Caspian Journal of Applied Sciences Research, 1(12), pp. 83-90, 2012 Available online at http://www.cjasr.

com ISSN: 2251-9114, 2012 CJASR

Full Length Research Paper Destructive Effect of Silver Nanoparticles on Biocontrol Agent Fungi Trichoderma viride and T. harzianum
Shahin Gavanji1*, Majid Shams2, Noushin Shafagh3, Alireza jalali Zand4, Behrouz Larki4, Mohsen Doost Mohammadi4, Amir Hosein Taraghian4, Seyyed vahid niknezhad5
2

Young Researchers Club, Khorasgan Branch, Islamic Azad University, Khorasgan, Isfahan, Iran. Department of Agronomy and Plant Breeding Khorasgan Branch, Islamic Azad University, Isfahan, Iran. 3 Isfahan Center for Research of Agricultural Science and Natural Resources. 4 Department of Plant protection, Khorasgan (Isfahan) Branch , Islamic Azad University, Isfahan , Iran . 5 Chemical Engineering-Biotechnology, Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran.
*Corresponding Author: Tel.: (+98)-09138072585; E-mail: shahin.gavanji@yahoo.com
Received 23 August 2012; Accepted 14 October 2012

Trichoderma spp. serves as biocontrol and growth promoting agents for many crop plants due to their antagonistic properties against plant pathogens. Nanoparticles have become of an interest relatively recently, mostly because of their possible use in diverse technologies. In this research we have investigated the role of silver nanoparticles on growth rate of two biocontrol agent species of Trichoderma viride and T. harzianum by measuring the diameter of colonies of fungi on potato dextrose agar and the dry weight of mycelia culture medium. In this test in order to determine the minimum inhibitory concentration, 25, 50, 100 and 150 ppm of silver nanoparticles were used. General Regression Analysis was used to determine both the time and concentration variable and the possible interactions. The results showed that silver nanoparticles caused inhibited the growth of the two species of fungi. In this paper we suggest that silver nanoparticles, because of the harmful effects on environment, should be used less. Key word: Trichoderma viride, Trichoderma harzianum, Silver, Nanoparticle.

1. INTRODUCTION Trichoderma viride is a filamentous Ascomycota. Agricultural and orchard soil, which contain huge quantities of plant roots are the best places to live for these organisms (Samuels et al., 1999). Persoon almost 200 years ago described the genus Trichoderma that consists of anamorphic fungi isolated primarily from soil and decomposing organic matter. Isolates of Trichoderma are ubiquitous and their isolation and culture are very easy. These isolates can grow quickly on a wide range of substrates and produce metabolites with demonstrable antibiotic activity or mycoparasitic against a wide range of pathogens. Because of these properties, Trichoderma species have been used in biological control of fungi including plant pathogens (Inbar and Chet, 1992; Lumsden and Locke, 1989). Through nutrient competition and rhizosphere competence, mycoparasitism, enzyme, metabolism of germination stimulants and induced defense responses in plants. Trichoderma spp can control

root foliar pathogens (Howell, 2003; Haram et al., 1996; Zimand et al., 1996). Jin and Custis (2011) also showed that Trichoderma strains induce changes in the microbial composition on roots, enhance nutrient uptake, stabilize soil nutrients, promote root development, and increase root hair formation. Since mycoparasitic and antibiotic properties were demonstrated in 1932 and 1934 by Weindling, there have been biotechnological applications of these fungi as biocontrol agents (Chet, 1987; Haran et al., 1996; Schirmbock et al., 1994). Most species of the genus grow rapidly in articial culture and produce large numbers of small green or white conidia from conidiogenous cells of widely branched conidiophores which allow a relatively easy identication of Trichoderma as a genus (Rifai, 1969). Research is in progress on multifunctional materials containing silver nanoparticles (SNPs) in reactive or nonreactive polymer networks for applications as biocidal products and drug supports (Gaidau et al., 2009).

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Nanoparticles can be defined as objects ranging in size from 1-100 nm that due to their size may differ from the bulk material. The high surface to volume ratio increases their ability to penetrate cell membranes and possible biochemical activity. Nanoparticles are being used for diverse purposes, from medical treatments, using in various branches of industry production such as solar and oxide fuel batteries for energy storage, to wide incorporation into diverse materials of everyday use such as cosmetics or clothes (Dubchak et al., 2010). Silver ions have been used for decades as antimicrobial agents in various fields because of capacity to inhibit the growth of microorganisms. However since presence of Ag ions diminished rapidly, Ag ions from salts have only limited usefulness as antimicrobial agents. In contrast, silver nano particles provide a large surface of Ag for contact with microorganisms and Ag ions are released gradually (Vermerie et al., 1997).There are numerous reports of the antimicrobial activity of silver. However, antifungal properties of different forms of silver, which often coexist in solution, have not been investigated fully. Furthermore, there have been few studies that test the antifungal activity of silver for plant-pathogenic fungi (Park et al., 2006). Silver displays multiple modes of inhibitory action to microorganisms (Rifai, 1969) and so may be used to control various plant pathogens in a relatively safer manner compared to synthetic fungicides. Stable colloidal solutions containing nanoparticles were found to be effective against Aspergillus and Penicillium (Petica et al., 2008). Nano-Ag affected yeast by attacking cell membranes, thus disrupting membrane potential. Transmission electron microscopy analysis showed that Nano-Ag exposure to the membranes of Candida albicans, caused the formation of pits on the membrane surfaces and the formation of pores and subsequent cell death (Gajbhiye et al., 2009). The objective of this study is to determine the destructive and inhibitory properties of silver nanoparticles on colony formation of the beneficial fungi T. viride and T.harzianum. 2. MATERIAL AND METHODS Isolates of Trichoderma harzianum (T8) and T. viridae (PTCC5157) were obtained from the Agricultural Research Center of Isfahan, Department of Plant Pathology. Isolates were sub cultured on potato dextrose agar plates (Merck Company) from 5 mm disks of Trichoderma spp. and maintained in an incubator at 35 C for 7 to 10

days until spores developed. We used distilled water to prepare a spore suspension with a concentration of 5 106 spores/ml. These water suspensions of Trichoderma spp. spores were mixed with silver nanoparticles at different concentrations. Control samples were prepared by using a sterile water suspension of Trichoderma spp spores with the same concentrations of spores as the samples which contained silver nanoparticles. The control samples and solutions containing spores and silver nanoparticles were inoculated after 2 and 24 h of incubation on sterile Petri dishes containing PDA culture medium. These cultures were grown on (PDA) medium at 25C for 3 days. Benomyl was used as positive control. The fungicide was solved in the PDA medium combined with nanoparticles (Togashi et al., 1999) and it was used at g/liter of PDA culture medium. The fungicide was added after sterilization of PDA. All the experiments were replicated 5 times. 2.1. Ag-NPs Ag nanoparticles (Nanocid L2000, Nano Nasb Pars Company) were used. The concentration of 4000 ppm was used in form of colloidal suspension to provide stability in cultural medium. The Ag-NPs were between 18 to 34 nm, and the Zeta potential of silver nanoparticles was -33.5 that showed the average stability of this compound. All dilutions of the Ag-NPs were made with distilled water. Concentrations of 25, 50, 100 and 150 ppm were used and for each concentration 5 repetitions of fungus isolates were used. Nanoparticle solutions were mixed with the sterilized PDA medium before pouring into the Petri dishes. The fungi were incubated at 25C for one week. After incubation the diameter of fungal colonies was measured. The assays were carried out every two days and recording continued until 14 days after cultivation. Fungus treated with distilled water without nanoparticles was used for negative controls and fungicide Benomyl incorporated into the PDA combined with nanoparticles was used for positive controls. 2.2. Studying inhibition effect of nanosilver on growth of T. viride and T. harzianum PDB medium inoculated with T. viride and T. harzianum was grown 7 days of incubation, then the culture was filtered onto Watman paper No 1 using vacuum pump. Preweighed papers were

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dried in oven for 2 hours at 60 C and the dry weight of fungi was measured by using a balance with 0.0001 precision. 2.3. Statistical analysis General Regression Analysis was used to determine both the time and concentration variable and the effect of these two on each other. For a

95% confidence level, the p-value was less than or equal to 0.05 3. RESULTS The analysis of variance is presented in Tables 1 and 2. All of the factors and their interactions were significant for concentrations. Although the confidence seems less than 95% the P-value of regression is 0.000, indicating that the data are significant. (Tables 1 and 2)

Table 1: Analysis of variance table for Trichoderma viride Source DF Seq SS Adj SS AdJ MS F Regression 3 58.4430 58.4430 19.4810 66.0310 Time1 1 28.0357 27.3313 27.3313 92.6390 Concentrion1 1 24.6361 0.7058 0.7058 2.3920 Time1*Concentration1 1 5.7711 5.7711 5.7711 19.5610 Error 36 10.6210 10.6210 0.2950 Lack-of-Fit 16 10.5610 10.5610 0.6601 220.0220 Pure Error 20 0.0600 0.0600 0.0030 Total 39 69.0640

P 0.0000 0.0000 0.13067 0.0001 0.0000

DF: Degrees of freedom; Seq SS : Sequential sums of squares; Adj SS: Adjusted sums of squares; AdJ MS: adjusted mean of squares; F: F- test; P: P-value

Table 2: Analysis of variance table for Trichoderma harzianum Source DF Seq SS Adj SS AdJ MS F Regression 3 45.2035 45.2035 15.0678 61.3102 Time1 1 20.7765 19.8232 19.8232 80.6595 Concentrion1 1 23.5927 4.0220 4.0220 16.3654 Time1*Concentration1 1 0.8344 0.8344 0.8344 3.3950 Error 36 8.8475 8.8475 0.2458 11.1763 Lack-of-Fit 16 7.9575 7.9575 0.4973 Pure Error 20 0.8900 0.8900 0.0445 Total 39 54.0510

P 0.0000 0.0000 0.0003 0.0736 0.0000

3.1. Normal Probability Plot of Residuals One of the key assumptions for the statistical analysis of data from experiments is that the data come from a normal distribution. The normality of the data can be checked by plotting a normal probability plot of the residuals. If the points on the plot fall fairly close to a straight line then the data are normally distributed (Antony, 2003). In Figures

1-1 and 1-2 for ensuring that our data are normal we checked the data by plotting a normal probability plot of the residuals. The normal probability plot of the residual for fungi T. viride and T. harzianum are shown in Figures 1-1 and 12. According to these Figures we see that the points fall fairly close to the straight line. Therefore the data from the experiments come from a normally distributed population.

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Gavanji et al. Destructive Effect of Silver Nanoparticles on Biocontrol Agent Fungi Trichoderma viride and T. harzianum

Figure A

Figure B

Fig. 1: A & B: Normal probability plot of the residual for T. viride and T. harzianum 3.2. Plot of Interaction Effects The interaction effects plots are shown in Figures 2 and 3 for T. viridae and T. harzianum respectively. The plots provide the mean response of two factors at all possible combinations of their settings. If the lines are not parallel, it is an indication of interaction between the two factors (Srinivasan and Viraraghavan, 2010). In Figures 2 and 3, the effect of time and concentration on growth rate of T. viridae and T. harzianum is shown. Based on statistical analysis the results show that the growth rate of T. viridae in low concentration of nanoparticles is higher than T. harzianum. At 50 ppm, the rate of changes of concentration effect on T. viridae is lower than T. harzianum. In this concentration T. viridae shows a higher rate of growth. In concentration of 100 ppm, the time and concentration effects on T. viridae were higher than T. harzianum. But in concentration of 150 ppm, the effect of silver nanoparticles was considerable and showed an equal effect on both species. Increasing the concentration from 0 to 25 ppm in T. viridae was more effective and the concentration changing from 25 to 50 ppm showed a good inhibitory effect on T. harzianum. Figures 2a and 3a show the effect of time and concentration so that as they increase, the diameter of fungal colonies decreases. Data analysis using General Regression Analysis showed that 25 ppm of silver nanoparticles has an effect on the growth of T. viride and T. harzianum and the concentration 150 ppm has the most inhibiting growth effect at 120, 216 and 360 hours after exposure to the fungi (Figures2 and 3). Another way to understand this problem is to use Regression Equation 1 and 2 which show the variant effects of concentration and time separately and the effect of two parameters on each other on growth rate of fungi. Time has a positive effect on growth rate of T.viride and T.harzianum. The effect of silver nanoparticle can be observed based on equation coefficients on growth of T.viride and T.harzianum. Considering the equations and the curve in Figure 1, the main factor in decreasing the growth rate of T.viride and T.harzianum is the concentration and the time and concentration interaction is another factor in growth decreasing (Figures 2 and 3). 3.3. Regression Equation T. viride (D1) = 0.698411 + 0.0104381 Time 1 0.00434507 Concentrion1 5.68234e-005 Time1*Concentration1 T. harzianum (D2) = 0.796981 + 0.0088895 Time2- 0.00472422 concentration2 - 4.74373e-005 Time2*Concentration2 Based on statistical analysis at different times with increasing in concentration, the growth rate of fungi T.viride and T.harzianum also decreased. Silver nanoparticles had an inhibitory effect on the fungi. The fungal growth rate decreased with increasing time of exposure and concentration of silver nanoparticles. The concentration 150 ppm of silver nano particles has equal effect with Benomyl (Figure 4).

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inhibiting growth cm

Concentrations

ppm m

Figure2a

Figure2b

Fig. 2: The growth rate of T. viridae as a function of concentration and time.

inhibiting growth cm

Concentrations

ppm

Figure3b Figure3a Fig. 3: The growth rate of T. harzianum as a function of concentration and time.
0.7 Diameter of Fungal (mm2/h) 0.6 Figure 4. The growth rate of T. harzianum as a function of concentration and time. 0.5 0.4 0.3 0.2 0.1 0 0 25 50 100 150 Fungicide Nano Silver Concentration (ppm) T. harzianum T. viride

Fig. 4: Effect of different concentration of nanoparticles on diameter of fungal T. viride and T. harzianum. Zero is negative control and fungicide is positive control.

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3.4. Effect of different concentrations of nano silver on growth of myceliums: Nano silver at a concentration of 150 ppm, compared to other concentrations, had a significant effect on dry weight of these two fungi (Table 3). The microscopic observations with 100X zoom show that there is shrinkage in b1, b2 and b3 of

mycelium of T. harzianum. At 150 ppm similar results were shown in d1, d2 and d3 for T. viride. These findings clearly indicate that the nanoparticles have a detrimental effect on mycelia of T. harzianum and T. viride. Control groups (A and C), did not receive silver nanoparticles (Figures 5 and 6).

Fungi T. viride T. harzianum

Table 3: Main dry weight of mycelium (mg) control 25ppm 50ppm 100ppm 230 210 193 157 190 182 161 143

150ppm 101 92

Fig. 5:T. harzianum; A: control (without using nanoparticles); B: 150 ppm of silver nanoparticles.

Fig. 6: T. viride; C: control (without using nanoparticles); D: 150 ppm of silver nanoparticles.

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4. DISCUSSION Little is known about the destructive and inhibitory effect of silver nanoparticles on beneficial fungi since most studies have focused on pathogenic fungi. Multiple lines of evidence support the hypothesis that silver nanoparticles enhance antimicrobial activity. According to Morones et al. (2005), silver nanoparticles are highly reactive because they generate Ag+ ions. Nano-silver is an effective and fast-acting fungicide against a broad spectrum of common fungi and yeasts including genera such as Aspergillus, Candida, and Saccharomyces (Wright et al., 1999). Moreover, silver nanoparticles are effective against yeast (Kim et al., 2007). In this test we determined the inhibitory effect of silver nanoparticles on two beneficial fungi. The different concentrations of nanoparticles inhibited the growth rate of T. viride and T. harzianum. In this test beside the effect of different concentrations on fungi growth, the time factor was also evaluated. Growth rate was examined for 360 hours. The exact mechanisms of action of silver nanoparticles against fungi are still not clear, but mechanisms similar to that of the antibacterial actions have been proposed for fungi (Wright et al., 1999). Based on studies that show that silver nanoparticles anchor to and penetrate the cell wall of Gram negative bacteria (Morones et al., 2005; Sondi and Salopek-Sondi, 2004), it has also been proposed that the antibacterial mechanism of silver nanoparticles is related to the formation of free radicals and subsequent free radical-induced membrane damage (Danilczuk et al., 2006; Kim et al., 2007). Unlike other studies that investigated the side effects of silver nanoparticles against pathogenic bacteria, this research studied the effect of silver nanoparticles on fungicidal effect of these nanoparticles against T. viride and T. harzianum. The results showed that growth rate of the fungi decreased with increasing time of exposure and concentration of silver nanoparticles. Also in this study the fungicidal effect of antifungal Benomyl was compared with Nano silver effect. 150 ppm of silver Nano particles had equal effect to that of Benomyl. Different species of fungi Trichoderma are spread in different environments. Trichoderma secretes Chitinase and can control biological diseases caused by pathogenic fungi. The benefits of Trichoderma species are clear and by using silver nanoparticles inappropriately, this relation will be disrupted. Despite the wide range of application of Nano silver, little research has been done on the toxicity and fate of Nano silver within

the ecosystem. The extensive use of Nano silver can lead to development of resistance among pathogens and creating a threat to microbial diversity (Faunce and Watal, 2010). The bactericidal dose of silver nanoparticles differs for each microbe. It is recently showed that some concentrations of Nano silver are toxic to several nitrifying bacterial species which play an important role in nitrification (Benn and Westerhoff, 2008). Silver nanoparticles cause harmful effects to beneficial fungi, so use it less in sensitive environments. Based on obtained results it is clear that the usage of silver nanoparticles should be in a good management in environment because microbes have an important role in environment and any changes in their growth can cause many problems for biological cycles. ACKNOWLEDGEMENTS This project with number 04-14-5-57494 was approved in Young Researchers Club of Islamic Azad University of Khorasgan Branch which had done by financial supports of this club. And the efforts of Dr.Foroughi, the head of Islamic Azad University of Khorasgan branch, Dr.PayamNajafi and Mr.Khaje are very considerable. REFERENCE Antony J (2003). Design of Experiments for Engineers and Scientists, ButterworthHeinemann, New York. Benn TM, Westerhoff P (2008). Nanoparticle silver released into water from commercially available sock fabrics. Environ. Sci. Technol., 42(11): 4133-4139. Chet I ( 1987). Innovative approaches to plant disease control. John Wiley and Sons, New York, N.Y. p.137160. Dubchak S, Ogar A, Mietelski JW, Turnau K (2010). Influence of silver and titanium nanoparticles on arbuscular mycorrhiza colonization and accumulation of radiocaesium in Helianthus annuus. Span. J. Agric. Res., 8(1): 103-108. Danilczuk M, Lund A, Sadlo J, Yamada H, Michalik J (2006). Conduction electron spin resonance of small silver particles. Spectrochim. Acta A Mol. Biomol. Spectrosc., 63(1):189- 191. Faunce T, Watal A (2010). Nanosilver and global public health: international regulatory issues. Nanomedicine., 5(4): 617-632.

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Gajbhiye M, Kesharwani J, Ingle, A, Gade A and Rai M (2009). Fungus mediated synthesis of silver nanoparticles and their activity against pathogenic fungi in combination with fluconazole. Nanomedicine: NBM., 5: 382386. Gaidau C, Petica A, Ciobanu C, Martinescu T (2009). Investigations on antimicrobial activity of collagen and keratin based materials doped with silver nanoparticles. Biotechnol. Lett., 14(5):4665-4672. Haran S, Schickler H and Chet I (1996). Molecular mechanisms of lytic enzymes involved in the biocontrol activity of Trichoderma harzianum. Microbiology., 142: 23212331. Howell CR (2003). Mechanisms employed by Trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant Dis., 87: 4-10. Inbar J, Chet I (1992). Bionomics of fungi cell wall recognition by use of lectin-coated nylon fibres. J. Bacteriol., 174: 1055-1059. Jin X, Custis D (2011). Microencapsulating aerial conidia of Trichoderma harzianum through spray drying at elevated temperatures. Biol. control., 56: 202208. Kim JS, Kuk E, Yu KN, Kim JH, Park SJ, Lee HJ, Kim SH, Park YK, Park YH, Hwang CY, Kim YK, Lee YS, Jeong DH, Cho MH (2007). Antimicrobial effects of silver nanoparticles. Nanomedicine: NBM., 3(1):95-101. Lumsden RD, Locke JC (1989). Biological control of damping-off caused by Pythium ultimum and Rhizoctonia solani in soiless mix. Phytopathology., 79:361-366. Morones JR, Elechiguerra A, Camacho k, Holt JB, Kouri JT, Ramirez and Yacaman MJ ( 2005). The bactericidal effect of silver nanoparticles. Nanotechnology., 16)10): 2346-2353. Park HJ, Kim SH, Kim HJ, Choi SH (2006). A new composition of nanosized silica-silver for control of various plant diseases. Plant Pathol. J., 22(3):295-302. Petica A, Gavriliu S, Lungua M, Burunteaa N, Panzarub C (2008).Colloidal silver solutions with antimicrobial properties. Mater. Sci. Eng. B., 152: 22-27.

Rifai MA ( 1969). A revision of the genus Trichoderma. Mycol. Papers. 116: 156. Samuels GJ, Lieckfeldt E, Nirenberg HI(1999). Trichoderma asprellum a new species with warted conidia and redescription of T. viride. Sydowia, 51: 71-88. Sondi I, Salopek, Sondi B (2004). Silver nanoparticles as antimicrobial agent: A case study on E. coli as a model for Gram negative bacteria. J. Colloid Interface Sci., 275:177-182. Schirmbock M, LoritoYL, Wang CK, Hayes I, Scala F, Harman Ge, Kubicek CP (1994). Parallel formation and synergism of hydrolytic enzymes and peptaibol antibiotics, molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi. Appl. Environ. Microbiol., 60:43644370. Srinivasan A, Viraraghavan T ( 2010). Oil removal from water by fungal biomass: A factorial design analysis. J. Hazard Mater., 175(1): 695-702. Togashi I, Gisusi S, Harada A ( 1999). Effects of Benomyl wettable powder autoclaved on the mycelial growth of Trichoderma spp and Lentinula edodes. J. Hokkaido Forest Prod. Res. Inst., 13(1): 1-5. Vermerie N, Malbrount C, Azar M, Arnaud P (1997). Stability of nystatin in mouthrinses; Effect of pH, temperature, concentration and colloidal silver addition, studied using an in vitro antifungal activity. Pharm. World Sci., 19(4): 197 - 201. Wright JB, Lam K, Hansen D, Burrell RE (1999). Efficacy of topical silver against fungal burn wound pathogens. Am. J. Infect. Control., 27:344-350. Weindling R (1932). Trichoderma lignorum as a parasite of the soil fungi. Phytopathology, 22: 837-845. Weindling, R (1934). Studies on lethal principle effective in the parasitic action of Trichoderma lignorum on Rhizoctonia solani and other fungi. Phytopathology. 24: 1153- -1179 Zimand G, Elad Y, Chet I (1996). Effect of Trichoderma harzianum on Botrytis cinerea pathogenicity. Phytopathology, 86:12551260.

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