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International Biodeterioration & Biodegradation 62 (2008) 116–124

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Effect of chitosan on physiological, morphological, and ultrastructuralcharacteristics of wood-degrading fungi

Tripti Singha,�, Damiano Vesentinia, Adya P. Singha, Geoffrey Danielb

aEnsis, Wood Processing, Private Bag 3020, Rotorua, New ZealandbWood Ultrastructure Research Centre, Swedish University of Agricultural Sciences, Uppsala, Sweden

Received 14 June 2007; received in revised form 17 August 2007; accepted 26 September 2007

Available online 19 February 2008

Abstract

An investigation was undertaken to understand the mechanism(s) by which chitosan exerts its antifungal effects against the wood-

degrading fungi Sphaeropsis sapinea and Trichoderma harzianum. Exposure to increasing concentrations of chitosan caused an increase in the

amount of hydrogen peroxide accumulation in cultures of S. sapinea, which was accompanied by a decrease in superoxide formation. The

same effect was not observed in T. harzianum. Potassium ion leakage was an early event for both test fungi, leakage being more pronounced

for S. sapinea than T. harzianum for the first 5min, particularly at higher concentrations of chitosan treatment. Fluorescence microscopy

provided evidence that the effect of chitosan on fungal hyphae was mediated through alterations in the plasma membrane properties.

Chitosan also severely affected fungal morphology. Increasing concentrations of chitosan induced excessive branching, vacuolation, and a

reduction in hyphal diameter. Transmission electron microscopy, which showed more severe ultrastructural changes in S. sapinea hyphae

from chitosan treatment as compared to T. harzianum, provided valuable complementary information. The data suggest that the plasma

membrane may be the primary target of chitosan action, and that the two fungi differ in the extent to which they are affected.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Bioactive molecule; Mode(s) of action; Sphaeropsis sapinea; Trichoderma harzianum; Wood degradation

1. Introduction

After harvest from the forest, logs become susceptible toinvasion by a variety of microorganisms, which includeboth wood-discolouring and wood-decaying fungi. Wood-discolouring fungi, which are commonly known assapstaining fungi, are early colonisers and can infect logswithin few hours to few days of their harvest. Therefore,logs have to be protected from such fungi, particularly ifthey are destined for export. Traditional protectionmethods involve treating logs with synthetic chemicalsthat are toxic, raising concern for the environment. Thedevelopment we are undertaking to protect logs of Pinus

radiata, the most important plantation forest species inNew Zealand, from sapstaining fungi will involve treat-ment of harvested logs with environmentally compatiblenatural products. Our initial laboratory studies involving

e front matter r 2007 Elsevier Ltd. All rights reserved.

iod.2007.09.006

ing author. Tel.: +647 3435329; fax: +64 7 3435507.

ess: tripti.singh@ensisjv.com (T. Singh).

treatment of some common sapstaining fungi withchitosan, a derivative of the natural amino polysaccharidechitin (Kurita et al., 1993), have shown considerablepromise in controlling the growth of these fungi. Thus,chitosan has the potential for application in field trials onlog protection.Chitosan (2-amino-2-deoxy-beta-D-glucan), a polymer of

D-glucosamine, can be obtained by the deacetylation ofchitin (2-acetamido-2-deoxy-beta-D-glucan), which is oneof the most abundant natural polymers. Chitosan isbiodegradable and has excellent biocompatibility andalmost no toxicity to humans and animals (Rabea et al.,2003). For these reasons, chitosan has had practicalapplications in many areas important for human well-being and health, such as pharmaceutical, nutritional,crop-protection, and a range of other processes (Simionatoet al., 2006; Wang et al., 2006).The role of chitosan in plant elicitation responses has

been well documented (Radman et al., 2003). Severalstudies have shown chitosan to be a potent antibacterial

ARTICLE IN PRESST. Singh et al. / International Biodeterioration & Biodegradation 62 (2008) 116–124 117

(Kittur et al., 2005) and antifungal agent (Roller andCovill, 1999; Alfredsen et al., 2004; Chittenden et al., 2004)and it has been shown that chitosan can inhibit the growthof plant pathogenic fungi (Benhamou, 1992; El Ghaouthet al., 1994). Several hypotheses have been proposed for theantimicrobial action of chitosan, for example, that it playsa role in the inhibition of mRNA synthesis (Hadwigerand Loschke, 1981), acts as a chelating agent (Skjak-Braeket al., 1989), and induces membrane permeability (Younget al., 1982). However, it is not possible to identify theprimary cellular target of chitosan action on the basis ofinformation available to date.

In probing the mode of action of chitosan, ourhypothesis has been that chitosan’s inhibitory effect onfungal growth may be mediated by oxidative stress,resulting from over-production of reactive oxygen species(ROS) in fungal cells in response to chitosan treatment.During normal cell metabolism, organisms produce ROS,but their amount within cells is tightly regulated for normalgrowth to occur. However, when the generation of ROSexceeds the cell’s ability (antioxidant defence system) toneutralize ROS, oxidative stress occurs (Angelova et al.,2005). The effects of oxidative stress include damage to allmajor cell components, namely proteins, lipids, and DNA(Fridovich, 1998). In fungal systems, cadmium toxicity inAspergillus nidulans was found to be related to increasedformation of ROS within the mycelium (Guelfi et al.,2003). Similar effects were also observed in Saccharomyces

cerevisiae treated with copper ions (Ogita et al., 2005). Riceplant treated with chitosan to stimulate elicitationresponses showed browning of the leaves (Agrawal et al.,2002), which might have been caused by the accumulationof ROS. Nevertheless, the direct evidence linking chitosan’sinhibitory effect on fungal growth to production of ROSis missing.

The study reported here focuses on understanding earlyresponses of a common sapstaining fungus, Sphaeropsis

sapinea, which we have found to be susceptible to chitosantreatment, compared to a common mould fungus, Tricho-

derma harzianum, which has shown greater resistance tochitosan treatment and which can potentially be used as abiocontrol agent (Dumas et al., 1996). Understanding themode of action of chitosan should help us to optimizeconditions for the treatment of logs with this compound forsapstain control.

2. Materials and methods

2.1. Fungal culture and chitosan preparation

Sphaeropsis sapinea (Fr.) Dyko and Sutton isolate 2/94 and

T. harzianum Rifai isolate ]CBS 597.91 were used throughout the study.

The fungi were grown and maintained at 25 1C and 75% RH on 90-mm

petri dishes containing approximately 20ml 2% malt extract agar (2%

malt extract, 1.2% agar; Merck & Co., Inc., Whitehouse Station, NJ).

A 2% chitosan stock solution was prepared aseptically using low-

molecular-weight chitosan (Sigma-Aldrich Corp., St. Louis, MO, USA).

This was solubilized in 1% acetic acid and blended for 20min at high

speed. The effect of chitosan on fungal growth was assessed by

supplementing the culture medium with the chitosan stock solution to a

final concentration of 0.01%, 0.05%, 0.1%, 0.15% chitosan.

2.2. Evaluation of oxidative stress

Oxidative stress is damage to cellular components due to a buildup of

ROS such as hydrogen peroxide (H2O2) and superoxide, resulting from an

imbalance between their formation and neutralisation. Hydrogen peroxide

is the longest-lived member of ROS, and is a good indicator of oxidative

stress. Therefore, in our work H2O2 has been the main focus among

ROS, as in many other previous studies of ROS-related stresses (Yoshinga

et al., 2005).

Three replicate cultures of each fungal isolate were incubated for 48 h,

as described. The accumulation of H2O2 and the formation of superoxide

radicals were monitored according to the method described by Munkres

(1990) with some modifications. Briefly, after incubation the plates were

flooded with 5ml of either 2.5mM Nitro Blue Tetrazolium (NBT) in

5mM (N-morpholino) propane sulphonate-NaOH at pH 7.6 to detect

superoxide that reacts with NBT to form a blue precipitate, or with

2.5mM dimaminobenzidine and 5 purpurogallin unitsml�1 of horseradish

peroxidase (Sigma-Aldrich Corp.) in 100mM potassium phosphate buffer

at pH 6.9 to detect peroxide that triggers the accumulation of a red

precipitate. The 5-ml solution was spread with a sterile glass rod to ensure

a homogeneous contact with all the hyphae. The supernatant was removed

after 45 and 30min, respectively, and the plates incubated for 24 h at the

conditions described. Scans were taken at various incubation times with a

Canon CanoScan N640 P and saved in an uncompressed ‘‘tif’’ format.

Quantifications of the colour intensity, assumed to be reflective of the

accumulation of precipitates, were made using V++ (Total Turnkey

Solutions, Mona Vale, NSW, Australia). Five replicate measurements

were obtained by randomly selecting areas from the three replicate

cultures within each treatment. Data are expressed as average colour

intensity and standard errors for each treatment, in arbitrary units on the

red, blue, and green channels. The software analysed the colour intensity

in a manner inversely proportional to the ‘‘darkness’’ of the colour. Thus,

a dark colour, associated with high accumulation of precipitates, was

expressed as a low-intensity value, and vice versa.

2.3. Effect of chitosan and hydrogen peroxide in the presence/

absence of catalase on fungal growth

This part of the study aimed to investigate the effect of H2O2 on fungal

growth; H2O2 was applied with and without catalase to exclude

interference from other factors. Catalase was also applied alone and in

combination with chitosan to investigate the effect of catalase itself on

fungal growth as well as to find out if the growth inhibition effect of

chitosan was reversed by catalase, and if so to what extent. Cultures of S.

sapinea and T. harzianum were inoculated and grown as described on 2%

malt extract agar supplemented with either 20mM H2O2 or 0.1%

chitosan. A set of controls containing neither H2O2 nor chitosan was

included. The effect of H2O2 on fungal growth was monitored in all

treatments by the addition of 50 units of catalase in each plate immediately

prior to inoculation. Radial growth was measured daily for up to 7 days

along four pre-marked axes crossing perpendicularly at the centre of the

inoculum plug. The experiment was carried out in five replicates and

average rates of growth in mm�1 were calculated for each day of the

experiment.

2.4. Evaluation of potassium ion leakage

This part of the study aimed to investigate the permeability properties

of cell membranes. Some of the bioactive molecules act upon the cell

membrane, either by inhibiting active transport or by increasing cell

permeability, which leads to electrolyte leakage. The latter effect can be

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detected by measuring intracellular changes in K+ ion concentration (Rui

and Morrell, 1994; Cox et al., 2001).

Fungal inoculum for T. harzianum was prepared by adding distiled

water (2ml) to a 7-day-old culture, which was subsequently shaken gently

to release spores from the mycelia. The spore suspension was adjusted to

106 sporesml�1. As S. sapinea does not readily produce spores on malt

agar, distiled water (2ml) was added to a 7-day-old culture and the surface

of the mycelium was lightly scratched with a wire loop to release hyphal

fragments. The suspension was blended for 5min, filtered through a 30-mmmembrane, and adjusted to a concentration of 106 hyphal fragmentsml�1.

The test fungi were grown in liquid medium (containing 0.2% yeast extract

and 1.5% malt extract) for 5 days in the dark under stationary conditions at

25 1C. Fungal biomass was harvested following the method described by Rui

and Morrell (1994) with some modification as reported by Singh et al. (2006).

The mycelium was suspended in 50mMNa-maleate buffer (pH 5.5) containing

1mM glucose and 0.01%, 0.05%, and 0.1% chitosan, and incubated at 28 1C.

Aliquots (1.5ml) were removed from each liquid culture after 0, 5, 15, and

30min; aliquots were centrifuged for 5min at 10,000 rpm, and the supernatant

was discarded. The fungal pellet was extracted in 1ml of 6N HNO3 for 30min

at 95 1C. The solution was centrifuged for 5min at 10,000 rpm (using a Sorvall

T-1270 rotor) and the supernatant was diluted with distiled sterile water and

3N HNO3. Diluted supernatant was analysed for potassium using an atomic

absorption spectrophotometer with an air-acetylene flame and a wavelength of

324.8nm. Three replicates per sample were analysed. Control samples (without

chitosan) were also included. The intracellular level of the K+ ion was

expressed in mMmg�1 oven dry weight of fungus and plotted against time.

2.5. Fungal branch frequency and morphology

Microscope slides were coated with a thin (500-ml) layer of water-agar(2% agar) amended with 0.01%, 0.05%, 0.1%, and 0.15% chitosan. After

the agar film had solidified, 10 ml of the fungal suspension described above

were inoculated in the centre of the agar film. Chitosan was excluded from

the controls. Inoculated slides were incubated at 25 1C and 75% RH in the

dark. Three replicates were prepared for each concentration. After 4 days

of incubation, differential interference contrast microscopy was used to

observe the fungal morphology and branching pattern from all treatments.

The total number of branches was recorded within a 200-mm peripheral

length of the mycelium. For each slide, five hyphal tips (peripheral zone)

were selected at random and the mean branching frequency was calculated

for each concentration. Micrographs were taken for each hyphal element

using a Zeiss Axioplan 2 light microscope with a 10� or 20� objective.

Hyphal thickness was also measured at different chitosan concentrations.

2.6. Epifluorescence microscopy to assess damage

to plasma membrane

Microscope slides were prepared and inoculated as described above. A

live/dead viability kit, which utilizes a mixture of two nucleic acid stains,

SYTO 9 green-fluorescent stain and the propidium iodide (PI) red-

fluorescent stain, was used to determine the integrity of the plasma

membrane for both test fungi (Cat ]L-7012, Molecular Probes, Eugene,

OR, USA). Hyphae with an intact plasma membrane stained fluorescent

green and those with a damaged membrane stained fluorescent red. The

method supplied by the manufacturer was followed. Both reagents were

prepared in a solution of anhydrous dimethylsulphoxide. A mixture of

3034mM SYTO 9 dye and 20mM PI was prepared in an Eppendorf tube.

The dyes were vortexed for a few seconds and incubated at room

temperature in the dark for 15min. A small amount (10ml) of the stain

mixture was placed over the fungal colony. After 15min incubation at

room temperature in the dark, fungal hyphae were examined under blue

light excitation, using a 470-nm excitation and a 490–700-nm emission

filters set and a Zeiss Axioplan 2 light microscope. The live/dead stain

mixture was used in repeated experiments with test fungi exposed to

different concentrations of chitosan. Fungal mycelia that has not been

exposed to chitosan was used as a control.

2.7. Transmission electron microscopy

Erlenmeyer flasks (100-ml) containing 9ml of yeast-malt (0.2% yeast

extract and 1.5% malt extract) were inoculated with 100ml of individualtest fungal inoculum (106 spores or propagulesml�1). After 5 days

incubation under stationary conditions at 25 1C and 75% RH in the dark,

0.01%, 0.05%, and 0.1% chitosan was added. Each concentration was

prepared by adding an appropriate amount of chitosan stock solution to

distiled water to make up a total volume of 900ml. This solution was

subsequently added to the culture medium to obtain a total volume of

10ml. For the controls, sterile distiled water was included instead of

chitosan. After 24 h of incubation, 100ml of culture was taken out from

both treated and untreated flasks. Fungal hyphae were fixed in 3%

glutaraldehyde (prepared in 0.05M sodium cacodylate buffer) at 4 1C for

4 h, and then post-fixed in 1% osmium tetroxide (prepared in the same

buffer) overnight at 4 1C. After dehydration in acetone, the samples were

embedded in Spurr’s low-viscosity resin. Ultrathin sections (ca. 80 nm)

were cut with a diamond knife on a RMC MTX or a Reichert Ultracut E

ultramicrotome. The sections were sequentially stained with uranyl acetate

and lead citrate prior to examination with JEOL 1010 and Philips CM/12

transmission electron microscopes.

2.8. Statistical analysis

The data obtained after the production of H2O2 and superoxide at

different treatments was analysed using ANOVA followed by Dunnett’s

post-test analysis in order to compare treatments with their respective

controls. Bonferroni post-test analysis was performed to compare selected

pairs of treatments when assessing the effect of H2O2 and catalase on

fungal growth. Data analysis was implemented using GraphPad InStat

3.06 statistical software (GraphPad Software Inc., San Diego, CA, USA).

A two-way ANOVA was conducted and LSD tests were used to

compare the mean of total K+ ion levels recorded at different incubation

times and for treatment concentrations. Differences were considered to be

significant at Pp0.05.

3. Results

3.1. Evaluation of oxidative stress

The occurrence of oxidative stress caused by the presenceof ROS, such as H2O2 and superoxide, was evaluatedduring the initial stages of fungal growth. Sphaeropsis

sapinea showed visible decrease in the intensity of a blue/purple colour after staining with NBT due to superoxideformation as the concentration of chitosan in the mediumincreased. Inversely, colour intensity measurement usingthe software showed a significant (Po0.01) increase inintensity values in chitosan-treated samples as recorded forthe blue green and red channels (Fig. 1a).Visual assessment of the intensity of red colour, which

develops from precipitation of the dimaminobenzidinereagent in the presence of H2O2, showed deepening of thecolour with increasing chitosan concentrations. Inversely,colour intensity measurements using the software showed adecrease in intensity values (Po0.01, Fig. 1b). Theincreased accumulation of H2O2 (Fig. 1b) also correlatedwith a decrease in superoxide amount (Fig. 1a).For T. harzianum, there was no significant difference

(P40.05) in superoxide production across the range ofchitosan concentrations tested (Fig. 2a). Likewise, therewas no significant difference (P40.05) in the production of

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Fig. 1. Effect of increasing concentrations of chitosan on production of

superoxide (a) and peroxide (b) in S. sapinea after 2 days of incubation.

Error bars represent standard error (n ¼ 15) of the mean values recorded

for the red, green, and blue channels (black, grey, and white, respectively).

Fig. 2. Effect of increasing concentrations of chitosan on production of

superoxide (a) and peroxide (b) in T. harzianum after 2 days of incubation.

Error bars represent standard error (n ¼ 15) of the mean values recorded

for the red, green, and blue channels (black, grey, and white, respectively).

Fig. 3. Effect of peroxide and chitosan on the growth of S. sapinea in the

presence and absence of catalase. Error bars represent standard error

(n ¼ 20) of the mean hourly rate of growth.

T. Singh et al. / International Biodeterioration & Biodegradation 62 (2008) 116–124 119

H2O2 across the range of chitosan concentrations tested(Fig. 2b). Little difference was observed between thechitosan concentrations tested with regard to the intensitiesof the blue/purple colour associated with superoxide(Fig. 2a) and the red colour associated with H2O2 (Fig. 2b).

3.2. Effect of chitosan and hydrogen peroxide in the

presence/absence of catalase on fungal growth

The growth rate of both fungal species was affected uponaddition of 20mM H2O2 to the medium. As compared tocontrols, the rate of growth of H2O2 treated S. sapinea

was lower during the first 4 days (Fig. 3). However, theeffect of H2O2 on S. sapinea growth was less severecompared to chitosan (Fig. 3). Application of catalasefollowing peroxide treatment resulted in a significantrecovery (Po0.001) of growth to levels observed forcultures treated with catalase alone (i.e., without H2O2 orchitosan). However, the growth recovery following catalasetreatment in the chitosan-amended cultures did not reachthe same level as the recovery for H2O2-treated cultures(Fig. 3). Nevertheless, the catalase treatment overcame thegrowth-inhibitory effect of chitosan as early as the secondday from inoculation (Po0.05).

Hydrogen peroxide treatment caused a marked reduc-tion in the rate of growth also in T. harzianum after all

growth periods (Fig. 4). Initially, growth was retarded byday 1, and only started on day 2 post-inoculation. Uponaddition of catalase, the inhibitory effect induced by H2O2

was alleviated significantly (Po0.001), with growth return-ing to levels similar to those observed in the controlcultures after day 3. Chitosan treatment also caused growthreduction as compared to the control, but not to the sameextent as the H2O2 treatment, and addition of catalaseimproved growth only marginally (Fig. 4). This is in sharp

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Fig. 4. Effect of peroxide and chitosan on the growth of T. harzianum in

the presence and absence of catalase. Error bars represent standard error

(n ¼ 20) of the mean hourly rate of growth.Fig. 5. Intracellular levels of K+ in S. sapinea over time following

exposure to increasing concentrations of chitosan. Control (—), 0.01%

(– –), 0.05% (??), 0.1% (– � –).

Fig. 6. Intracellular levels of K+ in T. harzianum over time following

exposure to increasing concentrations of chitosan. Control (—), 0.01%

(– –), 0.05% (??), 0.1% (– � –).

T. Singh et al. / International Biodeterioration & Biodegradation 62 (2008) 116–124120

contrast to S. sapinea (Fig. 3), where the growth rate wasmore severely affected by chitosan treatment, leveling offfrom the fourth day post-inoculation.

3.3. Evaluation of potassium ion leakage

Monitoring of the concentration of intracellular potas-sium ion (K+) indicated that the presence of chitosan in thegrowth medium caused the leakage of K+ from fungal cellsof both test fungi (Figs. 5 and 6). This effect was observedas early as 5min after chitosan treatment, with K+ leakagesteadily increasing over a period of 30min after treatment.The lowest concentration of chitosan tested (0.01%) wassufficient to initiate the leakage of K+ and the effect wasmore pronounced at higher concentrations (Pp0.05).However, K+ ion leakage was higher for S. sapinea ascompared to T. harzianum in the first 5min at higherconcentrations of chitosan (Figs. 5 and 6).

3.4. Fungal branch frequency and morphology

Microscopic observations revealed that chitosan affectedthe morphology of both test fungi, with greater morpho-logical changes observable for S. sapinea. Little effect wasobserved on mycelia exposed to the lowest concentration ofchitosan (0.01%), but the effect was apparent at the higherconcentrations (X0.05%). Hyphae of S. sapinea exposed to0.1% chitosan showed pronounced vacuolation; sometimeshyphae appeared almost empty. Although an increase invacuolation and reduction in hyphal thickness was alsoobserved for T. harzianum, particularly at the higherchitosan concentration of 0.1%, T. harzianum hyphae wereless severely affected than were S. sapinea hyphae.

Chitosan induced excessive branching in both test fungi,and the effect was evident even at the lowest concentrationof chitosan. The increase in branching was directlyproportional to concentrations of chitosan used, with thehighest concentration showing the greatest affect. Areduction in hyphal diameter and length was also found,

the general trend being a reduction in hyphal diameter andlength with increasing chitosan concentrations.

3.5. Epifluorescence microscopy

Untreated hyphae of both test fungi emitted an intensegreen fluorescence immediately after the application of themixture of SYTO 9 and PI (Fig. 7a). Hyphae of S. sapinea,exposed to 0.1% and higher concentrations of chitosan,showed red fluorescence as a result of complete entry ofpropidium iodide stain into the cytoplasm (Fig. 7b).However, S. sapinea hyphae treated with lower concentra-tions of chitosan showed a combination of both green andred fluorescence, some hyphae fluorescing green whereasothers fluoresced red (not illustrated). At the highestconcentration of chitosan, S. sapinea hyphae showed cellcollapse in addition to showing red fluorescence.For T. harzianum, red fluorescence was observed only at

higher chitosan concentrations, and some hyphae stillexhibited green fluorescence even at 0.15% chitosanconcentration (not illustrated).

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Fig. 7. Hyphae of S. sapinea after application of the mixture of SYTO 9 and PI. (a) Untreated hyphae showing green fluorescence. (b) Hyphae treated

with 0.1% chitosan showing red fluorescence. Scale bar ¼ 50mm.

T. Singh et al. / International Biodeterioration & Biodegradation 62 (2008) 116–124 121

3.6. Transmission electron microscopy

A comparison of untreated with chitosan-treatedS. sapinea hyphae provided evidence of ultrastructuralchanges in hyphal cells at all concentrations of chitosanused, with higher concentrations causing more severealterations. Untreated hyphae contained a finely granularcytoplasm that was well preserved and enclosed by anintact plasma membrane. Organelles, such as mitochondriaand endoplasmic reticulum, were readily recognisable andoccurred throughout the cytoplasm. In addition, a fewdense bodies of unknown identity were also present. Theenclosing cell wall was thin but well defined (Fig. 8a).Pronounced cellular changes in S. sapinea were observableeven at the lowest concentration of chitosan, which causeddisruptions in plasma membrane, vesiculation of mem-brane, aggregation of cytoplasmic contents and residues,and a change in the density of cytoplasm (Fig. 8b). Higherchitosan concentrations caused even greater destruction ofcytoplasmic constituents, including excessive vesiculationof membranous structures, which tended to aggregate,increased vacuolation, and complete disruption of plasmamembrane (Fig. 8c). Treatment with 0.15% chitosanresulted in complete loss in the integrity of the cytoplasm.At this concentration, only residues of disintegratedcytoplasmic constituents were visible as irregular, denselycompacted masses within a highly vacuolated cell interior.Cell walls were also greatly modified at 0.15% chitosan,remaining as irregular skeins of fibrillar residues aroundthe hyphal cells (Fig. 8d).

Chitosan treatment caused less severe ultrastructuralchanges in T. harzianum. Untreated T. harzianum hyphaecontained well-defined organelles and an intact plasmamembrane (Fig. 9a). After treatment with 0.01% chitosan,there was a noticeable loss in the cytoplasmic density, butthe organelles kept their integrity and the plasmamembrane was also not disrupted (Fig. 9b). Changes weremore pronounced after treatment with 0.1% chitosan,which resulted in vesiculation of internal membranoussystems; however, the plasma membrane still appearedintact although it was undulated and had lost contact with

the cell wall in places (Fig. 9c). There was further loss in thecytoplasmic density, and degeneration of cell organelleswas also apparent. Another striking feature of the hyphaetreated with 0.1% chitosan was abundant presence of anextracellular material, which appeared fibrillar and formeda prominent sheath around the cell wall (Fig. 9c).

4. Discussion

The information obtained using a combination oftechniques employed in this study indicates that bothS. sapinea and T. harzianum were affected by chitosantreatment. However, S. sapinea was considerably moresensitive to chitosan than was T. harzianum. Whereas allchitosan concentrations induced changes in S. sapinea

hyphae, pronounced changes in T. harzianum hyphaeoccurred only at higher chitosan concentrations. Previousstudies have shown the inhibitory effect of chitosan todiffer among the fungi tested (Laflamme et al., 1999).Greater resistance of T. harzianum to chitosan as comparedto S. sapinea may be related to its greater ability to detoxifyROS, as has been suggested recently for a higher plantsystem, where detoxification of hydrogen peroxide wasconsidered to be a factor in maintaining the watertransport activity in a root system that had been subjectedto low-temperature stress (Lee et al., 2007). Trichoderma

spp. are known for their competitive ability (Murmaniset al., 1988), which is facilitated also by the production ofantibiotics and lytic enzymes (Dennis and Webster, 1971;Burgel et al., 1994; Brown and Bruce, 1999; Brown et al.,1999). Trichoderma’s greater tolerance of imposed externalstresses, such as chitosan treatment, may confer upon it acompetitive advantage over microorganisms that are lesstolerant of stressful conditions.Increased formation of superoxide and peroxide has

been observed during hyphal interaction between non-compatible filamentous fungi, suggesting that ROS mightbe secreted as signal and interference molecules betweenincompatible fungi (Silar, 2005). Interspecific recognition iscertainly a very important step during fungal interactionsand might also include carbohydrate-related chemical

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Fig. 8. (a–d) TEM micrographs of S. sapinea hyphae. (a) Untreated hyphae showing intact plasma membrane, well-preserved cell organelles, and a

granular cytoplasm. (b) Hyphae treated with 0.01% chitosan showing disrupted plasma membrane and disorganized cell organelles and cytoplasmic

materials. (c) Hyphae treated with 0.1% chitosan showing ruptured plasma membrane, membranous vesicles, and degenerated cytoplasm. Scale

bar ¼ 200 nm. (d) Hyphae treated with 0.15% chitosan showing cell collapse. None of the cell organelles are recognisable. Scale bar ¼ 1mm.

T. Singh et al. / International Biodeterioration & Biodegradation 62 (2008) 116–124122

signaling (Boddy, 2000). Such carbohydrate-related signal-ing might well occur through chitin recognition, as this is aspecific polymer found only in fungi and arthropods. Fungiare composed of varying amounts of chitin in their cellwall, the amount being species-specific (Nilsson andBjurman, 1998). Since chitosan is a product of chitindeacetylation, it could trigger both non-specific and specificresponses in fungi, depending upon its concentration. Inlow concentration ranges, chitosan may function as asignaling molecule, while higher chitosan concentrationsmay trigger oxidative stress, measurable by the concentra-tion of H2O2 in cells, as our work has shown.

Chitosan treatments induced changes to cellular systemsthat are vital to maintaining physiological environmentsimportant for normal fungal growth. Leakage of K+

occurring within the first few minutes of chitosan treatmentindicated that changes in the plasma membrane, gatewayto the external environment and one that maintains ionichomeostasis within living cells by regulating ionic move-

ments, must be rapid and sufficient to render it leaky to K+

ions. Disruption in the plasma membrane in S. sapinea maybe a reason for K+ leakage, where the lowest concentra-tion of chitosan used resulted in the disruption of plasmamembrane. However, K+ leakage also occurred inT. harzianum from chitosan treatments, albeit much lessin the first 5min at higher chitosan concentrations ascompared to S. sapinea, yet the plasma membrane was notdisrupted by lower concentrations of chitosan used. Thissuggests that there must also be other reasons for K+

leakage. Dysfunction in the regulation of plasma mem-brane based K+ channel may be another important cause,and this could happen if the plasma membrane composi-tion/architecture is altered. We think that chitosan treat-ments induce alterations in the composition/properties ofhyphal cell plasma membrane and this could be mediatedby ROS, judging by the rapid fungal response to chitosantreatment in terms of K+ leakage. ROS is known toadversely affect many cell components, including plasma

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Fig. 9. (a–c) TEM micrographs of T. harzianum hyphae. (a) Untreated hyphae showing intact plasma membrane and organelles. (b) Hyphae treated with

0.01% chitosan showing intact plasma membrane and cell organelles but a loss in cytoplasmic density. (c) Hyphae treated with 0.1% chitosan showing

disorganisation of cytoplasmic contents and the presence of extracellular materials around the cell wall. Scale bar ¼ 500 nm.

T. Singh et al. / International Biodeterioration & Biodegradation 62 (2008) 116–124 123

membrane through peroxidation of lipids (Garnier et al.,2006), and resulting physiological responses are rapid.

Induction of hyphal branching and alterations in hyphalcell ultrastructure from chitosan treatment are manifesta-tions of what occurs within fungal cells early on.Alterations in fungal morphology can be indicative ofcompromised plasma membrane, among a host of othercellular changes that are no doubt induced by chitosan.Protein secretion in filamentous fungi occurs around theapical and subapical region of the advancing hyphal tip(Gordon et al., 2000; Muller et al., 2002). It has beenshown that hyperbranching is apparently the result of atleast one unprocessed protein. Muller et al. (2002)suggested that an unprocessed dysfunctional chitinsynthase B may contribute to the abnormal branchingphenotype in Aspergillus sp., which is an outer cellmembrane targeted protein (Record et al., 2001). It ispossible that chitosan indirectly influences the properties/function of chitin synthase by affecting plasma membrane,

which may lead to an alteration in cell wall composition/architecture.In conclusion, we have identified K+ leakage as an early

response, which no doubt disturbs the ionic balance neededfor normal fungal growth and development. This and otherchitosan-induced cellular responses are likely to be at leastpartly mediated by ROS.

Acknowledgement

We would like to thank Dr. Ahmed Osman for his timeand helpful discussion.

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