The mycelial response of the white-rot fungus, Schizophyllum commune to the biocontrol agent, Trichoderma viride Victor C. UJOR a, *, Monia MONTI b , Diluka Gayani PEIRIS a , Mark Owen CLEMENTS a , John Norman HEDGER a a School of Life Sciences, University of Westminster, 115 New Cavendish Street, London, UK b 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 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 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). Interspecific mycelial 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 them to detect and respond to nonself mycelia (Rayner 1991; 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 species parasitize other fungi, making them po- 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 * Corresponding author. Department of Animal Sciences, The Ohio State University, OARDC, Wooster, OH 44691, USA. Tel.: þ1 330263 3803; fax: þ1 330263 3949. E-mail address: [email protected]journal homepage: www.elsevier.com/locate/funbio fungal biology 116 (2012) 332 e341 1878-6146/$ e see front matter ª 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2011.12.008
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journa l homepage : www.e lsev ier . com/ loca te / funb io
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: [email protected]/$ 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
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
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