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The mycelial response of the white-rot fungus, Schizophyllum commune to the biocontrol agent, Trichoderma viride
Victor C. UJORa,*, Monia MONTIb, Diluka Gayani PEIRISa, Mark Owen CLEMENTSa, John Norman HEDGERa
a b

School of Life Sciences, University of Westminster, 115 New Cavendish Street, London, UK Dipartimento di Biologia Molecolare, Cellulare e Animale University of Camerino, Via Gentile III da Varano, 62032 Camerino, MC, Italy

article info
Article history: Received 16 October 2011 Received in revised form 13 December 2011 Accepted 14 December 2011 Available online 26 December 2011 Corresponding Editor: Stephen W. Peterson Keywords: Biocontrol agents Combative interactions Metabolomics Scizophyllum commune Trichoderm viride

abstract
In this study, agar plate interaction between Schizophyllum commune and Trichoderma viride was investigated to characterise the physiological responses occurring during interspecic mycelial combat. The metabolite proles and morphological changes in both fungi paired on agar were studied relative to the modulation of phenoloxidase activity in S. commune. The calcium ionophore A23187 was incorporated in self-paired cultures of S. commune to explore possible involvement of calcium inux in the response of S. commune to T. viride. The levels of lipid peroxides and protein carbonyls in the confronted mycelia of S. commune were also measured. Contact with T. viride induced pigmentation and cell wall hydrolysis in S. commune with concomitant increase in phenoloxidase activity, rise in the levels of oxidative stress indicators and increased levels of phenolic compounds, antioxidant g-amino butyric acid, and pyridoxine and osmo-protective sugar alcohols. Calcium ionophore mimicked the pigmentation in the T. viride-confronted mycelia of S. commune, implicating calcium inux in the response to T. viride. The changes in S. commune are indicative of targeted responses to osmotic and oxidative stresses and phenoloxidase-mediated detoxication of noxious compounds in the contact interface with T. viride, which may confer resistance in natural environments. 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction
Fungusefungus combative interactions have been extensively studied, leading to the use of more antagonistic species to control plant pathogenic fungi and to some measure, woodrot in commercial logging (Bruce & Highley 1991; Boddy 2000; Adomas et al. 2006). Interspecic mycelial combat is characterised by physiological responses including cessation of mycelial extension, pigmentation, barrage formation, and increased secretion of phenoloxidases, leading to the premise that fungi possess a recognition mechanism that allows

them to detect and respond to nonself mycelia (Rayner 1991; Grifth et al. 1994; Boddy 2000). Such mechanisms allow fungi to defend their territories, thereby restricting access to captured nutrients by opposing species (Rayner 1991; Boddy 2000). Trichoderma species parasitize other fungi, making them potent biocontrol agents of specic fungal plant pathogens in the eld (Bruce et al. 1995; Boddy 2000; Howell 2003; Adomas et al. 2006). When mycoparasites are paired against less combative species, oversecretion of some metabolites and enzymes, which participate in pH regulation, host cell wall hydrolysis, and adjustment of moisture content of the growth medium

* Corresponding author. Department of Animal Sciences, The Ohio State University, OARDC, Wooster, OH 44691, USA. Tel.: 1 330263 3803; fax: 1 330263 3949. E-mail address: ujor.1@osu.edu 1878-6146/$ e see front matter 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2011.12.008

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has been reported (Woodward & Boddy 2008). Although Trichoderma species are used as biocontrol agents of plant pathogenic fungi, they have been less efcacious for the control of woodrot caused by white-rot fungi (Bruce & Highley 1991). To gain further insight into the mechanisms underlying interspecic mycelial combat involving a white-rot fungus, we studied the mycelia of the competitive white-rot fungus, Schizophyllum commune when paired against the biocontrol fungus, Trichoderma viride on agar. In addition to its relatively competitive capacity, S. commune is well characterised, hence, is used as a model fungus in the study of fungal biology (Ohm et al. 2010). Since the antagonistic properties of Trichoderma species have been well described, this work concentrated on the response of S. commune to the antagonist, including metabolite proling, changes in lipid peroxidation, protein carbonylation, and calcium inux.

lens on a Leica DM microscope (Leica, Germany). For unstained preparations, the interactions zones of whole cultures were viewed directly by phase contrast and light microscopy, while stained preparations were viewed using uorescence microscopy. Images were acquired with a Leica camera and LAZ-EZ software (Leica, Germany). Staining with Nile Red and Congo Red was performed according to the method described by Kimura et al. (2003), and Slifkin & Cumbie (1988) respectively. Both test and control cultures were microscopically viewed in triplicate.

Metabolite extraction
Interaction assay was set up in replicates of ten for both control and test cultures. Mycelial strips (3.5 g) were cut off 10 mm away from the contact interface for each fungus. Strips were freezedried for 48 h, crushed with a glass rod in 50 ml tubes and extracted in methanol (10 ml). Extraction was carried out overnight at 4  C. Excess methanol was removed by drying under vacuum in a centrifugal evaporator (Genevac Ltd, UK). Extracts were stored at 20  C in glass vials. Extraction was carried out with ten biological replicate samples for both test and control prior to gas chromatographyemass spectrometry (GCeMS).

Materials and methods

Fungal cultures
Both fungi were obtained from the culture collection of the School of Life Sciences, University of Westminster (UK). Stocks were maintained as single 5 mm mycelial plugs in 1 ml of sterilized distilled water in 1.5 ml vials (Nalgene Ltd, UK) at room temperature.

GC-time of ight (TOF)-MS

Ten extracts each from separate control and test cultures were analysed according to a previously described method (Peiris et al. 2008).

Agar plate interaction assay

Self (control) and nonself (test) interactions were set up on potato dextrose agar (PDA; SigmaeAldrich, UK), in 9 cm (diameter) Petri dishes according to the method described by Peiris et al. (2008). Because of the fast growth rate of Trichoderma viride, Schizophyllum commune was inoculated 4 d prior to the inoculation of former. All cultures were incubated at 28  C. Interactions were monitored for 14 d to evaluate the outcome of combat between both species.

Zymogram assay
Laccase and manganese peroxidase (MnP) activities were assayed in-gel (12 % polyacrylamide), under native conditions. Gels were run in triplicate using 30 mg of protein at 120 V for 3 h at 4  C. Laccase activity was stained for, using 2.5 mM; 2,20 -azinobis (3-ethylbenzathiazoline-6-sulfonic acid) (ABTS; SigmaeAldrich, UK), in 0.1 M sodium tartrate (pH 3). MnP staining buffer consisted of 1 mM 2,6-dimethoxy phenol (Sigmae Aldrich, UK), 0.4 mM hydrogen peroxide and 1 mM manganese sulphate in 0.1 mM sodium tartrate (pH 4.5). Samples were incubated for 10 min at room temperature and 30  C for laccase and MnP activities respectively. Gel images were acquired and densitometric analysis performed using Bio-Rad Quantity 1 software.

In situ detection of phenoloxidase activity

To detect phenoloxidase activity in situ, interactions were set up as above on PDA saturated with 0.01 % (w/v) Remazol brilliant blue (RBB). Phenoloxidases decolourise RBB from blue to yellow, allowing the visualisation of enzyme activity in situ.

Incorporation of calcium ionophore in self-paired cultures of Schizophyllum commune

S. commune was self-paired on PDA containing calcium ionophore A23187 (SigmaeAldrich, UK), to a nal concentration of 6 mM. Calcium ionophore A23187 was predissolved in dimethyl sulfoxide (DMSO) before incorporation in agar and an equal amount of DMSO was added to control cultures. Assay plates were incubated as above.

Lipid peroxidation assay

Lipid peroxidation assay was carried out in triplicate using Oxis Bioxytech LPO-586 colourimetric lipid peroxidation assay kit (Oxis international, CA, USA), following the manufacturers protocol. This assay directly measures lipid peroxide (LPO) levels as a function of the amounts of Malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE) produced in a given sample. Schizophyllum commune mycelial strips (0.2 g) were cut off every 24 h as described above. Strips were homogenised in a Fastprep-24 homogeniser (MP, UK), reconstituted in sterile distilled water (1 ml) and spun at 10 000g at room temperature for 8 min. Two hundred micro-litres of homogenate was used for assay.

Microscopy
Both stained (with Nile Red and Congo Red) and unstained preparations were viewed with 100 (oil immersion) objective

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Protein carbonylation assay

Protein carbonyl content of the Schizophyllum commune mycelia was measured over the same period as lipid peroxidation. Mycelial strips (0.2 g) homogenised as above were reconstituted in 1 phosphate-buffered saline (PBS) (1 ml) containing fungal protease inhibitor cocktail (25 ml) (SigmaeAldrich, UK) and protein content was quantied according to the method of Bradford (1976). Homogenates (100 mg) were mixed with 500 ml of 10 mM 2,4-dinitrophenyl hydrazine (DNPH), dissolved in 2 M HCL and incubated at 37  C for 1 h. Proteins were then precipitated with 20 % trichloroacetic acid for 20 min at 4  C and centrifuged at 12 000g for 15 min. The resulting pellet was washed three times with ethanol: ethylacetate (1:1) to

remove excess DNPH. The pellets were dissolved in 6 M guanidine hydrochloride (1.5 ml, pH 2) and absorbance was read at 380 nm (Novaspec II spectrophotometer; Amersham, UK). Protein carbonyl content was calculated as nanomoles of DNPH incorporated per milligramme of protein (molar absorption coefcient (3 ) 22 000 M1 cm1).

Statistical analysis
All experiments were carried out in triplicate, and separate biological samples were used for analyses except where otherwise stated. Assay results were analysed by unpaired t-test using the SPSS software. The means of enzyme activity, LPO, and protein carbonyl levels in control mycelia were compared against those

Fig 1 e Representative plates depicting morphological changes in the mycelia of S. commune and T. viride interacting on agar. (A) Initial mycelial rejection between S. commune and T. viride before contact. (B) Sealing-off of S. commune mycelial front 24 h after contact with T. viride. (C) Barrage formation by S. commune after 48 h of contact with T. viride. (D) Bottom side of Petri dish showing the inception of pigmentation in the mycelia of S. commune after 16 h of interaction with T. viride. (E) & (F) Increase in the intensity of pigmentation after 30 and 96 h of contact respectively. (G) Overgrowth of S. commune by T. viride after 120 h of contact. (H) Self-paired culture of S. commune after 120 h of mycelial contact. (I) Bottom side of self-paired culture of S. commune 120 h post mycelial contact.

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from test cultures, as variables to test for signicance. Nonparametric KruskaleWallis test (Kruskal & Wallis 1952) was used to estimate the level of signicance of the differences in the means of peak areas of metabolites detected in test samples compared to the controls as previously described (Peiris et al. 2008), using the Matlab Version 7.1 (http://www.mathworks.com) running on Windows XP on an IBM-compatible PC.

when the mycelia of S. commune were self-paired at 60 h postinoculation (Fig 3). As in the case of mycelial combat, the intensity of calcium ionophore-induced pigmentation increased with time. However, calcium ionophore-induced pigmentation occurred at both the top and bottom of spreading mycelia, and appeared to originate from the point of inoculation.

Microscopy

Results
Agar plate interaction assays
The interaction of Schizophyllum commune with Trichoderma viride was studied using an agar plate interaction assay (Peiris et al. 2008). The early response of S. commune to T. viride was characterised by a reduction in mycelial extension rate (Fig 1A) which occurred prior to gross mycelial contact. Subsequently, sealing-off of the mycelial front (Fig 1B) and formation of mycelial barrage were observed in S. commune (Fig 1C), at 24 and 48 h postcontact respectively. A brownish pigment developed at the bottom of the plate within the domain occupied by S. commune at 8 h postcontact. The intensity of this pigmentation increased with the duration of contact (Fig 1DeF). Schizophyllum commune was eventually overgrown by T. viride after 5 d of contact (Fig 1G). The incorporation of RBB into the PDA agar resulted in dye degradation (blue-to-yellow decolourisation) within the contact zone at 48 h after contact indicative of the production of phenoloxidases at the site of mycelial interaction (Fig 2). The addition of calcium ionophore A23187 to the PDA agar induced the development of pigmentation

The observation of unstained preparations of Schizophyllum commune mycelia by phase contrast microscopy revealed the degeneration of protoplasmic components in S. commune around the points of contact with Trichoderma viride (Fig 4A). Light microscopy showed that contact elicited profuse mycelial extension and coiling in T. viride towards, and subsequently around S. commune mycelia. The development of brownish pigments, predominantly around the encircled mycelia was also observed (Fig 4B). Staining of mycelial preparations with Nile Red and Congo Red revealed extensive cell wall lysis, relative enlargement of hyphae, and protoplasmic degeneration, in S. commune 48 h after mycelial contact (Fig 5A & C) when compared to self-paired mycelia (Fig 5B & D).

Metabolite proling
Changes in the metabolite proles during mycelial interaction were analysed by extracting mycelial strips 10 mm away from the contact interface for each fungus followed by GC-TOF-MS analysis. A total of 108 peaks were detected by MS with potential identication obtained for 38 (35 %) of these. The identied metabolites were classied as mainly either sugar alcohols

Fig 2 e Decolourisation of RBB in the interaction zone, following contact between S. commune and T. viride. (A) Underside of interaction plate of both fungi 24 h after contact showing minimal dye decolourisation. (B) Increased decolourisation of RBB at 48 h post mycelial contact. (C) & (D) Self-paired S. commune and T. viride respectively 48 h postcontact.

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Fig 3 e Pigmentation in cultures of S. commune growing on calcium ionophore A23187-containing PDA, 120 h postinoculation. (A) & (B) Top and bottom sides of control plates containing DMSO. (C) & (D) Brownish pigmentation in plates containing calcium ionophore A23187 dissolved in DMSO.

(including cyclitols), fatty acids, phenolic compounds, organic acids, amino acids, aldehydes or vitamins. The observed patterns of metabolite abundance were expressed as a function of increased/decreased peak area for both fungi (Tables 1 and 2). Peaks, which showed !30 % increase in peak area (P < 0.05), were considered to be up-regulated/down-regulated respectively upon interspecic mycelial contact. Contact with Trichoderma viride resulted in the up-regulation of g-amino butyric acid (GABA), organic acids, sugar alcohols (erythritol/isomer and hexanetetrol), myo-inositol phosphate, N-acetylglucosamine, and pyridoxine in Schizophyllum commune. On the other hand, pyruvic acid, glycerol, and alanine were all downregulated in S. commune. Conversely, contact of T. viride with S. commune resulted in up-regulation of organic acids and sugar alcohols (galactosylglycerol, xylitol, and an unspecied sugar alcohol) in T. viride. In addition, both tropic and mandelic acid increased in abundance in both fungal domains, while 4-hydroxyphenyl ethanol was only up-regulated in T. viride.

Laccase and MnP were not detected in samples taken from the mycelial domain of T. viride (data not shown). However the activities of laccase and MnP increased 2.9-fold (P < 0.0001) and 7.6-fold (P < 0.0001) respectively, after 24 h of contact with T. viride (Fig 6). While the activity of laccase in the interacting mycelia reduced after 48 h of contact, the activity of MnP remained stable for the same time period. Liquid-based assay for both enzymes conrmed the results from in-gel assay, where laccase activity decreased after 48 h of contact, while MnP activity did not decrease signicantly until 72 h post mycelial contact (data not shown).

Levels of oxidative stress indicators in the mycelia of Schizophyllum commune paired against Trichoderma viride
Levels of LPO and protein carbonyls in the mycelia of S. commune were quantied during interaction with T. viride (Fig 7). The level of LPO increased 2.6-fold (P 0.003) in the mycelia of S. commune confronted by T. viride after 24 h compared to self-paired mycelia of S. commune, but decreased with increasing duration of mycelial contact although levels were still signicantly higher after 48 h (3.7-fold; P 0.022) and 72 h (2.0-fold; P 0.033) compared to self-paired cultures. A similar pattern was observed for protein carbonylation.

Modulation of laccase and MnP activity in the domain of Schizophyllum commune interacting with Trichoderma viride
The activity levels of laccase and MnP activity were quantied during mycelial interaction between S. commune and T. viride.

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Fig 4 e Micrographs depicting morphological changes in S. commune (SC) at points of contact with T. viride (TV; bar [ 10 mm). (A) Phase contrast micrograph showing the degeneration of protoplasmic content in S. commune (arrows), 48 h postcontact with T. viride. (B) Entwining of T. viride around S. commune after 48 h of contact. Protein carbonyl content was signicantly higher in the mycelia of S. commune interacting with T. viride during the rst 3 d of interaction; 1.3-fold, 2.7-fold, and 2.2-fold (P 0.0002; P < 0.0001; P < 0.0001) respectively than in self-paired cultures.

Discussion
The aim of this study was to investigate the interaction of the white-rot fungus, Schizophyllum commune with the biocontrol

Fig 5 e Fluorescent micrographs of S. commune mycelia stained with Congo Red and Nile Red (bar [ 10 mm). (A) Nile Redstained mycelia of S. commune after 48 h of contact with T. viride. (B) Nile Red-stained mycelia of self-paired S. commune, 48 h postcontact. (C) Mycelia of S. commune after 48 h of conict with T. viride, stained with Congo Red. (D) Self-paired mycelia of S. commune stained with Congo Red, 48 h postcontact.

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Table 1 e Metabolites that showed statistically signicant differences in peak area (P < 0.05) in the mycelial domain of S. commune paired against T. viride, in comparison to its self-paired mycelia. Peak number
5 4 6 9 14 19 28 33 38 48 53 57 69 74 76 89

% Increase/ decrease in peak area

60 67 60 99 52 60 41 72 77 97 44 99 81 70 99 99

P-value

Metabolite identity

0.020 0.001 0.0005 <0.0001 0.001 <0.0001 0.0003 0.002 0.019 <0.0001 0.0002 <0.0001 0.0001 <0.0001 0.0002 <0.0001

3-Hydroxyporpanoic acid Pyruvic acid Glycerol GABA Alanine Erythritol/isomer Malic acid Citramalic acid Mandelic acid Hexanetetrol 2-Furancarboxylic acid Tropic acid Pyridoxine Unidentied N-Acetylglucosamine Myo-inositol phosphate

Negative sign () represents decrease in peak area.

agent, Trichoderma viride at the metabolomic level. We analysed the interplay between predominating metabolites, phenoloxidases, morphological variations, and oxidative damage in the contact zone, particularly in S. commune as it was outcompeted by T. viride. In addition, we explored the possible links between cell wall-related stress and calcium inux using the calcium ionophore A23187. Although reactions observed on laboratory media may not be replicated to the same extent in the eld due to varying environmental conditions, the use of agar-based media remains the most suitable strategy for studying fungal conicts (Grifth et al. 1994; Peiris et al. 2008; Woodward & Boddy 2008).

Table 2 e Metabolites that showed statistically signicant differences in peak area (P < 0.05) in the mycelial domain of T. viride paired against S. commune, in comparison to self-paired mycelia. Peak number
5 38 40 43 44 46 50 53 57 58 74 81

% Increase/ decrease in peak area

60 71 90 57 80 61 33 41 60 55 * 50

P-value

Metabolite identity

0.0001 0.019 0.005 0.0004 <0.0001 0.005 0.001 <0.0001 0.021 0.0003 * 0.001

3-Hydroxyporpanoic acid Mandelic acid 2-Hydroxyglutaric acid Xylitol Sugar alcohol 4-Hydroxyphenyl ethanol 2,3,4-Trihyroxybutanal 2-Furancarboxylic acid Tropic acid Unidentied (b) Unidentied () Galatosylglycerol

*Metabolite was detected only in cultures of T. viride paired against S. commune, but not in the self-paired cultures of the former. b e molecular weight: 98; e molecular weight: 69.

The observed cell wall lysis in S. commune was associated with a rise in the levels of N-acetylglucosamine (which is the product of cell wall hydrolysis), in the combat zone. In addition, sugar alcohols were up-regulated in both interacting species. Synthesis of protective osmolytes such as sugar alcohols in response to osmotic, oxidative or heat stresses is a wellknown microbial response, especially in yeasts and lamentous fungi (Davis et al. 2000). In light of this, accumulation of sugar alcohols in both fungi postcontact points to the possibility of increased local stress in the mycelial conict zone. Osmotic stress response has been shown to inuence mycoparasitic behaviour in Trichoderma harzianum (DelgadoJarana et al. 2006). This is logical given that Trichoderma species can metabolise a variety of cell wall polymers and different intracellular metabolites during mycelial combat, thereby altering the solute concentration of the immediate environment, relative to its cytoplasm (Delgado-Jarana et al. 2006). This triggers a biochemical response to counterbalance the resulting osmotic stress. In addition, the ability of Trichoderma species to coil around their hosts (also observed in this study) requires high inner hydrostatic turgour pressure, generated by the accumulation of molar concentrations of sugar alcohols (Thines et al. 2000). Taken together, it is plausible that up-regulation of sugar alcohols in T. viride could be an adaptation to rising osmotic stress, as well as to aid parasitic coiling around the host. The observed loss of cell wall in S. commune upon extended contact with T. viride would exert pressure on the cell membrane and drastically impair membrane transport mechanisms that regulate cytosolic composition. In other studies, this has been reported to trigger the synthesis of osmoprotectants, such as sugar alcohols, to cushion the resulting pressure on the cell membrane (Davis et al. 2000; Ramirez et al. 2004). The observed increase in sugar alcohols in S. commune in this study could be a similar response to that seen in an earlier study of combative interactions among wood-rot fungi, particularly for erythritol (Peiris et al. 2008). The increase in both lipid peroxidation and protein carbonylation in S. commune in response to contact with T. viride indicated that oxidative damage was occurring. The cessation of mycelial growth and the degeneration of protoplasmic organelles we observed are similar to complex deteriorations seen in stationary phase of growth of other fungi. Increased carbonylation in yeasts has been linked to both pronounced production of reactive oxygen species by ageing mitochondria and starvation (Yan et al. 1997; Aguilaniu et al. 2003). As cell wall damage can limit nutrient acquisition (Casadevall et al. 2009), it is possible that this may have impaired nutrient absorption in the confronted mycelia of S. commune. Cumulatively, cell wall damage and the resulting starvation might have led to a switch of mycelial growth to secondary phase with resultant oxidative stress, explaining rise in the levels of LPO and carbonylated proteins in S. commune. The assumption that contact with T. viride caused oxidative stress in S. commune is further supported by the up-regulation of GABA, pyridoxine, and to some extent sugar alcohols. Synthesis of sugar alcohols can rebalance the redox state of the cell by increasing NADPH levels, hence, reducing the production of reactive oxygen radicals in the respiratory chain (Lee et al. 2003). Furthermore, pyridoxine has been repeatedly implicated in antioxidation reactions during which it quenches

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Fig 6 e Representative 12 % SDS-PAGE run under native conditions and stained with substrates for laccase (A) and MnP (B). Gels were loaded with 30 mg of protein extracts of S. commune. 1 & 2: Protein extract from self-paired S. commune, after 24 and 48 h of contact respectively. 3 & 4: Protein sample from S. commune paired against T. viride after 24 and 48 h of interaction respectively.

singlet oxygen and hydrogen peroxide (Ehrenshaft & Daub 2001; Ristila et al. 2006). Activation of GABA synthesis is triggered by the down-regulation or repression of a-ketoglutarate dehydrogenase, an enzyme known to be sensitive to redox im balance (Bouche et al. 2003; Panagiotou et al. 2005). Interestingly, most enzymes involved in GABA synthesis require pyridoxine as a cofactor. Phenoloxidases are thought to play a defence role during combat, by oxidizing phenolic compounds into hypha-sealing

polymers (Grifth et al. 1994; Rayner et al. 1994; Boddy 2000). In this study, we observed increase in phenoloxidase activity in S. commune within the interaction zone suggesting a specic function for this activity at the contact interface. This was associated with increases in mandelic and tropic acid (both phenolic compounds) in the domains of both fungi near the interaction interface. The increase of laccase and MnP activity in S. commune could be a response to detoxify these compounds as well as 4-hydroxyphenyl ethanol (another phenolic

Fig 7 e (A) Combined levels of MDA and 4-HNE (indicators of LPO levels) in the mycelia of S. commune paired against T. viride (SCTR) in comparison to levels in self-paired cultures (SCSC). (B) Comparative levels of intracellular protein carbonyl content in the mycelia of S. commune paired against self and mycelia paired against T. viride. All experiments were carried out in triplicate, using three biological samples (cultures) for both test and control pairings. Error bars represent standard deviation.

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compound), which was signicantly up-regulated in T. viride during combat. The down-regulation of the key metabolic intermediate, pyruvic acid in S. commune during interaction with T. viride was also observed. This down-regulation is likely to be associated with reduced metabolic ux (secondary metabolism). In addition, we also observed changes in the levels of inositol, which also has been implicated in osmotic stress response, signalling, (Perera et al. 2004) and maintenance of cytoskeletal integrity (Homma et al. 1998). The treatment of S. commune with the calcium ionophore A23187 mimicked the pigmentation typical of combative interactions. Although we did not assay for phenoloxidase activity in calcium ionophore A23187-treated cultures, others have reported an increase in laccase activity in liquid cultures of Rhizoctonia solani challenged with calcium ionophore A23187 (Crowe & Olsson 2001). Calcium ionophores mobilise calcium across cell membranes (Abott et al. 1979; Crowe & Olsson 2001) and have been reported to also uncouple oxidative phosphorylation (Abott et al. 1979). It is unlikely that the latter is responsible for the phenotype we observed with calcium ionophore, as mycelial growth was not inhibited. Hence, the resulting phenotype is ascribable to calcium inux resulting from compromised cell integrity, which was also reported in R. solani (Crowe & Olsson 2001). The responses observed in S. commune paired against T. viride suggest the up-regulation of mechanisms specically targeted at alleviating the stresses stemming from the antagonistic machinery of the latter. Although the extent to which S. commune recruits these reactions in natural environments warrants further investigation, overall, it is conceivable that these mechanisms may be amplied in the natural environment perhaps contributing in part to the resistance of S. commune to T. viride.

Acknowledgements
This research was funded by the University of Westminster, London UK. Victor Ujor and Diluka Peiris were recipients of University of Westminster Biosciences Research Scholarship and Cavendish Research Scholarship respectively.

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