International Journal of Biological Macromolecules 67 (2014) 452–457
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
A study on antifungal activity of water-soluble chitosan against Macrophomina phaseolina Sudipta Chatterjee a,b,∗ , Bishnu P. Chatterjee c , Arun K. Guha b a b c
School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India Department of Natural Science, West Bengal University of Technology, Saltlake, Kolkata 700 064, India
a r t i c l e
i n f o
Article history: Received 16 November 2013 Received in revised form 28 March 2014 Accepted 5 April 2014 Available online 18 April 2014 Keywords: Chitosan Antifungal activity Fungus
a b s t r a c t The objective of this study was to evaluate antifungal effect of water-soluble chitosan (s-chitosan) on Macrophomina phaseolina (M. phaseolina) causing jute seedling infection and monitor the change in activity of released enzymes during infection. The minimum inhibitory concentration (MIC) of s-chitosan for M. phaseolina was found at 12.5 g/l and s-chitosan exhibited fungistatic mode of action against this pathogen. The application of s-chitosan (12.5 g/l) during infection of jute seedlings by M. phaseolina inhibited fungal infection and length of the seedlings was found almost similar to seedlings without infection. M. phaseolina infected jute seedlings showed length of 22 mm over 10 days of incubation and it increased to 58 mm in presence of s-chitosan (12.5 g/l) during incubation for 10 days. TEM study indicated presence of hyphae in the cortical and epidermal cells of fungus infected jute seedlings indicating colonization by the fungus and it disappeared after treatment with s-chitosan. The changes in enzyme profiles of jute seedling during prevention of fungal infection using s-chitosan helped in proper understanding of mode of action of s-chitosan as antifungal agent. The activity of defense related enzymes like chitosanase and peroxidase in infected seedlings was observed to be enhanced after treatment with s-chitosan. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cash crops like jute and cotton are infected by Macrophomina phaseolina (M. phaseolina) causing stem rotting [1]. Besides jute, the fungus can infect several other plants such as peanut, corn, sorghum [2]. Different methods have been employed to control this soil borne pathogen. The available methods are crop rotation [3], application of pathogen resistant cultivars [4], and regular irrigation [5]. Considerable research has been performed with plant extracts to control seed-borne fungi. Garlic bulb extracts can inhibit spore germination and mycelia growth of seed-borne fungal pathogens of jute, including M. phaseolina [6]. Chitosan, a linear polymer of -1,4-d-glucosamine is derived by deacetylation of chitin, a naturally occurring biopolymer and second most abundant polysaccharides after cellulose. Chitin is present in exoskeleton of crustacean such as crab, shrimp, lobster, cray fish, and insects. Chitosan can also be found in certain groups of
∗ Corresponding author at: Nanyang Technological University, School of Chemical and Biomedical Engineering, 62 Nanyang Drive, Singapore 637459, Singapore. Tel.: +65 63168790; fax: +65 67947553. E-mail address: [email protected] (S. Chatterjee). http://dx.doi.org/10.1016/j.ijbiomac.2014.04.008 0141-8130/© 2014 Elsevier B.V. All rights reserved.
fungi particularly zygomycetes [7]. Chitosan being nontoxic, polycationic and biodegradable finds numerous applications especially in the agricultural food and pharmaceutical industries [8]. Recently chitosan, its derivative and oligomers find numerous applications as antimicrobial agent to inhibit the growth of different microorganisms [9,10]. Much of the interest in the antimicrobial properties of chitosan has been focused on its possible role in plant protection [11]. Infection of plants by viruses, fungi or bacteria induces some pathogenesis related proteins such as chitosanase and peroxidase [12]. The products of these enzymes act as elicitors for further induction of the enzymes and for activation of other defense related biochemical such as phytoalexin and lignification [13]. Plant defensive role against pathogens can be elicited by adding chitosan oligosaccharides [14]. Chitosan plays potential dual role by directly inhibiting fungal growth [15] and inducing defense response of the host plant by eliciting production of pathogenesis related proteins and phytoalexin [16]. Infection of seeds by fungi also changes nutritional profiles and activity of amylases and protease etc [17]. The present study describes the antifungal activity of watersoluble chitosan (s-chitosan) against soil borne pathogenic fungus M. phaseolina causing infection to jute seedlings. The changes in the activities of chitosanase, peroxidase and amylase in
S. Chatterjee et al. / International Journal of Biological Macromolecules 67 (2014) 452–457
M. phaseolina infected jute seedlings after treatment with schitosan are described here.
453
2. Experimental
examined for visible growth. Absence of visible colony on agar plate within 48 h could be considered as fungicidal effect of s-chitosan. The appearance of visible growth was an indication of its fungistatic nature.
2.1. Materials
2.7. Infection of jute seeds
The phytopathogenic fungus M. phaseolina (MTCC 166) was obtained from The Institute of Microbial Technology, Chandigarh, India. Jute seed (JRO 524) was obtained from National Seeds Corporation, Kolkata, India. Chitosan (>85% deacetylation) was purchased from Sigma Chemical Co., USA. All other chemicals were procured from E. Merck, Germany.
One hundred jute seeds were surface sterilized with 0.1% HgCl2 solution, washed thoroughly with double distilled water for several times to remove HgCl2 and the seeds were steeped in sterile water for 6 h. The surface sterilized seeds were transferred in a germination plate containing a filter paper which was previously soaked with Richard’s medium. The plate was incubated at 30 ◦ C in a humidified chamber (relative humidity 100%). Elongation of germ tubes of jute seeds (seedlings) was measured at four different conditions using slide calipers every other day up to 10 days. The conditions were normal seeds, infected seeds, s-chitosan (12.5 g/l s-chitosan in Richard’s medium) treated non-infected seeds, and schitosan treated infected seeds. Spore suspension (100 l; 2.9 × 106 spore/ml) was added separately to the filter paper in the germination plate containing infected seedlings and s-chitosan treated infected seedlings.
2.2. Preparation of water-soluble chitosan Chitosan was dissolved in 7% acetic acid (v/v), and hydrolyzed by refluxing at 95 ◦ C for 20 h [18]. The hydrolyzed product was dried by rotary evaporator and extracted with de-ionized water. To the aqueous solution containing chitosan, acetone–alcohol mixture (60:40) was added. Water-soluble chitosan was precipitated, triturated with acetone, dried at 60 ◦ C [19], and designated s-chitosan.
2.8. Assay of enzyme activity in jute seedlings 2.3. Growth kinetics Different concentrations of s-chitosan (0.5, 1.0, 2.0, 5.0, 7.5, 10.0 and 12.5 g/l, pH 5.0) were added to 50 ml Richard’s medium (3% sucrose, 1% KNO3 , 0.5% KH2 PO4 , 0.25% MgSO4 and 2% agar). 100 l of fungal spore suspension (spore density 2.9 × 106 spore/ml) was added and inoculated at 30 ◦ C. The growth of M. phaseolina was observed in every alternate day up to 20 days, and the growth kinetics was determined by measuring dry weight of fungal biomass. Richard’s medium containing spores only was used as control. 2.4. Effect of s-chitosan on spore germination To Richard’s medium (5 ml) containing different concentrations of s-chitosan (0.5–12.5 g/l, pH 5.0), 100 l of spore suspension (2.9 × 106 spore/ml) was inoculated. Germ tube elongation of the fungus was measured up to 48 h using phase contrast microscope (Microstar American optical at 100× magnification). The same experiment was performed without s-chitosan serving as control. Germination was indicated by visible germ tube elongation. 2.5. Effect of s-chitosan on radial growth Agar plates containing Richard’s medium were prepared with different concentrations of s-chitosan (5, 7.5, 10, 12.5, and 15.0 g/l). The agar plate without s-chitosan was used as control. Fungal mass of 8 mm diameter previously grown in potato dextrose agar (PDA) plate for 2 days was placed in the Richard’s agar medium plates and were incubated at 30 ◦ C for 3 days. Radial growth of the fungus was determined by measuring the diameter at three points across the mycelia colony. 2.6. Determination of antifungal property of s-chitosan To determine whether s-chitosan is fungistatic or fungicidal against M. phaseolina, spore suspension was inoculated to Richard’s medium in presence of s-chitosan at 30 ◦ C for 24 h. The germination was examined microscopically. A portion of this solution was centrifuged under aseptic condition, washed thoroughly with sterile water, and again suspended in Richard’s medium without s-chitosan. A minute amount of grown mass was streaked on Richard’s agar plate and incubated at 30 ◦ C for 48 h, and finally
One hundred germinated jute seeds were harvested after 120 h, frozen in liquid nitrogen, lyophilized, and powdered with a mortar and pestle. The powder (0.1 g) was extracted with 5 ml 0.05 M sodium phosphate buffer (pH 6.5) for 8 h at 4 ◦ C and centrifuged at 16,000 × g for 30 min at 4 ◦ C. The protein content of the supernatant was determined by Bradford method [20]. Chitosanase activity was assayed in 0.2 ml seed extract containing 50 g protein and 0.2% s-chitosan as a substrate. After incubation for 10, 20 and 30 min, respectively, at 37 ◦ C the reactions were terminated followed by the addition of 0.8 ml of dinitrosalicylic acid and the mixture was immediately boiled for 10 min and cooled. The reducing sugar, glucosamine (GlcN) released by chitosanse was measured spectrophotometrically at 575 nm [21]. Chitosanse activity was expressed as g of glucosamine (GlcN)/min/mg protein. To determine peroxidase activity in jute seed extract, an aliquot of the crude seed extract containing 50 g of protein was added to 1 ml assay mixture containing 0.05 (M) sodium acetate buffer (pH 6.0), 0.2 ml 0.1 M pyrogallol, 0.1 ml 90 mM H2 O2 . The reaction was allowed to proceed for 6 min and optical density was measured spectrometrically at 300 nm at an interval of 1 min after the addition of crude seed extract to the substrate [22]. Peroxidase enzyme activity was expressed as absorbance (OD) difference at 300 nm per min per mg protein or OD300 nm/min/mg protein. Activity of amylase was assayed as follows. To crude seed extract containing 50 g of protein and 10 l of 1% NaCl was added 80 l of 0.5% soluble starch in 0.5 M phosphate buffer at pH 6.7 and 0.2 ml sample of assay mixture was incubated at 37 ◦ C for 10, 20 and 30 min, respectively. Thereafter 0.8 ml of dinitrosalicylic acid was added and the reaction mixture was immediately boiled for 10 min. After cooling the amylase activity was measured spectrophotometrically at 540 nm [21]. Amylase activity was expressed as g of glucose/min/mg protein. 2.9. Transmission electron microscopy (TEM) After 5 days of germination, segments from non-infected, infected, and s-chitosan treated jute seedlings were primarily fixed with 3% (v/v) glutaraldehyde in 10 mM phosphate buffer (pH 7.2) for 4 h at 4 ◦ C. After rinsing with the same buffer, secondary fixation of tissue was done with 1% (w/v) aqueous solution of osmium
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S. Chatterjee et al. / International Journal of Biological Macromolecules 67 (2014) 452–457
12
Inhibition of spore germination (%)
100
10
8
6
4
2
10 80 8 60 6 40 4 20
2
Length of the germ tube ( μ m)
Dry weight of mycellia / litre of broth (g/l)
12
0 0
0
2
4
6
8
10
12
14
16
18
20
22
Number of days
0 0
2
4
6
8
10
12
Concentration of chitosan (g/l)
Fig. 1. Anti fungal activity of s-chitosan at different concentrations 0 g/l, --; 0.5 g/l, -䊉-; 1.0 g/l, --; 2.0 g/l, --; 5.0 g/l, --; 7.5 g/l, -+-; 10 g/l, -×-; 12.5 g/l, -*-; against M. Phaseolina.
Fig. 2. Anti fungal activity of s-chitosan on inhibition of spore germination, -䊉-; length of germ tube, --; of M. phaseolina.
tetroxide for 1 h. After washing with water for three times, samples were dehydrated using different concentrations of ethanol (30, 50, 70, 90 and 100%; 10 min each step) at room temperature. After treating the sample with propylene oxide (PO) as transitional solvent for three times (10 min each step), segments were infiltrated in 1:2 (v/v) PO-resin for 1 h, 2:1(v/v) PO-resin for 4 h and pure resin for overnight [23]. Samples were embedded in pure resin and cured at 60 ◦ C for 3 days. The ultrathin sections were cut in an ultramicrotome and picked up on uncoated grid. The sections were usually stained with a saturated solution of uranyl acetate for 10–15 min at room temperature, washed with water, stained again with lead citrate for 5–10 min at room temperature, washed again with water and was blotted dry. TEM was done in a JEM-2010 electron microscope.
3. Results and discussion 3.1. Antifungal activity of s-chitosan Chitosan, which is a biodegradable, non-toxic, ecofriendly natural biopolymer, is frequently used as an antimicrobial agent. In this study, it was found that s-chitosan inhibited the growth of plant pathogen M. phaseolina. The minimum inhibitory concentration (MIC) of s-chitosan against this fungus was found to be 12.5 g/l. The MIC as low as 0.075 g/l for particulate chitosan and 0.018 g/l for soluble chitosan against Fusarium solani, a plant pathogenic fungus in liquid broth had been reported [24,25]. However, Botrytis cinerea and Rhizopus stolanifers [26] required chitosan for MIC as high as 10 g/l. Some authors suggested that Mucor sp. having
Fig. 3. Effect of s-chitosan at different concentration Control, C; 5 g/l, (I) 7.5 g/l, (II) 10 g/l, (III) 12.5 g/l, (IV) on radial growth of M. phaseolina.
S. Chatterjee et al. / International Journal of Biological Macromolecules 67 (2014) 452–457
a)
70
non infected Infected
6
60
Chitosanase activity
50 40 30 20 10 0 1
2
3
4
5
6
7
8
9
10
11
( μ g GlcN/min/mg of protein)
Length of jute seedlings (mm)
455
Day of culture
0
Chitosan untreated
Chitosan treated
18
b) Peroxidase activity Δ OD300 nm /min/mg of protein
chitosan within the cell walls are more resistant to the antimicrobial action of chitosan [24]. The change in germination time, germ tube length and dry weight of mycelia of M. phaseolina, a soil borne pathogen was noticed during its growth in s-chitosan supplemented Richard’s agar medium at 30 ◦ C for a definite period of time. Addition of s-chitosan up to 5 g/l in growth medium of fungus, germination time fungus did not markedly change. However the reduction in dry weight of mycelia and germ tube length indicated an inhibitory effect of s-chitosan on fungal growth. Further increase in concentration of s-chitosan (up to 10 g/l) showed a pronounced inhibitory effect as mycelial growth was found to reduce from 12 to 6.45 g/l (Fig. 1), Similar effect was observed in germ tube elongation of fungus and it was found to reduce from 12.9 to 2.9 m with increase in s-chitosan concentration. 80% inhibition of spore germination was also observed with s-chitosan supplementation in the growth medium (Fig. 2). Germination time of fungus at 10 g/l of s-chitosan was found to be 48 h where as control without s-chitosan showed germination time of 24 h. There was no growth of fungus at 12.5 g/l of s-chitosan. Therefore, it was concluded that the MIC of s-chitosan against M. phaseolina was 12.5 g/l. It was observed that diameter of the fungal mycelia was reduced markedly with increase in s-chitosan concentration (Fig. 3). Chitosan due to its positive charge on the C-2 of the glucosamine monomer is more stable and showed better antimicrobial activity than chitin [27]. The exact mechanism of the antimicrobial action of chitin, chitosan and their derivatives is not clearly understood but different mechanisms have been proposed [28]. The interaction between positively charged chitosan molecule and negatively charged microbial cell membrane leads to the leakage of proteinaceous and other intracellular constituents [29]. Chitosan acts mainly on the outer surface of the bacteria. At lower concentration (
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
A study on antifungal activity of water-soluble chitosan against Macrophomina phaseolina Sudipta Chatterjee a,b,∗ , Bishnu P. Chatterjee c , Arun K. Guha b a b c
School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India Department of Natural Science, West Bengal University of Technology, Saltlake, Kolkata 700 064, India
a r t i c l e
i n f o
Article history: Received 16 November 2013 Received in revised form 28 March 2014 Accepted 5 April 2014 Available online 18 April 2014 Keywords: Chitosan Antifungal activity Fungus
a b s t r a c t The objective of this study was to evaluate antifungal effect of water-soluble chitosan (s-chitosan) on Macrophomina phaseolina (M. phaseolina) causing jute seedling infection and monitor the change in activity of released enzymes during infection. The minimum inhibitory concentration (MIC) of s-chitosan for M. phaseolina was found at 12.5 g/l and s-chitosan exhibited fungistatic mode of action against this pathogen. The application of s-chitosan (12.5 g/l) during infection of jute seedlings by M. phaseolina inhibited fungal infection and length of the seedlings was found almost similar to seedlings without infection. M. phaseolina infected jute seedlings showed length of 22 mm over 10 days of incubation and it increased to 58 mm in presence of s-chitosan (12.5 g/l) during incubation for 10 days. TEM study indicated presence of hyphae in the cortical and epidermal cells of fungus infected jute seedlings indicating colonization by the fungus and it disappeared after treatment with s-chitosan. The changes in enzyme profiles of jute seedling during prevention of fungal infection using s-chitosan helped in proper understanding of mode of action of s-chitosan as antifungal agent. The activity of defense related enzymes like chitosanase and peroxidase in infected seedlings was observed to be enhanced after treatment with s-chitosan. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cash crops like jute and cotton are infected by Macrophomina phaseolina (M. phaseolina) causing stem rotting [1]. Besides jute, the fungus can infect several other plants such as peanut, corn, sorghum [2]. Different methods have been employed to control this soil borne pathogen. The available methods are crop rotation [3], application of pathogen resistant cultivars [4], and regular irrigation [5]. Considerable research has been performed with plant extracts to control seed-borne fungi. Garlic bulb extracts can inhibit spore germination and mycelia growth of seed-borne fungal pathogens of jute, including M. phaseolina [6]. Chitosan, a linear polymer of -1,4-d-glucosamine is derived by deacetylation of chitin, a naturally occurring biopolymer and second most abundant polysaccharides after cellulose. Chitin is present in exoskeleton of crustacean such as crab, shrimp, lobster, cray fish, and insects. Chitosan can also be found in certain groups of
∗ Corresponding author at: Nanyang Technological University, School of Chemical and Biomedical Engineering, 62 Nanyang Drive, Singapore 637459, Singapore. Tel.: +65 63168790; fax: +65 67947553. E-mail address: [email protected] (S. Chatterjee). http://dx.doi.org/10.1016/j.ijbiomac.2014.04.008 0141-8130/© 2014 Elsevier B.V. All rights reserved.
fungi particularly zygomycetes [7]. Chitosan being nontoxic, polycationic and biodegradable finds numerous applications especially in the agricultural food and pharmaceutical industries [8]. Recently chitosan, its derivative and oligomers find numerous applications as antimicrobial agent to inhibit the growth of different microorganisms [9,10]. Much of the interest in the antimicrobial properties of chitosan has been focused on its possible role in plant protection [11]. Infection of plants by viruses, fungi or bacteria induces some pathogenesis related proteins such as chitosanase and peroxidase [12]. The products of these enzymes act as elicitors for further induction of the enzymes and for activation of other defense related biochemical such as phytoalexin and lignification [13]. Plant defensive role against pathogens can be elicited by adding chitosan oligosaccharides [14]. Chitosan plays potential dual role by directly inhibiting fungal growth [15] and inducing defense response of the host plant by eliciting production of pathogenesis related proteins and phytoalexin [16]. Infection of seeds by fungi also changes nutritional profiles and activity of amylases and protease etc [17]. The present study describes the antifungal activity of watersoluble chitosan (s-chitosan) against soil borne pathogenic fungus M. phaseolina causing infection to jute seedlings. The changes in the activities of chitosanase, peroxidase and amylase in
S. Chatterjee et al. / International Journal of Biological Macromolecules 67 (2014) 452–457
M. phaseolina infected jute seedlings after treatment with schitosan are described here.
453
2. Experimental
examined for visible growth. Absence of visible colony on agar plate within 48 h could be considered as fungicidal effect of s-chitosan. The appearance of visible growth was an indication of its fungistatic nature.
2.1. Materials
2.7. Infection of jute seeds
The phytopathogenic fungus M. phaseolina (MTCC 166) was obtained from The Institute of Microbial Technology, Chandigarh, India. Jute seed (JRO 524) was obtained from National Seeds Corporation, Kolkata, India. Chitosan (>85% deacetylation) was purchased from Sigma Chemical Co., USA. All other chemicals were procured from E. Merck, Germany.
One hundred jute seeds were surface sterilized with 0.1% HgCl2 solution, washed thoroughly with double distilled water for several times to remove HgCl2 and the seeds were steeped in sterile water for 6 h. The surface sterilized seeds were transferred in a germination plate containing a filter paper which was previously soaked with Richard’s medium. The plate was incubated at 30 ◦ C in a humidified chamber (relative humidity 100%). Elongation of germ tubes of jute seeds (seedlings) was measured at four different conditions using slide calipers every other day up to 10 days. The conditions were normal seeds, infected seeds, s-chitosan (12.5 g/l s-chitosan in Richard’s medium) treated non-infected seeds, and schitosan treated infected seeds. Spore suspension (100 l; 2.9 × 106 spore/ml) was added separately to the filter paper in the germination plate containing infected seedlings and s-chitosan treated infected seedlings.
2.2. Preparation of water-soluble chitosan Chitosan was dissolved in 7% acetic acid (v/v), and hydrolyzed by refluxing at 95 ◦ C for 20 h [18]. The hydrolyzed product was dried by rotary evaporator and extracted with de-ionized water. To the aqueous solution containing chitosan, acetone–alcohol mixture (60:40) was added. Water-soluble chitosan was precipitated, triturated with acetone, dried at 60 ◦ C [19], and designated s-chitosan.
2.8. Assay of enzyme activity in jute seedlings 2.3. Growth kinetics Different concentrations of s-chitosan (0.5, 1.0, 2.0, 5.0, 7.5, 10.0 and 12.5 g/l, pH 5.0) were added to 50 ml Richard’s medium (3% sucrose, 1% KNO3 , 0.5% KH2 PO4 , 0.25% MgSO4 and 2% agar). 100 l of fungal spore suspension (spore density 2.9 × 106 spore/ml) was added and inoculated at 30 ◦ C. The growth of M. phaseolina was observed in every alternate day up to 20 days, and the growth kinetics was determined by measuring dry weight of fungal biomass. Richard’s medium containing spores only was used as control. 2.4. Effect of s-chitosan on spore germination To Richard’s medium (5 ml) containing different concentrations of s-chitosan (0.5–12.5 g/l, pH 5.0), 100 l of spore suspension (2.9 × 106 spore/ml) was inoculated. Germ tube elongation of the fungus was measured up to 48 h using phase contrast microscope (Microstar American optical at 100× magnification). The same experiment was performed without s-chitosan serving as control. Germination was indicated by visible germ tube elongation. 2.5. Effect of s-chitosan on radial growth Agar plates containing Richard’s medium were prepared with different concentrations of s-chitosan (5, 7.5, 10, 12.5, and 15.0 g/l). The agar plate without s-chitosan was used as control. Fungal mass of 8 mm diameter previously grown in potato dextrose agar (PDA) plate for 2 days was placed in the Richard’s agar medium plates and were incubated at 30 ◦ C for 3 days. Radial growth of the fungus was determined by measuring the diameter at three points across the mycelia colony. 2.6. Determination of antifungal property of s-chitosan To determine whether s-chitosan is fungistatic or fungicidal against M. phaseolina, spore suspension was inoculated to Richard’s medium in presence of s-chitosan at 30 ◦ C for 24 h. The germination was examined microscopically. A portion of this solution was centrifuged under aseptic condition, washed thoroughly with sterile water, and again suspended in Richard’s medium without s-chitosan. A minute amount of grown mass was streaked on Richard’s agar plate and incubated at 30 ◦ C for 48 h, and finally
One hundred germinated jute seeds were harvested after 120 h, frozen in liquid nitrogen, lyophilized, and powdered with a mortar and pestle. The powder (0.1 g) was extracted with 5 ml 0.05 M sodium phosphate buffer (pH 6.5) for 8 h at 4 ◦ C and centrifuged at 16,000 × g for 30 min at 4 ◦ C. The protein content of the supernatant was determined by Bradford method [20]. Chitosanase activity was assayed in 0.2 ml seed extract containing 50 g protein and 0.2% s-chitosan as a substrate. After incubation for 10, 20 and 30 min, respectively, at 37 ◦ C the reactions were terminated followed by the addition of 0.8 ml of dinitrosalicylic acid and the mixture was immediately boiled for 10 min and cooled. The reducing sugar, glucosamine (GlcN) released by chitosanse was measured spectrophotometrically at 575 nm [21]. Chitosanse activity was expressed as g of glucosamine (GlcN)/min/mg protein. To determine peroxidase activity in jute seed extract, an aliquot of the crude seed extract containing 50 g of protein was added to 1 ml assay mixture containing 0.05 (M) sodium acetate buffer (pH 6.0), 0.2 ml 0.1 M pyrogallol, 0.1 ml 90 mM H2 O2 . The reaction was allowed to proceed for 6 min and optical density was measured spectrometrically at 300 nm at an interval of 1 min after the addition of crude seed extract to the substrate [22]. Peroxidase enzyme activity was expressed as absorbance (OD) difference at 300 nm per min per mg protein or OD300 nm/min/mg protein. Activity of amylase was assayed as follows. To crude seed extract containing 50 g of protein and 10 l of 1% NaCl was added 80 l of 0.5% soluble starch in 0.5 M phosphate buffer at pH 6.7 and 0.2 ml sample of assay mixture was incubated at 37 ◦ C for 10, 20 and 30 min, respectively. Thereafter 0.8 ml of dinitrosalicylic acid was added and the reaction mixture was immediately boiled for 10 min. After cooling the amylase activity was measured spectrophotometrically at 540 nm [21]. Amylase activity was expressed as g of glucose/min/mg protein. 2.9. Transmission electron microscopy (TEM) After 5 days of germination, segments from non-infected, infected, and s-chitosan treated jute seedlings were primarily fixed with 3% (v/v) glutaraldehyde in 10 mM phosphate buffer (pH 7.2) for 4 h at 4 ◦ C. After rinsing with the same buffer, secondary fixation of tissue was done with 1% (w/v) aqueous solution of osmium
454
S. Chatterjee et al. / International Journal of Biological Macromolecules 67 (2014) 452–457
12
Inhibition of spore germination (%)
100
10
8
6
4
2
10 80 8 60 6 40 4 20
2
Length of the germ tube ( μ m)
Dry weight of mycellia / litre of broth (g/l)
12
0 0
0
2
4
6
8
10
12
14
16
18
20
22
Number of days
0 0
2
4
6
8
10
12
Concentration of chitosan (g/l)
Fig. 1. Anti fungal activity of s-chitosan at different concentrations 0 g/l, --; 0.5 g/l, -䊉-; 1.0 g/l, --; 2.0 g/l, --; 5.0 g/l, --; 7.5 g/l, -+-; 10 g/l, -×-; 12.5 g/l, -*-; against M. Phaseolina.
Fig. 2. Anti fungal activity of s-chitosan on inhibition of spore germination, -䊉-; length of germ tube, --; of M. phaseolina.
tetroxide for 1 h. After washing with water for three times, samples were dehydrated using different concentrations of ethanol (30, 50, 70, 90 and 100%; 10 min each step) at room temperature. After treating the sample with propylene oxide (PO) as transitional solvent for three times (10 min each step), segments were infiltrated in 1:2 (v/v) PO-resin for 1 h, 2:1(v/v) PO-resin for 4 h and pure resin for overnight [23]. Samples were embedded in pure resin and cured at 60 ◦ C for 3 days. The ultrathin sections were cut in an ultramicrotome and picked up on uncoated grid. The sections were usually stained with a saturated solution of uranyl acetate for 10–15 min at room temperature, washed with water, stained again with lead citrate for 5–10 min at room temperature, washed again with water and was blotted dry. TEM was done in a JEM-2010 electron microscope.
3. Results and discussion 3.1. Antifungal activity of s-chitosan Chitosan, which is a biodegradable, non-toxic, ecofriendly natural biopolymer, is frequently used as an antimicrobial agent. In this study, it was found that s-chitosan inhibited the growth of plant pathogen M. phaseolina. The minimum inhibitory concentration (MIC) of s-chitosan against this fungus was found to be 12.5 g/l. The MIC as low as 0.075 g/l for particulate chitosan and 0.018 g/l for soluble chitosan against Fusarium solani, a plant pathogenic fungus in liquid broth had been reported [24,25]. However, Botrytis cinerea and Rhizopus stolanifers [26] required chitosan for MIC as high as 10 g/l. Some authors suggested that Mucor sp. having
Fig. 3. Effect of s-chitosan at different concentration Control, C; 5 g/l, (I) 7.5 g/l, (II) 10 g/l, (III) 12.5 g/l, (IV) on radial growth of M. phaseolina.
S. Chatterjee et al. / International Journal of Biological Macromolecules 67 (2014) 452–457
a)
70
non infected Infected
6
60
Chitosanase activity
50 40 30 20 10 0 1
2
3
4
5
6
7
8
9
10
11
( μ g GlcN/min/mg of protein)
Length of jute seedlings (mm)
455
Day of culture
0
Chitosan untreated
Chitosan treated
18
b) Peroxidase activity Δ OD300 nm /min/mg of protein
chitosan within the cell walls are more resistant to the antimicrobial action of chitosan [24]. The change in germination time, germ tube length and dry weight of mycelia of M. phaseolina, a soil borne pathogen was noticed during its growth in s-chitosan supplemented Richard’s agar medium at 30 ◦ C for a definite period of time. Addition of s-chitosan up to 5 g/l in growth medium of fungus, germination time fungus did not markedly change. However the reduction in dry weight of mycelia and germ tube length indicated an inhibitory effect of s-chitosan on fungal growth. Further increase in concentration of s-chitosan (up to 10 g/l) showed a pronounced inhibitory effect as mycelial growth was found to reduce from 12 to 6.45 g/l (Fig. 1), Similar effect was observed in germ tube elongation of fungus and it was found to reduce from 12.9 to 2.9 m with increase in s-chitosan concentration. 80% inhibition of spore germination was also observed with s-chitosan supplementation in the growth medium (Fig. 2). Germination time of fungus at 10 g/l of s-chitosan was found to be 48 h where as control without s-chitosan showed germination time of 24 h. There was no growth of fungus at 12.5 g/l of s-chitosan. Therefore, it was concluded that the MIC of s-chitosan against M. phaseolina was 12.5 g/l. It was observed that diameter of the fungal mycelia was reduced markedly with increase in s-chitosan concentration (Fig. 3). Chitosan due to its positive charge on the C-2 of the glucosamine monomer is more stable and showed better antimicrobial activity than chitin [27]. The exact mechanism of the antimicrobial action of chitin, chitosan and their derivatives is not clearly understood but different mechanisms have been proposed [28]. The interaction between positively charged chitosan molecule and negatively charged microbial cell membrane leads to the leakage of proteinaceous and other intracellular constituents [29]. Chitosan acts mainly on the outer surface of the bacteria. At lower concentration (
