed by the presence of a new peak at 1560 cm–1 which corresponds to an amide linkage between saponin and chitosan nanoparticles [28]. There was a sharp peak at 3430 cm–1 region which assures that interaction of saponin with chitosan is more of hydrogen bonding than ionic interactions (Fig. 2c). The peaks at 1636 cm–1 ( CONH2 ) and 1550 cm–1 ( NH2 ) in the spectrum of Cu-chitosan nanoparticles were sharper and shifted to 1631 and 1536 cm–1 . Therefore, Cu bonding with chitosan induces redistribution of vibration frequencies (Fig. 2d).
3.3. Structure analysis by TEM and SEM TEM image of single (Fig. 3a, in inset) and aggregate chitosan nanoparticles (Fig. 3a) shows spherical shaped nano structures, further SEM micrograph confirmed the nano-organization of chitosan formulation (Fig. 3b). Shape of chitosan-saponin nanoparticles were smooth-rounded with variable sizes as examined using a TEM (Fig. 3c), where as in SEM micrograph,
chitosan-saponin nanoparticles were quite similar to chitosan nanoparticles (Fig. 3d). TEM and SEM micrograph shows compact polyhedron shaped of Cu-chitosan nanoparticles (Fig. 3e and f).
4. Antifungal activity In vitro mycelial growth of A. alternata was comprehensively controlled by 0.06 and 0.1% concentrations of all the nanoparticles. A maximum 89.5% inhibition rate was recorded at 0.1% concentration of Cu-chitosan nanoparticles followed by 82.2% at 0.1% chitosan nanoparticles (Fig. 4). In the case of chitosan-saponin nanoparticles, 80.9% of mycelial growth was inhibited at 0.1% concentration (Table 1). Chitosan nanoparticles displayed strong inhibition of mycelial growth of M. phaseolina. A maximum 87.6% inhibition rate was recorded at 0.1% of chitosan nanoparticles. However, 0.01–0.1% concentrations showed similar growth inhibition rate as per the statistical analysis (Table 1). Chitosan-saponin nanoparticles showed a dose dependent effect on mycelial growth. The highest inhibition rate (66.2%) was recorded at 0.1% concentration while the other concentrations (0.001–0.06%) gave inhibition rate from 30.8 to 45.2% (Table 1). Cu-chitosan nanoparticles at 0.001 to 0.02% concentrations inhibited growth from 35.3 to 54.0% whereas 0.06 and 0.1% formulation inhibited 62.5–63.0% of mycelia growth (Table 1). The radial growth of R. solani was reduced by all concentrations of chitosan nanoparticles in a dose dependent manner. The highest growth inhibition (34.4%) was observed at 0.1% chitosan nanoparticles followed by 32.2% at 0.06% concentration. Chitosan-saponin nanoparticles also showed a dose dependent effect. The highest concentration (0.1%) of chitosan-saponin nanoparticles inhibited
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Fig. 3. TEM and SEM micrographs: (a) aggregated chitosan NPs; single NP in inset in TEM and, (b) SEM micrograph of chitosan NPs, (c) chitosan-saponin NPs in TEM, and (d) SEM, (e) Cu-chitosan NPs in TEM, and (f) SEM.
fungal growth by 27.7%, and the same inhibition rate was recorded at 0.06% concentration (Table 1). Among the various concentrations of Cu-chitosan nanoparticles, highest inhibition rate (60.1%) was recorded at a dose of 0.1%. The concentrations of Cu-chitosan nanoparticles ranging from 0.01 to 0.06% showed fairly stable growth inhibition from 38.1 to 43.1%. A maximum 87.4% spore germination was inhibited by 0.1% concentration of Cu-chitosan nanoparticles followed by chitosan nanoparticles (87.1%) at 0.1%. Taken as a whole, all the examined nanoparticles were found effective in controlling spore germination of A. alternata (Table 2). Bulk chitosan, bulk saponin and CuSO4 at 0.1% level were found less effective for inhibition of mycelial growth and spore germination as compared to our synthesized nanoparticles (Tables 1 and 2). Stable, non-toxic and organic solvent free synthesis of chitosan nanoparticles have been achieved using ionic gelation technique by various independent group of researchers [42,43]. This technique
is based on the ionic interactions between the positively charged primary amino groups of chitosan and the negatively charged TPP and avoids the use of toxic chemical and emulsifying agents which are often toxic to non-targeted organisms [43]. Until now, the synthesis of chitosan nanoparticles have been well documented for particle size and stability by modifying chitosan concentration, chitosan to TPP weight ratio, rate of mixing and pH of the reaction mixture [37]. In this study, the existing protocols for synthesis of chitosan nanoparticles were modified to successfully achieve stable and speedy fabrication of nanoparticles. We have standardized the synthesis process by precise control of cross linking reaction using Pediatric set (100 ml volume) under room temperature that produced 98 ± 1 mg of nanomaterials in a single reaction in 3 h which can further be scaled up by using higher capacity of Pediatric set. The chitosan nanoparticles were further modified into chitosan-saponin and Cu-chitosan nanoparticles and their effect on fungal growth was evaluated. None of the screened fungi showed
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Table 1 Effect of various chitosan NPs on in vitro mycelial growth of phytopathogenic fungi. Treatments (%)
% Inhibition ratea A. alternata
Chitosan NPs 0.001 0.005 0.01 0.02 0.06 0.1 Controlb Chitosan-saponin NPs 0.001 0.005 0.01 0.02 0.06 0.1 Controlb Cu-chitosan NPs 0.001 0.005 0.01 0.02 0.06 0.1 Controlb Chitosan (0.1%)c Saponin (0.1%)d CuSo4 (0.1%)d
M. phaseolina
R. solani
11.5 49.0 52.7 65.3 80.1 82.2 0.00
± ± ± ± ± ± ±
0.8 e 1.0 d 0.5 c 0.7 b 0.9 a 0.5 a 0.0 f
62.5 76.8 84.0 87.3 87.5 87.6 0.00
± ± ± ± ± ± ±
3.7 c 1.9 b 2.8 a 0.6 a 0.3 a 0.1 a 0.0 d
13.1 17.2 17.4 18.1 32.2 34.4 0.0
± ± ± ± ± ± ±
1.5 b 2.2 b 1.5 b 0.7 b 1.6 a 1.1 a 0.0 c
15.7 59.8 62.8 70.2 78.3 80.9 0.00
± ± ± ± ± ± ±
1.0 d 0.8 c 0.6 b 2.1 b 0.5 a 0.7 a 0.0 e
30.8 32.3 38.8 39.7 45.2 66.2 0.00
± ± ± ± ± ± ±
0.4 d 0.5 d 0.9 c 0.6 c 0.7 b 0.4 a 0.0 e
12.2 16.0 16.4 22.8 27.7 27.7 0.00
± ± ± ± ± ± ±
0.7 c 3.1 bc 1.7 bc 0.7 ab 0.6 a 0.5 a 0.0 d
9.9 40.3 53.0 69.3 82.1 89.5 0.00 21.3 31.1 20.8
± ± ± ± ± ± ± ± ± ±
1.2 e 0.1 d 0.9 c 0.5 b 1.5 a 0.7 a 0.0 f 0.3 0.5 0.0
35.3 45.2 45.8 54.0 62.5 63.0 0.00 18.0 25.6 16.7
± ± ± ± ± ± ± ± ± ±
0.4 d 2.6 c 2.4 c 2.5 b 0.9 a 0.3 a 0.0 e 0.2 0.6 0.0
22.3 28.5 38.1 38.7 43.1 60.1 0.00 16.7 13.2 18.4
± ± ± ± ± ± ± ± ± ±
0.5 c 2.4 c 0.3 b 2.4 b 1.6 b 1.5 a 0.0 d 0.7 0.3 0.0
a Each value is mean of 3 replicates from 2 experiments. Mean ± SE followed by same letter in column of each treatment is not significant difference at p = 0.05 by Tukey–Kramer HSD test, % inhibition rate was calculated compared to the germination of the control (0%). b Control without any formulation. c Dissolved in 0.1% acetic acid. d Dissolved in water, NPs-nanoparticles.
Table 2 Effect of various chitosan NPs on in vitro spore germination of A. alternata. Treatments (%) Chitosan NPs 0.001 0.005 0.01 0.02 0.06 0.1 Controlb Chitosan-saponin NPs 0.001 0.005 0.01 0.02 0.06 0.1 Controlb Cu-chitosan NPs 0.001 0.005 0.01 0.02 0.06 0.1 Controlb Chitosan (0.1%)c Saponin (0.1%)d CuSO4 (0.1%)d
% Inhibition ratea 14.6 51.0 57.3 68.5 84.4 87.1 0.00
± ± ± ± ± ± ±
1.0 e 0.8 d 0.7 c 0.4 b 0.6 a 0.1 a 0.0 f
13.2 62.8 63.6 73.1 78.3 82.9 0.00
± ± ± ± ± ± ±
1.2 c 0.6 b 2.6 b 5.1 ab 0.1 a 0.8 a 0.0 d
10.6 43.2 52.0 69.3 83.3 87.4 0.00 21.1 31.4 23.1
± ± ± ± ± ± ± ± ± ±
1.8 e 0.0 d 0.6 c 0.2 b 1.3 a 0.2 a 0.0 f 0.4 0.1 1.3
a Each value is mean of 3 replicates from 2 experiments. Mean ± SE followed by same letter in column of each treatment is not significant difference at p = 0.05 by Tukey–Kramer HSD test, % inhibition rate was calculated compared to the germination of the control (0%). b Control without any formulation. c Dissolved in 0.1% acetic acid. d Dissolved in water, NPs-nanoparticles.
Fig. 4. A representative photograph of antifungal bioassay. Effect of Cu-chitosan nanoparticles on mycelial growth of A. alternata.
significant improvement in growth inhibition rate by chitosansaponin treatment indicating that chitosan-saponin nanoparticles do not deserve a good antifungal agent in present study. Moreover, higher PDI value and lower zeta potential make chitosan-saponin nanoparticles less stable and less effective against fungi used in present study. Albeit, saponin-chitosan nanoparticle showed excellent growth inhibition on cancer cell in earlier report [28]. Copper compounds are known for their antifungal nature and are being effectively used to control fungal diseases. However, due to uncontrolled application, their concentrations have exceeded the limits specified for heavy metals [44]. Therefore, in present study Cuchitosan nanoparticles were prepared to improve the antifungal activity and overcome the existing problem of heavy dose. The protocol for synthesis of Cu-chitosan nanoparticles was standardized in the same way as chitosan nanoparticles and the developed method delivered highly stable nanoparticles with low PDI and high zeta potential as compared to previous reports [29,32]. AAS result shown that 70% of copper embedded in chitosan formulation in Cu-chitosan nanoparticles synthesis. Among all the treatments, Cu-chitosan nanoparticles were found very effective at 0.1% concentration and showed 89.5, 63.0 and 60.1% growth inhibition of A. alternata, M. phaseolina and R. solani, respectively in vitro. Further, the Cu-chitosan nanoparticles at 0.1% concentration showed the strongest growth inhibitory effects (87.4%) on spore germination of A. alternata. The observed higher antifungal activities of Cu-chitosan nanoparticles could be explained by the higher surface charge density (higher zeta potential) which provides them greater binding affinity for negatively charged fungal membrane. Further, Cu(II) undergoes reduction to Cu(I) in fungi which produces toxic H2 O2 which in turn destructs the cell viability [32]. In general, 0.06–1% concentrations of nanoparticles were found effective to control fungal growth in present study. In a prior report, higher doses ranging from 750 to 6000 mg/l of bulk chitosan were found effective against plant fungi [26], and these concentrations were higher compared to nanoparticles concentrations used in present study. The use of bulk chitosan has been restricted due to its poor solubility in water and poor antifungal activity [20]. Chemically modified chitosans viz. triethylene diamine dithiocarbamate chitosan and o-hydroxyphenylaldehyde thiosemicarbazone chitosan have previously been evaluated and found to show maximum 60.4 and 52.6% growth inhibition of F. oxysporum and R. solani at 500 and 50 mg/l concentration [26,45]. Therefore, the results of our study clearly indicate the pronounced effect of chitosan nanoparticles
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over bulk and chitosan derivatives on growth inhibition of important plant pathogenic fungi. 5. Conclusions Chitosan, chitosan-saponin and Cu-chitosan nanoparticles were prepared and their structures were well characterized by DLS, FTIR, TEM and SEM. The lower PDI value and higher zeta-potential of chitosan and Cu-chitosan nanoparticles proved their uniform size and stability which may contribute to their higher antifungal activity against A. alternata, M. phaseolina and R. solani in in vitro studies. Cu-chitosan nanoparticles also showed maximum inhibition rate of spore germination of A. alternata. Compared to chitosan and Cuchitosan nanoparticles, the chitosan-saponin nanoparticles were found poor in antifungal activity. The higher PDI value and lower zeta-potential decreased their uniform size and stability in aqueous solution. The efficacy of chitosan based nanoparticles mainly chitosan and Cu-chitosan could further be tested on other plant pathogenic fungi in in vitro and in vivo models. The present study is a effort to explore potential of chitosan based nanoparticles and the findings of present study could be utilized for development of nano fungicide for crop protection in future. Acknowledgements The authors wish to thank Directorate of Research, MPUAT for their support of this work. The kind co-operation extended by Dr. Ramesh Raliya, Research Scientist, CAZRI, Jodhpur and Dr. N. Ilaiyaraja, Scientist, Defence Food Research Laboratory, Mysore, India for their technical help in FTIR and DLS studies, respectively is highly acknowledged. References [1] V. Ghormade, M.V. Deshpande, K.M. Paknikar, Biotechnol. Adv. 29 (2010) 792–803. [2] R. Nair, S.H. Varghese, B.G. Nair, T. Maekawa, Y. Yoshida, D.S. Kumar, Plant Sci. 179 (2010) 154–163. [3] G.N. Agrios, Plant Pathology, Academic Press, London, 2000. [4] H. Bouwmeester, S. Dekkers, M.Y. Noordam, W.I. Hagens, A.S. Bulder, C.D. Heer, S.E.C.G.T. Voorde, S.W.P. Wijnhoven, H.J.P. Marvin, A.J.A.M. Sips, Regul. Toxicol. Pharmacol. 53 (2009) 52–62. [5] L.R. Khot, S. Sankaran, J.M. Maja, R. Ehsani, E.W. Schuster, Crop Prot. 35 (2012) 64–70. [6] H.J. Park, S.H. Kim, H.J. Kim, S.H. Choi, J. Plant Pathol. 22 (2006) 295–302. [7] Y.K. Jo, B.H. Kim, G. Jung, Plant Dis. 93 (2009) 1037–1043. [8] S.W. Kim, J.H. Jung, K. Lamsal, Y.S. Kim, J.S. Min, Y.S. Lee, Mycobiology 40 (2012) 53–58. [9] L. He, Y. Liu, A. Mustapha, M. Lin, Microbiol. Res. 166 (2011) 207–215.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Synthesis of chitosan based nanoparticles and their in vitro evaluation against phytopathogenic fungi Vinod Saharan a,∗ , Akanksha Mehrotra a , Rajesh Khatik a , Pokhar Rawal b , S.S. Sharma b , Ajay Pal c a Department of Molecular Biology and Biotechnology, Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur, Rajasthan, India b Department of Plant Pathology, Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur, Rajasthan, India c Department of Biochemistry, College of Basic Sciences and Humanities, CCS Haryana Agricultural University, Hisar, Haryana, India
a r t i c l e
i n f o
Article history: Received 8 July 2013 Received in revised form 24 September 2013 Accepted 11 October 2013 Available online 16 October 2013 Keywords: Antifungal activity Chitosan Plant fungi Saponin Nanoparticle
a b s t r a c t The main aim of present study was to prepare chitosan, chitosan-saponin and Cu-chitosan nanoparticles to evaluate their in vitro antifungal activities. Various nanoparticles were prepared using ionic gelation method by interaction of chitosan, sodium tripolyphosphate, saponin and Cu ions. Their particle size, polydispersity index, zeta potential and structures were confirmed by DLS, FTIR, TEM and SEM. The antifungal properties of nanoparticles against phytopathogenic fungi namely Alternaria alternata, Macrophomina phaseolina and Rhizoctonia solani were investigated at various concentrations ranging from 0.001 to 0.1%. Among the various formulations of nanoparticles, Cu-chitosan nanoparticles were found most effective at 0.1% concentration and showed 89.5, 63.0 and 60.1% growth inhibition of A. alternata, M. phaseolina and R. solani, respectively in in vitro model. At the same concentration, Cu-chitosan nanoparticles also showed maximum of 87.4% inhibition rate of spore germination of A. alternata. Chitosan nanoparticles showed the maximum growth inhibitory effects (87.6%) on in vitro mycelial growth of M. phaseolina at 0.1% concentration. From our study it is evident that chitosan based nanoparticles particularly chitosan and Cu-chitosan nanoparticles have tremendous potential for further field screening towards crop protection. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The advancement in nanotechnology has markedly extended the application of nanomaterials in plants. From past 5 to 8 years, nanomaterials have also been utilized in agriculture fields mostly in crop protection [1,2]. Plant pathogenic fungi cause plant and seed diseases to the most economically important crops and result in quantitative and qualitative losses in agriculture [3]. Due to excessive use of chemical fungicides, the environmental hazards to human, flora and fauna have raised major concerns over the years. Moreover, the uncontrolled use of chemical agents can cause development of resistance in phytopathogenic fungi against fungicides. More surface area, activation of novel reactive groups and unique physico-chemical properties enable nanoparticles more effective against microbes at very low dose [4,5]. Among the nanoparticles, use of metal-based nanoparticles were initiated widely in crop protection [6–13]. But the possible environmental toxicity due to unpredicted nature of metal nanoparticles have raised serious
∗ Corresponding author. Tel.: +91 9461180586; fax: +91 294 2420447. E-mail address: [email protected] (V. Saharan). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.012
questions of their application in crops [12–19,5]. These issues have ignited considerable interest among scientists and researchers in search for bio-based nanomaterials for crop protection [19,20]. Chitosan based nanoparticles are preferably used worldwide for various applications owing to their biodegradability, high permeability, non-toxicity to human and cost effectiveness [20]. It has been shown that chitosan has broad antifungal activities [21–24]. However, the insolubility of bulk chitosan in aqueous media limits its wide spectrum application as an antifungal agent. Moreover, it has relatively low antifungal activity in bulk form [20]. Therefore, various strategies have been employed to enhance its antifungal activity [23,25,26]. The chelating property of chitosan towards various organic and inorganic compounds makes it a suitable biopolymer for improvement in stability, solubility and biocidal activity [20]. Saponins, the complex glycosidic compounds, are known for their fungistatic activities [27] and their self assemble property in aqueous media have been successfully exploited in chitosan-saponin nanoformulation against cancer cells [28]. Copper (Cu) compounds are well known for their antifungal nature and have been used with chitosan for antibacterial [29] and antifungal activities [30]. Mainly, the biocidal studies of chitosan nanoparticles have been carried out on bacteria and fungi and a meagre
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study has been reported on pathogenic plant fungi [29,31,32]. In present investigation, chitosan based nanoparticles viz. chitosan, chitosan-saponin and Cu-chitosan nanoparticles were prepared by blending of saponins and Cu which were further examined against phytopathogenic fungi viz. Alternaria alternata, Macrophomina phaseolina and Rhizoctonia solani. 2. Materials and methods 2.1. Materials Chitosan (80% N-deacetylation) and saponin were obtained from HiMedia, Mumbai, India. Sodium tripolyphosphate was supplied by SRL, Mumbai, India. PVDF (Poly vinylidene difluoride) syringe filters (pore size 0.22 m) were purchased from HiMedia, Mumbai, India. Three fungal species, Alternaria alternate, Macrophomina phaseolina and Rhiszoctonia solani were obtained from Department of Plant Pathology, Rajasthan College of Agriculture, Maharan Pratap University of Agriculture, Udaipur. 2.2. Preparation of nanoparticles 2.2.1. Preparation of chitosan nanoparticles Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with TPP anions [33]. For scale-up synthesis of nanoparticles, the given method was modified. Chitosan was dissolved at 0.1% level (w/v) in 1% (v/v) acetic acid followed by overnight stirring on magnetic stirrer (Remi Laboratory Instruments, Mumbai, India) at 200 rpm and filtered through a PVDF syringe filter (pore size 0.22 m). TPP was dissolved at 1% level (w/v) in ultrapure water and filtered through PVDF membrane syringe filter (pore size 0.22 m). The cross-linking of chitosan with TPP at equal volume was performed by Pediatric set (Romsons, Agra, India) under magnetic stirrer at 700 rpm. The resulting formulation was subjected to centrifugation for 10 min at 10,000 rpm and the pellet was re-suspended in ultrapure water followed by ultra-sonication (with 3.0 mm probe, Sonicator, Lark Innovative, Chennai, India) at 80% pulser ration for 100 s at 4 ◦ C. Centrifugation followed by ultra-sonication was repeated three times and the precipitated nanoformulation was lyophilized and stored at 4 ◦ C for further analysis. 2.2.2. Preparation of chitosan-saponin nanoparticles Chitosan-saponin nanoparticles were prepared by ionic crosslinking using TPP, as described earlier with certain modifications [34]. Saponin was dissolved at 0.5% level (w/v) in ethanol and stirred mix with chitosan solution [0.1% (w/v) in 1% (v/v) acetic acid] in a volumetric ratio of 10:1. TPP [1% (w/v) in aqueous] was mixed with chitosan-saponin solution using Pediatric set (Romsons, Agra, India) under magnetic stirrer at 700 rpm. Further purification of nanoparticles was done as described in chitosan nanoparticles preparation. 2.2.3. Preparation of Cu-chitosan nanoparticles Cu-chitosan nanoparticles were prepared as described earlier by Jaiswal [35]. The procedure as used for preparation of chitosan nanoparticles was used with extra addition of 0.01% CuSO4 (HiMedia, Mumbai, India) solution into the formulation before the finishing of cross-linking reaction. The amount of entrapped Cu ions into the chitosan nanoparticles was determined by double beam Atomic Absorption Spectrophotometry (AAS4141 model, Electronics Corporation of India Ltd, India). After centrifugation steps, as discussed previously, the left-over supernatant was subjected to AAS analysis to quantify free copper [36].
2.3. Characterization of nanoparticles 2.3.1. Dynamic light scattering (DLS) measurements DLS was used for the measurement of average particle size, polydispersity index (PDI) and zeta potential of nanoparticles on high performance particle Zetasizer HPPS-5001 (Malvern, UK). Each sample was analyzed in triplicate at 25 ◦ C at a scattering angle of 90◦ [37]. Pure water was used as a reference for dispersing medium. The results are given as the average particle size obtained from the analysis of three different batches, each of them measured three times.
2.3.2. Fourier transform infrared (FTIR) analysis To confirm the synthesis of various nanoparticles, FTIR analysis was done. For FTIR each sample was prepared in potassium bromide (KBr) as a pellet under 1:99 ratio of sample and KBr, and it was recorded by ABB FTLA 2000-100 (Quebec, Canada) at a resolution limit of 16 cm–1 [29].
2.3.3. Transmission electron microscopy (TEM) observation In TEM, the various nanoparticles were diluted in ultrapure water and mixed with 2% uranyl acetate solution; a drop of the mixture was deposited onto a standard copper grid covered by a holey carbon film, and dried at ambient temperature before observation. After evaporation of the liquid, the samples were fixed on the carbon film of the grid, and visualized under transmission electron microscopy model H-7650 (Hitachi, Japan) with 40–120 kV accelerating voltage [38].
2.3.4. Scanning electron microscopy (SEM) observation Scanning electron microscope was used to study the surface morphology of nanoparticles. The samples were dried by critical point drying (CPD, Emitech) and mounted on aluminium stubs and then coated with gold using a Sputter coater model E-1010 (Emitech). The samples were examined using scanning electron microscope model S 2700 (Hitachi, Japan) with 15 kV accelerating voltage [28].
2.4. Antifungal activities Poison food technique was used to measure the antifungal activity [39]. Different concentrations (0.001, 0.005, 0.01, 0.02, 0.06 and 0.1%, w/v) of various nanoparticles in aqueous solution were used in antifungal activity test against three fungus species viz. A. alternate, M. phaseolina and R. solani. Potato dextrose agar medium (HiMedia, Mumbai, India) was prepared and poured in Petri dishes (90 mm × 15 mm, HiMedia, Mumbai, India) with above mentioned percentages of various nanoparticles, separately. Mycelial bit from peripheral end of uniform size (diameter, 5.0 mm) was taken from 7 days old culture of test pathogens and placed in the centre of test Petri dishes. All the Petri dishes were incubated at 28 ± 1 ◦ C for 7 days and the observation of radial mycelial growth was recorded when control Petri dish cover full growth (90 mm). All the treatments consisted of three replications and experiment was repeated twice. The inoculated plates were compared with control (without nanoparticles) to calculate the % inhibition rate of mycelia of the pathogen by using the formula given by Vincent [40] %Inhibition rate =
Mc − Mt × 100 Mc
where Mc is the mycelial growth in control, Mt is the mycelial growth in treatment.
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Fig. 1. The size distribution by intensity in aqueous solution. (a) Chitosan NPs, (b) Chitosan-saponin NPs, and (c) Cu-chitosan NPs.
2.5. Spore germination method The antifungal activities of nanoparticles (0.001, 0.005, 0.01, 0.02, 0.06 and 0.1%, w/v) on spore germination of A. alternata were tested. Spore suspension (1.0 × 103 spores/ml) of A. alternata was prepared aseptically from 7 days old pure culture; 50 l of spore suspension and 50 l of nanoparticle at above mentioned concentrations in aqueous were taken on glass slides (HiMedia, Mumbai, India) in 10 replicates. All the treatments were maintained at 28 ± 1 ◦ C for 8 h and the observations were made under microscope to calculate the % inhibition rate by counting the number of spore germinated compared to control. %Inhibition rate
=
Gc − Gt × 100 Gc
where Gc is the germination in control and Gt is the germination in treatment. 2.6. Statistical analysis Statistical analysis for the data was performed with JMP software version 8 [41] using Turkey–Kramer HSD test for determining significant differences among treatment at p = 0.05 level. Each
experiment was repeated twice and each treatment consists of three replicates.
3. Results and discussion 3.1. Particle size, polydispersity index and zeta potential The size distribution profile, shown in Fig. 1a–c, represents mean diameter of chitosan, 192.2 ± 2.5 nm (ranges from 150.1 to 390.2 nm); chitosan-saponin, 373.9 ± 4.1 nm (ranges from 200.8 to 990.6 nm) and Cu-chitosan nanoparticles, 196.4 ± 2.2 nm (range from 180.0 to 487.9 nm). Subsequently, chitosan, chitosan-saponin and Cu-chitosan nanoparticles showed 0.6, 1.0 and 0.5 PDI values, respectively. The PDI data indicate that chitosan and Cu-chitosan nanoparticles showed narrow size distribution compared to chitoan-saponin nanoparticles. Zeta potential of chitosan (+45.33 mV) and Cu-chitosan (+88 mV) was higher than that of chitosan-saponin nanoparticles (+31 mV). Zeta potential is an important parameter for stability of nanoparticles in aqueous media, therefore chitosan and Cu-chitosan nanoparticles with higher zeta potential showed stability in aqueous solution in present study [29,32].
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Fig. 2. FTIR spectra: (a) chitosan, (b) chitosan NPs, (c) chitosan-saponin NPs, and (d) Cu-chitosan NPs.
3.2. FTIR study FTIR analysis was performed to confirm the interaction of chitosan, TPP, saponin and Cu. Bare chitosan was characterized with some specific peaks located at 1643, 903 and 3456 cm–1 which relate to amide ( CONH2 ), anhydro glucosidic ring and primary amine ( NH2 ), respectively (Fig. 2a). In the case of chitosan nanoparticles, the observed peaks 1643 and 3456 cm–1 get shifted from higher wave number region to lower wave number region as 1636 and 3410 cm−1 (Fig. 2b). The reduction in stretching frequency could be attributed to the TPP interaction with ammonium group of chitosan and more hydrogen bonding in chitosan–TPP complex [33]. The saponin interaction with chitosan was confirmed by the presence of a new peak at 1560 cm–1 which corresponds to an amide linkage between saponin and chitosan nanoparticles [28]. There was a sharp peak at 3430 cm–1 region which assures that interaction of saponin with chitosan is more of hydrogen bonding than ionic interactions (Fig. 2c). The peaks at 1636 cm–1 ( CONH2 ) and 1550 cm–1 ( NH2 ) in the spectrum of Cu-chitosan nanoparticles were sharper and shifted to 1631 and 1536 cm–1 . Therefore, Cu bonding with chitosan induces redistribution of vibration frequencies (Fig. 2d).
3.3. Structure analysis by TEM and SEM TEM image of single (Fig. 3a, in inset) and aggregate chitosan nanoparticles (Fig. 3a) shows spherical shaped nano structures, further SEM micrograph confirmed the nano-organization of chitosan formulation (Fig. 3b). Shape of chitosan-saponin nanoparticles were smooth-rounded with variable sizes as examined using a TEM (Fig. 3c), where as in SEM micrograph,
chitosan-saponin nanoparticles were quite similar to chitosan nanoparticles (Fig. 3d). TEM and SEM micrograph shows compact polyhedron shaped of Cu-chitosan nanoparticles (Fig. 3e and f).
4. Antifungal activity In vitro mycelial growth of A. alternata was comprehensively controlled by 0.06 and 0.1% concentrations of all the nanoparticles. A maximum 89.5% inhibition rate was recorded at 0.1% concentration of Cu-chitosan nanoparticles followed by 82.2% at 0.1% chitosan nanoparticles (Fig. 4). In the case of chitosan-saponin nanoparticles, 80.9% of mycelial growth was inhibited at 0.1% concentration (Table 1). Chitosan nanoparticles displayed strong inhibition of mycelial growth of M. phaseolina. A maximum 87.6% inhibition rate was recorded at 0.1% of chitosan nanoparticles. However, 0.01–0.1% concentrations showed similar growth inhibition rate as per the statistical analysis (Table 1). Chitosan-saponin nanoparticles showed a dose dependent effect on mycelial growth. The highest inhibition rate (66.2%) was recorded at 0.1% concentration while the other concentrations (0.001–0.06%) gave inhibition rate from 30.8 to 45.2% (Table 1). Cu-chitosan nanoparticles at 0.001 to 0.02% concentrations inhibited growth from 35.3 to 54.0% whereas 0.06 and 0.1% formulation inhibited 62.5–63.0% of mycelia growth (Table 1). The radial growth of R. solani was reduced by all concentrations of chitosan nanoparticles in a dose dependent manner. The highest growth inhibition (34.4%) was observed at 0.1% chitosan nanoparticles followed by 32.2% at 0.06% concentration. Chitosan-saponin nanoparticles also showed a dose dependent effect. The highest concentration (0.1%) of chitosan-saponin nanoparticles inhibited
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Fig. 3. TEM and SEM micrographs: (a) aggregated chitosan NPs; single NP in inset in TEM and, (b) SEM micrograph of chitosan NPs, (c) chitosan-saponin NPs in TEM, and (d) SEM, (e) Cu-chitosan NPs in TEM, and (f) SEM.
fungal growth by 27.7%, and the same inhibition rate was recorded at 0.06% concentration (Table 1). Among the various concentrations of Cu-chitosan nanoparticles, highest inhibition rate (60.1%) was recorded at a dose of 0.1%. The concentrations of Cu-chitosan nanoparticles ranging from 0.01 to 0.06% showed fairly stable growth inhibition from 38.1 to 43.1%. A maximum 87.4% spore germination was inhibited by 0.1% concentration of Cu-chitosan nanoparticles followed by chitosan nanoparticles (87.1%) at 0.1%. Taken as a whole, all the examined nanoparticles were found effective in controlling spore germination of A. alternata (Table 2). Bulk chitosan, bulk saponin and CuSO4 at 0.1% level were found less effective for inhibition of mycelial growth and spore germination as compared to our synthesized nanoparticles (Tables 1 and 2). Stable, non-toxic and organic solvent free synthesis of chitosan nanoparticles have been achieved using ionic gelation technique by various independent group of researchers [42,43]. This technique
is based on the ionic interactions between the positively charged primary amino groups of chitosan and the negatively charged TPP and avoids the use of toxic chemical and emulsifying agents which are often toxic to non-targeted organisms [43]. Until now, the synthesis of chitosan nanoparticles have been well documented for particle size and stability by modifying chitosan concentration, chitosan to TPP weight ratio, rate of mixing and pH of the reaction mixture [37]. In this study, the existing protocols for synthesis of chitosan nanoparticles were modified to successfully achieve stable and speedy fabrication of nanoparticles. We have standardized the synthesis process by precise control of cross linking reaction using Pediatric set (100 ml volume) under room temperature that produced 98 ± 1 mg of nanomaterials in a single reaction in 3 h which can further be scaled up by using higher capacity of Pediatric set. The chitosan nanoparticles were further modified into chitosan-saponin and Cu-chitosan nanoparticles and their effect on fungal growth was evaluated. None of the screened fungi showed
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Table 1 Effect of various chitosan NPs on in vitro mycelial growth of phytopathogenic fungi. Treatments (%)
% Inhibition ratea A. alternata
Chitosan NPs 0.001 0.005 0.01 0.02 0.06 0.1 Controlb Chitosan-saponin NPs 0.001 0.005 0.01 0.02 0.06 0.1 Controlb Cu-chitosan NPs 0.001 0.005 0.01 0.02 0.06 0.1 Controlb Chitosan (0.1%)c Saponin (0.1%)d CuSo4 (0.1%)d
M. phaseolina
R. solani
11.5 49.0 52.7 65.3 80.1 82.2 0.00
± ± ± ± ± ± ±
0.8 e 1.0 d 0.5 c 0.7 b 0.9 a 0.5 a 0.0 f
62.5 76.8 84.0 87.3 87.5 87.6 0.00
± ± ± ± ± ± ±
3.7 c 1.9 b 2.8 a 0.6 a 0.3 a 0.1 a 0.0 d
13.1 17.2 17.4 18.1 32.2 34.4 0.0
± ± ± ± ± ± ±
1.5 b 2.2 b 1.5 b 0.7 b 1.6 a 1.1 a 0.0 c
15.7 59.8 62.8 70.2 78.3 80.9 0.00
± ± ± ± ± ± ±
1.0 d 0.8 c 0.6 b 2.1 b 0.5 a 0.7 a 0.0 e
30.8 32.3 38.8 39.7 45.2 66.2 0.00
± ± ± ± ± ± ±
0.4 d 0.5 d 0.9 c 0.6 c 0.7 b 0.4 a 0.0 e
12.2 16.0 16.4 22.8 27.7 27.7 0.00
± ± ± ± ± ± ±
0.7 c 3.1 bc 1.7 bc 0.7 ab 0.6 a 0.5 a 0.0 d
9.9 40.3 53.0 69.3 82.1 89.5 0.00 21.3 31.1 20.8
± ± ± ± ± ± ± ± ± ±
1.2 e 0.1 d 0.9 c 0.5 b 1.5 a 0.7 a 0.0 f 0.3 0.5 0.0
35.3 45.2 45.8 54.0 62.5 63.0 0.00 18.0 25.6 16.7
± ± ± ± ± ± ± ± ± ±
0.4 d 2.6 c 2.4 c 2.5 b 0.9 a 0.3 a 0.0 e 0.2 0.6 0.0
22.3 28.5 38.1 38.7 43.1 60.1 0.00 16.7 13.2 18.4
± ± ± ± ± ± ± ± ± ±
0.5 c 2.4 c 0.3 b 2.4 b 1.6 b 1.5 a 0.0 d 0.7 0.3 0.0
a Each value is mean of 3 replicates from 2 experiments. Mean ± SE followed by same letter in column of each treatment is not significant difference at p = 0.05 by Tukey–Kramer HSD test, % inhibition rate was calculated compared to the germination of the control (0%). b Control without any formulation. c Dissolved in 0.1% acetic acid. d Dissolved in water, NPs-nanoparticles.
Table 2 Effect of various chitosan NPs on in vitro spore germination of A. alternata. Treatments (%) Chitosan NPs 0.001 0.005 0.01 0.02 0.06 0.1 Controlb Chitosan-saponin NPs 0.001 0.005 0.01 0.02 0.06 0.1 Controlb Cu-chitosan NPs 0.001 0.005 0.01 0.02 0.06 0.1 Controlb Chitosan (0.1%)c Saponin (0.1%)d CuSO4 (0.1%)d
% Inhibition ratea 14.6 51.0 57.3 68.5 84.4 87.1 0.00
± ± ± ± ± ± ±
1.0 e 0.8 d 0.7 c 0.4 b 0.6 a 0.1 a 0.0 f
13.2 62.8 63.6 73.1 78.3 82.9 0.00
± ± ± ± ± ± ±
1.2 c 0.6 b 2.6 b 5.1 ab 0.1 a 0.8 a 0.0 d
10.6 43.2 52.0 69.3 83.3 87.4 0.00 21.1 31.4 23.1
± ± ± ± ± ± ± ± ± ±
1.8 e 0.0 d 0.6 c 0.2 b 1.3 a 0.2 a 0.0 f 0.4 0.1 1.3
a Each value is mean of 3 replicates from 2 experiments. Mean ± SE followed by same letter in column of each treatment is not significant difference at p = 0.05 by Tukey–Kramer HSD test, % inhibition rate was calculated compared to the germination of the control (0%). b Control without any formulation. c Dissolved in 0.1% acetic acid. d Dissolved in water, NPs-nanoparticles.
Fig. 4. A representative photograph of antifungal bioassay. Effect of Cu-chitosan nanoparticles on mycelial growth of A. alternata.
significant improvement in growth inhibition rate by chitosansaponin treatment indicating that chitosan-saponin nanoparticles do not deserve a good antifungal agent in present study. Moreover, higher PDI value and lower zeta potential make chitosan-saponin nanoparticles less stable and less effective against fungi used in present study. Albeit, saponin-chitosan nanoparticle showed excellent growth inhibition on cancer cell in earlier report [28]. Copper compounds are known for their antifungal nature and are being effectively used to control fungal diseases. However, due to uncontrolled application, their concentrations have exceeded the limits specified for heavy metals [44]. Therefore, in present study Cuchitosan nanoparticles were prepared to improve the antifungal activity and overcome the existing problem of heavy dose. The protocol for synthesis of Cu-chitosan nanoparticles was standardized in the same way as chitosan nanoparticles and the developed method delivered highly stable nanoparticles with low PDI and high zeta potential as compared to previous reports [29,32]. AAS result shown that 70% of copper embedded in chitosan formulation in Cu-chitosan nanoparticles synthesis. Among all the treatments, Cu-chitosan nanoparticles were found very effective at 0.1% concentration and showed 89.5, 63.0 and 60.1% growth inhibition of A. alternata, M. phaseolina and R. solani, respectively in vitro. Further, the Cu-chitosan nanoparticles at 0.1% concentration showed the strongest growth inhibitory effects (87.4%) on spore germination of A. alternata. The observed higher antifungal activities of Cu-chitosan nanoparticles could be explained by the higher surface charge density (higher zeta potential) which provides them greater binding affinity for negatively charged fungal membrane. Further, Cu(II) undergoes reduction to Cu(I) in fungi which produces toxic H2 O2 which in turn destructs the cell viability [32]. In general, 0.06–1% concentrations of nanoparticles were found effective to control fungal growth in present study. In a prior report, higher doses ranging from 750 to 6000 mg/l of bulk chitosan were found effective against plant fungi [26], and these concentrations were higher compared to nanoparticles concentrations used in present study. The use of bulk chitosan has been restricted due to its poor solubility in water and poor antifungal activity [20]. Chemically modified chitosans viz. triethylene diamine dithiocarbamate chitosan and o-hydroxyphenylaldehyde thiosemicarbazone chitosan have previously been evaluated and found to show maximum 60.4 and 52.6% growth inhibition of F. oxysporum and R. solani at 500 and 50 mg/l concentration [26,45]. Therefore, the results of our study clearly indicate the pronounced effect of chitosan nanoparticles
V. Saharan et al. / International Journal of Biological Macromolecules 62 (2013) 677–683
over bulk and chitosan derivatives on growth inhibition of important plant pathogenic fungi. 5. Conclusions Chitosan, chitosan-saponin and Cu-chitosan nanoparticles were prepared and their structures were well characterized by DLS, FTIR, TEM and SEM. The lower PDI value and higher zeta-potential of chitosan and Cu-chitosan nanoparticles proved their uniform size and stability which may contribute to their higher antifungal activity against A. alternata, M. phaseolina and R. solani in in vitro studies. Cu-chitosan nanoparticles also showed maximum inhibition rate of spore germination of A. alternata. Compared to chitosan and Cuchitosan nanoparticles, the chitosan-saponin nanoparticles were found poor in antifungal activity. The higher PDI value and lower zeta-potential decreased their uniform size and stability in aqueous solution. The efficacy of chitosan based nanoparticles mainly chitosan and Cu-chitosan could further be tested on other plant pathogenic fungi in in vitro and in vivo models. The present study is a effort to explore potential of chitosan based nanoparticles and the findings of present study could be utilized for development of nano fungicide for crop protection in future. Acknowledgements The authors wish to thank Directorate of Research, MPUAT for their support of this work. The kind co-operation extended by Dr. Ramesh Raliya, Research Scientist, CAZRI, Jodhpur and Dr. N. Ilaiyaraja, Scientist, Defence Food Research Laboratory, Mysore, India for their technical help in FTIR and DLS studies, respectively is highly acknowledged. References [1] V. Ghormade, M.V. Deshpande, K.M. Paknikar, Biotechnol. Adv. 29 (2010) 792–803. [2] R. Nair, S.H. Varghese, B.G. Nair, T. Maekawa, Y. Yoshida, D.S. Kumar, Plant Sci. 179 (2010) 154–163. [3] G.N. Agrios, Plant Pathology, Academic Press, London, 2000. [4] H. Bouwmeester, S. Dekkers, M.Y. Noordam, W.I. Hagens, A.S. Bulder, C.D. Heer, S.E.C.G.T. Voorde, S.W.P. Wijnhoven, H.J.P. Marvin, A.J.A.M. Sips, Regul. Toxicol. Pharmacol. 53 (2009) 52–62. [5] L.R. Khot, S. Sankaran, J.M. Maja, R. Ehsani, E.W. Schuster, Crop Prot. 35 (2012) 64–70. [6] H.J. Park, S.H. Kim, H.J. Kim, S.H. Choi, J. Plant Pathol. 22 (2006) 295–302. [7] Y.K. Jo, B.H. Kim, G. Jung, Plant Dis. 93 (2009) 1037–1043. [8] S.W. Kim, J.H. Jung, K. Lamsal, Y.S. Kim, J.S. Min, Y.S. Lee, Mycobiology 40 (2012) 53–58. [9] L. He, Y. Liu, A. Mustapha, M. Lin, Microbiol. Res. 166 (2011) 207–215.
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