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The mycelial response of the white-rot fungus, Schizophyllumcommune to the biocontrol agent, Trichoderma viride

Victor C. UJORa,*, Monia MONTIb, Diluka Gayani PEIRISa, Mark Owen CLEMENTSa,John Norman HEDGERa

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

a r t i c l e i n f o

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

* Corresponding author. Department of Anim3803; fax: þ1 330263 3949.

E-mail address: ujor.1@osu.edu1878-6146/$ e see front matter ª 2011 Britisdoi:10.1016/j.funbio.2011.12.008

a b s t r a c t

In this study, agar plate interaction between Schizophyllum commune and Trichoderma viride

was investigated to characterise the physiological responses occurring during interspecific

mycelial combat. The metabolite profiles 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 influx 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 oxida-

tive stress indicators and increased levels of phenolic compounds, antioxidant g-amino bu-

tyric acid, and pyridoxine and osmo-protective sugar alcohols. Calcium ionophore

mimicked the pigmentation in the T. viride-confronted mycelia of S. commune, implicating

calcium influx in the response to T. viride. The changes in S. commune are indicative of tar-

geted responses to osmotic and oxidative stresses and phenoloxidase-mediated detoxifica-

tion of noxious compounds in the contact interface with T. viride, which may confer

resistance in natural environments.

ª 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction them to detect and respond to nonself mycelia (Rayner 1991;

Fungusefungus combative interactions have been extensively

studied, leading to the use of more antagonistic species to

control plant pathogenic fungi and to some measure, wood-

rot in commercial logging (Bruce & Highley 1991; Boddy 2000;

Adomas et al. 2006). Interspecificmycelial combat is character-

ised 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

al Sciences, The Ohio St

h Mycological Society. Pu

Griffith et al. 1994; Boddy 2000). Such mechanisms allow fungi

to defend their territories, thereby restricting access to cap-

tured nutrients by opposing species (Rayner 1991; Boddy 2000).

Trichoderma speciesparasitize other fungi,making thempo-

tent biocontrol agents of specific fungal plant pathogens in the

field (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

ate University, OARDC, Wooster, OH 44691, USA. Tel.: þ1 330263

blished by Elsevier Ltd. All rights reserved.

The mycelial response of S. commune 333

has been reported (Woodward & Boddy 2008). Although Tricho-

derma species areusedasbiocontrol agentsof plantpathogenic

fungi, they have been less efficacious for the control of wood-

rot caused by white-rot fungi (Bruce & Highley 1991).

To gain further insight into themechanisms underlying in-

terspecific mycelial combat involving a white-rot fungus, we

studied themycelia of the competitivewhite-rot fungus, Schiz-

ophyllum commune when paired against the biocontrol fungus,

Trichoderma viride on agar. In addition to its relatively compet-

itive 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 re-

sponse of S. commune to the antagonist, including metabolite

profiling, changes in lipid peroxidation, protein carbonylation,

and calcium influx.

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.

Agar plate interaction assay

Self (control) and nonself (test) interactions were set up on po-

tato dextrose agar (PDA; SigmaeAldrich, UK), in 9 cm (diame-

ter) Petri dishes according to the method described by Peiris

et al. (2008). Because of the fast growth rate of Trichoderma vir-

ide, Schizophyllum commune was inoculated 4 d prior to the in-

oculation of former. All cultures were incubated at 28 �C.Interactions were monitored for 14 d to evaluate the outcome

of combat between both species.

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 bril-

liant 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 ofSchizophyllum commune

S. commune was self-paired on PDA containing calcium iono-

phore A23187 (SigmaeAldrich, UK), to a final concentration

of 6 mM. Calcium ionophore A23187 was predissolved in di-

methyl sulfoxide (DMSO) before incorporation in agar and an

equal amount of DMSO was added to control cultures. Assay

plates were incubated as above.

Microscopy

Both stained (with Nile Red and Congo Red) and unstained

preparations were viewed with 100� (oil immersion) objective

lens on a LeicaDMmicroscope (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 fluorescence micros-

copy. 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 assaywas set up in replicates of ten for both control

and test cultures.Mycelial strips (3.5 g)werecut off 10mmaway

from the contact interface for each fungus. Strips were freeze-

dried for 48 h, crushed with a glass rod in 50 ml tubes and

extracted inmethanol (10 ml). Extraction was carried out over-

night 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).

GC-time of flight (TOF)-MS

Ten extracts each from separate control and test cultures

were analysed according to a previously described method

(Peiris et al. 2008).

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 0.1M sodium tartrate (pH 3). MnP stain-

ing buffer consisted of 1 mM 2,6-dimethoxy phenol (Sigmae

Aldrich, UK), 0.4 mM hydrogen peroxide and 1 mMmanganese

sulphate in0.1mMsodiumtartrate (pH4.5). Sampleswere incu-

bated for 10min 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.

Lipid peroxidation assay

Lipid peroxidation assay was carried out in triplicate using

Oxis Bioxytech� LPO-586� colourimetric lipid peroxidation as-

say kit (Oxis international, CA, USA), following the manufac-

turer’s 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 homoge-

nised 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 homoge-

nate was used for assay.

334 V. C. Ujor et al.

Protein carbonylation assay

Protein carbonyl content of the Schizophyllum commune myce-

lia was measured over the same period as lipid peroxidation.

Mycelial strips (0.2 g) homogenised as above were reconsti-

tuted in 1� phosphate-buffered saline (PBS) (1 ml) containing

fungal protease inhibitor cocktail (25 ml) (SigmaeAldrich, UK)

and protein content was quantified according to the method

of Bradford (1976). Homogenates (100 mg) were mixed with

500 ml of 10mM2,4-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 �Cand centrifuged at 12000g for 15 min. The resulting pellet

was washed three times with ethanol: ethylacetate (1:1) to

Fig 1 e Representative plates depicting morphological changes in

(A) Initialmycelial rejection between S. commune and T. viride befor

contact with T. viride. (C) Barrage formation by S. commune after 48

the inceptionofpigmentation in themyceliaof S. communeafter 16

pigmentationafter30and96hof contact respectively. (G)Overgrow

culture ofS. communeafter 120hofmycelial contact. (I) Bottomside

remove excess DNPH. The pellets were dissolved in 6 M guani-

dine hydrochloride (1.5 ml, pH 2) and absorbance was read at

380 nm (Novaspec II spectrophotometer; Amersham, UK). Pro-

tein carbonyl content was calculated as nanomoles of DNPH

incorporated per milligramme of protein (molar absorption

coefficient ( 3) ¼ 22000 M�1 cm�1).

Statistical analysis

All experimentswere carried out in triplicate, and separate bio-

logical sampleswere used for analyses exceptwhere otherwise

stated. Assay resultswere analysedbyunpaired t-test using the

SPSS software. Themeans of enzyme activity, LPO, and protein

carbonyl levels in controlmyceliawere comparedagainst those

the mycelia of S. commune and T. viride interacting on agar.

e contact. (B) Sealing-off of S. communemycelial front 24 h after

h of contact with T. viride. (D) Bottom side of Petri dish showing

hof interactionwithT. viride. (E)& (F) Increase in the intensityof

thofS. communebyT. virideafter120hof contact. (H)Self-paired

of self-paired culture ofS. commune120hpostmycelial contact.

The mycelial response of S. commune 335

fromtest cultures, asvariables to test for significance.Nonpara-

metric KruskaleWallis test (Kruskal &Wallis 1952) was used to

estimate the level of significanceof thedifferences in themeans

of peak areas ofmetabolites 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

onWindows XP on an IBM-compatible PC.

Results

Agar plate interaction assays

The interaction of Schizophyllum communewith Trichoderma vir-

ide 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 grossmycelial contact. Subse-

quently, sealing-off of themycelial 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 devel-

oped at the bottom of the plate within the domain occupied by

S. commune at 8 h postcontact. The intensity of this pigmenta-

tion increased with the duration of contact (Fig 1DeF). Schizo-

phyllum 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 decolourisa-

tion) within the contact zone at 48 h after contact indicative

of the production of phenoloxidases at the site of mycelial in-

teraction (Fig 2). The addition of calcium ionophore A23187 to

the PDA agar induced the development of pigmentation

Fig 2 e Decolourisation of RBB in the interaction zone, following

interaction plate of both fungi 24 h after contact showing minim

at 48 h post mycelial contact. (C) & (D) Self-paired S. commune a

when the mycelia of S. communewere self-paired at 60 h post-

inoculation (Fig 3). As in the case ofmycelial combat, the inten-

sity of calcium ionophore-induced pigmentation increased

with time. However, calcium ionophore-induced pigmenta-

tion occurred at both the top and bottomof spreadingmycelia,

and appeared to originate from the point of inoculation.

Microscopy

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 myce-

lial extension and coiling in T. viride towards, and subse-

quently around S. commune mycelia. The development of

brownish pigments, predominantly around the encircled my-

celia was also observed (Fig 4B). Staining of mycelial prepara-

tionswith Nile Red and Congo Red revealed extensive cell wall

lysis, relative enlargement of hyphae, and protoplasmic de-

generation, in S. commune 48 h after mycelial contact (Fig 5A

& C) when compared to self-paired mycelia (Fig 5B & D).

Metabolite profiling

Changes in the metabolite profiles 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 108peaksweredetectedbyMSwithpotential

identification obtained for 38 (35 %) of these. The identifiedme-

tabolites were classified as mainly either sugar alcohols

contact between S. commune and T. viride. (A) Underside of

al dye decolourisation. (B) Increased decolourisation of RBB

nd T. viride respectively 48 h postcontact.

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.

336 V. C. Ujor et al.

(including cyclitols), fatty acids, phenolic compounds, organic

acids, amino acids, aldehydes or vitamins. The observed pat-

terns 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 respec-

tively upon interspecific mycelial contact. Contact with Tricho-

derma viride resulted in the up-regulation of g-amino butyric

acid (GABA), organic acids, sugar alcohols (erythritol/isomer

and hexanetetrol), myo-inositol phosphate, N-acetylglucos-

amine, and pyridoxine in Schizophyllum commune. On the other

hand, pyruvic acid, glycerol, and alanine were all down-

regulated 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 unspecified 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.

Modulation of laccase and MnP activity in the domain ofSchizophyllum commune interacting with Trichodermaviride

The activity levels of laccase and MnP activity were quantified

during mycelial interaction between S. commune and 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 confirmed the results

from in-gel assay, where laccase activity decreased after

48 h of contact, while MnP activity did not decrease signifi-

cantly until 72 h post mycelial contact (data not shown).

Levels of oxidative stress indicators in the mycelia ofSchizophyllum commune paired against Trichodermaviride

Levels of LPO and protein carbonyls in the mycelia of S. com-

mune were quantified during interaction with T. viride (Fig 7).

The level of LPO increased 2.6-fold (P ¼ 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 in-

creasing duration of mycelial contact although levels were

still significantly higher after 48 h (3.7-fold; P ¼ 0.022) and

72 h (2.0-fold; P ¼ 0.033) compared to self-paired cultures. A

similar pattern was observed for protein carbonylation.

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.

The mycelial response of S. commune 337

Protein carbonyl content was significantly higher in the my-

celia of S. commune interacting with T. viride during the first

3 d of interaction; 1.3-fold, 2.7-fold, and 2.2-fold (P ¼ 0.0002;

P < 0.0001; P < 0.0001) respectively than in self-paired

cultures.

Fig 5 e Fluorescent micrographs of S. commune mycelia stained

stained mycelia of S. commune after 48 h of contact with T. viride

postcontact. (C) Mycelia of S. commune after 48 h of conflict with T

commune stained with Congo Red, 48 h postcontact.

Discussion

The aim of this study was to investigate the interaction of the

white-rot fungus, Schizophyllum commune with the biocontrol

with Congo Red and Nile Red (bar [ 10 mm). (A) Nile Red-

. (B) Nile Red-stained mycelia of self-paired S. commune, 48 h

. viride, stained with Congo Red. (D) Self-paired mycelia of S.

Table 1eMetabolites that showed statistically significantdifferences in peak area (P < 0.05) in the mycelial domainof S. commune paired against T. viride, in comparison to itsself-paired mycelia.

Peaknumber

% Increase/decrease inpeak area

P-value Metabolite identity

5 60 0.020 3-Hydroxyporpanoic acid

4 �67 0.001 Pyruvic acid

6 �60 0.0005 Glycerol

9 99 <0.0001 GABA

14 �52 0.001 Alanine

19 60 <0.0001 Erythritol/isomer

28 41 0.0003 Malic acid

33 72 0.002 Citramalic acid

38 77 0.019 Mandelic acid

48 97 <0.0001 Hexanetetrol

53 44 0.0002 2-Furancarboxylic acid

57 99 <0.0001 Tropic acid

69 81 0.0001 Pyridoxine

74 70 <0.0001 Unidentified

76 99 0.0002 N-Acetylglucosamine

89 99 <0.0001 Myo-inositol phosphate

Negative sign (�) represents decrease in peak area.

338 V. C. Ujor et al.

agent, Trichoderma viride at the metabolomic level. We ana-

lysed the interplay between predominating metabolites, phe-

noloxidases, morphological variations, and oxidative damage

in the contact zone, particularly in S. commune as it was out-

competed by T. viride. In addition, we explored the possible

links between cell wall-related stress and calcium influx using

the calcium ionophore A23187. Although reactions observed

on laboratory media may not be replicated to the same extent

in the field due to varying environmental conditions, the use

of agar-based media remains the most suitable strategy for

studying fungal conflicts (Griffith et al. 1994; Peiris et al. 2008;

Woodward & Boddy 2008).

Table 2eMetabolites that showed statistically significantdifferences in peak area (P < 0.05) in the mycelial domainof T. viride paired against S. commune, in comparison toself-paired mycelia.

Peaknumber

% Increase/decrease inpeak area

P-value Metabolite identity

5 60 0.0001 3-Hydroxyporpanoic acid

38 71 0.019 Mandelic acid

40 90 0.005 2-Hydroxyglutaric acid

43 57 0.0004 Xylitol

44 80 <0.0001 Sugar alcohol

46 61 0.005 4-Hydroxyphenyl ethanol

50 33 0.001 2,3,4-Trihyroxybutanal

53 41 <0.0001 2-Furancarboxylic acid

57 60 0.021 Tropic acid

58 �55 0.0003 Unidentified (b)

74 * * Unidentified (ʒ)

81 50 0.001 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; ʒ 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 addi-

tion, sugar alcohols were up-regulated in both interacting spe-

cies. Synthesis of protective osmolytes such as sugar alcohols

in response to osmotic, oxidative or heat stresses is a well-

known microbial response, especially in yeasts and filamen-

tous fungi (Davis et al. 2000). In light of this, accumulation of

sugar alcohols in both fungi postcontact points to the possibil-

ity of increased local stress in the mycelial conflict zone.

Osmotic stress response has been shown to influence

mycoparasitic behaviour in Trichoderma harzianum (Delgado-

Jarana et al. 2006). This is logical given that Trichoderma species

canmetabolise a variety of cell wall polymers and different in-

tracellular metabolites during mycelial combat, thereby alter-

ing the solute concentration of the immediate environment,

relative to its cytoplasm (Delgado-Jarana et al. 2006). This trig-

gers 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 ac-

cumulation 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 os-

motic 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 mem-

brane and drastically impair membrane transport mecha-

nisms that regulate cytosolic composition. In other studies,

this has been reported to trigger the synthesis of osmoprotec-

tants, such as sugar alcohols, to cushion the resulting pres-

sure 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 ear-

lier study of combative interactions among wood-rot fungi,

particularly for erythritol (Peiris et al. 2008).

The increase in both lipid peroxidation and protein carbon-

ylation in S. commune in response to contact with T. viride indi-

cated that oxidative damage was occurring. The cessation of

mycelial growth and the degeneration of protoplasmic organ-

elles we observed are similar to complex deteriorations seen

in stationary phase of growth of other fungi. Increased car-

bonylation in yeasts has been linked to both pronounced pro-

duction 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 ab-

sorption in the confronted mycelia of S. commune. Cumula-

tively, 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 contactwith T. viride caused oxidative

stress in S. commune is further supported by the up-regulation

of GABA, pyridoxine, and to some extent sugar alcohols. Syn-

thesis of sugar alcohols can rebalance the redox state of the

cell by increasing NADPH levels, hence, reducing the produc-

tion of reactive oxygen radicals in the respiratory chain (Lee

et al. 2003). Furthermore, pyridoxine has been repeatedly im-

plicated in antioxidation reactions during which it quenches

Fig 6 e Representative 12%SDS-PAGE rununder native conditions and stainedwith substrates for laccase (A) andMnP (B). Gels

were loadedwith 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.

The mycelial response of S. commune 339

singlet oxygen and hydrogen peroxide (Ehrenshaft & Daub

2001; Ristil€a et al. 2006). Activation of GABA synthesis is trig-

gered by the down-regulation or repression of a-ketoglutarate

dehydrogenase, an enzyme known to be sensitive to redox im-

balance (Bouch�e et al. 2003; Panagiotou et al. 2005). Interest-

ingly, 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

Fig 7 e (A) Combined levels of MDA and 4-HNE (indicators of LPO

(SCTR) in comparison to levels in self-paired cultures (SCSC). (B)

in the mycelia of S. commune paired against self and mycelia pa

triplicate, using three biological samples (cultures) for both test a

polymers (Griffith et al. 1994; Rayner et al. 1994; Boddy 2000).

In this study, we observed increase in phenoloxidase activity

in S. communewithin the interaction zone suggesting a specific

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

levels) in the mycelia of S. commune paired against T. viride

Comparative levels of intracellular protein carbonyl content

ired against T. viride. All experiments were carried out in

nd control pairings. Error bars represent standard deviation.

340 V. C. Ujor et al.

compound), which was significantly 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 associ-

ated with reduced metabolic flux (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 in-

teractions. Althoughwe did not assay for phenoloxidase activ-

ity 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 phosphor-

ylation (Abott et al. 1979). It is unlikely that the latter is respon-

sible for the phenotype we observed with calcium ionophore,

as mycelial growth was not inhibited. Hence, the resulting

phenotype is ascribable to calcium influx resulting from com-

promised 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 mecha-

nisms specifically targeted at alleviating the stresses stem-

ming from the antagonistic machinery of the latter.

Although the extent to which S. commune recruits these reac-

tions in natural environments warrants further investigation,

overall, it is conceivable that these mechanisms may be am-

plified 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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