ORI GI NAL ARTI CLE

Growth inhibition and antioxidative response of wood

decay fungi exposed to plant extracts of Casearia species
T.S. Bento
1
, L.M.B. Torres
2
, M.B. Fialho
2
and V.L.R. Bononi
1
1 Instituto de Bot^anica de S~ao Paulo, N ucleo de Pesquisa em Micologia, S~ao Paulo, Brazil
2 Instituto de Bot^anica de S~ao Paulo, N ucleo de Pesquisa em Fisiologia e Bioqumica de Plantas, S~ao Paulo, Brazil
Signicance and Impact of the Study: The Casearia plant extracts exhibited important antifungal activ-
ity on wood decay fungi and triggered oxidative stress process, an inhibitory mechanism rarely studied
in lamentous fungi exposed to plant extracts. Therefore, a starting point was provided for the devel-
opment of natural compounds-based products as an alternative to chemical fungicides. In addition, sub-
sidies were given to further studies in order to elucidate in more detail how compounds present in
extracts of native tropical plants affect the physiology of fungi.
Keywords
antifungal activity, natural compounds,
oxidative stress, Pycnoporus, Trametes, white
rot.
Correspondence
Mauricio B. Fialho, Instituto de Bot^anica de
S~ao Paulo, N ucleo de Pesquisa em Fisiologia e
Bioqumica de Plantas, 04301-012, S~ao Paulo,
SP, Brazil.
E-mail: alhomb@hotmail.com
2013/1281: received 27 June 2013, revised
28 August 2013 and accepted 9 September
2013
doi:10.1111/lam.12159
Abstract
Ligninolytic fungi take part in critical processes in ecosystems such as nutrient
recycling; however, some fungal species can be pathogenic to forest and urban
trees and deteriorate wood products. The tropical ora is an important source
of antimicrobial compounds environmentally safer than traditional wood
preservatives. Therefore, this study aimed to evaluate the inhibitory activity of
ethanol plant extracts of Casearia sylvestris and Casearia decandra on the
white-rot wood decay basidiomycetes Trametes villosa and Pycnoporus
sanguineus. In addition, the effect of the extracts on the fungal antioxidative
metabolism was studied. Among the different substances present in the
extracts, the phytochemical analyses identied a clerodane diterpenoid
(C. sylvestris) and cinnamic acid, hydroquinone and b-sitosterol (C. decandra).
The extracts inhibited the fungi up to 70% and caused hyphal morphology
changes. The extracts triggered oxidative stress process as indicated by the
increased levels of the antioxidant enzymes catalase and glutathione reductase.
Therefore, the Casearia extracts are a potential source of natural biocides to
control wood decay fungi, and one of the mechanisms of action is the
oxidative stress.
Introduction
Fungi play an essential role in nutrient cycling in ecosys-
tems; however, under certain conditions, many fungal
species can deteriorate timber products such as railway
sleepers, utility poles and fences, causing signicant eco-
nomic losses. In addition, these micro-organisms can act
as phytopathogens attacking trees used in urban forestry.
Urban trees with high levels of wood decay have
decreased strength and stability, imposing a high risk to
people and properties in areas of dense population.
Trametes villosa and Pycnoporus sanguineus are among
the most important saprophytic white-rot wood decay
basidiomycetes able to completely degrade all components
of lignocellulosic materials in nature. However, these
fungi are also responsible for important economic losses
in timber products and are also associated with wood
decay in living trees in tropical and subtropical areas
(Harsh and Bisht 1997; Luley 2005).
The wood quality maintenance is a constant concern in
timber industry, and there is an increased demand for
products extracted or produced with minimum damage
to human health and environment. Currently, chemical
preservatives are efcient to control wood decay fungi;
however, their use has led to environmental impacts
(Singh and Singh 2012).
Letters in Applied Microbiology 2013 The Society for Applied Microbiology 1
Letters in Applied Microbiology ISSN 0266-8254
The tropical ora is rich in species, which may serve as
a source of new antifungal compounds. Several medicinal
properties have been reported in the plant genus Casearia
(Salicaceae) (Santos et al. 2010; Ferreira et al. 2011).
Phytochemical analysis of C. sylvestris extracts showed the
predominance of clerodane-type diterpene called casea-
rins. Most of these diterpenes exhibit remarkable struc-
tural characteristics and are found only in a few plant
families (Oberlies et al. 2002; Wang et al. 2009). Chemical
and pharmacological knowledge on C. decandra is limited
to only one study, which analysed essential oils of owers
and leaves (Stefanello et al. 2010). Reports on the antimi-
crobial activity of Casearia extracts are scarce, which have
been restricted mainly to human pathogens. To our
knowledge, there are no studies reporting the effect of
these extracts on wood decay fungi.
The inhibitory mechanisms of natural compounds on
fungi are important for the development of efcient and
sustainable antifungal products. A possible mechanism of
action is the triggering of intracellular accumulation of
harmful levels of reactive oxygen species (ROS) resulting in
oxidative stress. These substances are potent oxidizing
agents reacting nonspecically and rapidly with macromol-
ecules, resulting in DNA mutations, protein oxidations and
lipid peroxidation. The cellular defence system against ROS
includes antioxidant enzymes such as catalases (CAT, EC
1.11.1.6) that detoxify the H
2
O
2
-producing molecular oxy-
gen and water (Aguirre et al. 2005; Heller and Tudzynski
2011). An antioxidant system based on the tripeptide gluta-
thione (GSH) is also involved in H
2
O
2
scavenging. In this
system, GSH is oxidized by H
2
O
2
to water by glutathione
peroxidases (GPx, EC 1.11.1.9) forming GSSG. The GSH is
regenerated by NADPH-dependent glutathione reductases
(GR, EC 1.6.4.2) by reduction of the GSSG (Pocsi et al.
2004). Therefore, the enhanced expression of antioxidant
enzymes such as CAT and GR is considered an important
oxidative stress marker.
This work aimed to evaluate the antimicrobial potential
of C. sylvestris and C. decandra extracts to control the
white-rot wood decay basidiomycetes T. villosa and
P. sanguineus. In addition, whether the extracts trigger a
process of oxidative stress was veried.
Results and discussion
The gas chromatographymass spectrometry (GC-MS)
analysis of the C. sylvestris extract detected 10 major
peaks (Table 1). It was not possible to identify the second
chromatographic peak at retention time (Rt) of 195 min
based on the NIST library; however, according to the lit-
erature, the ion fragments can be attributed to clerodane
diterpenoids with b-substituted furan ring. The base peak
at m/z = 82 can indicate a break in the beta position to
the furan ring (Wagner et al. 1978), suggesting that the
compound is hardwickiic acid (Santos et al. 2007). Fifteen
compounds were identied in the C. decandra extract,
including hydroquinone, b-sitosterol and cinnamic acid
(Table 1). The presence of large amount of sugars in the
extracts can be justied by the extraction method
employed. Phytochemical studies on C. decandra are
scarce, and their antimicrobial activity was unknown.
The biomass production by T. villosa and P. sanguineus
was signicantly inhibited from the rst 4 days of
exposure to the C. sylvestris and C. decandra extracts at
Table 1 Compounds identied in the Casearia sylvestris and Casearia decandra extracts according to the NIST library
Peak
Casearia sylvestris Casearia decandra
Rt (min) m/z* Possible compound MW** Rt (min) m/z* Possible compound MW**
1 1516 147 Trimethylsilyl ether glycerol 308 1523 147 Trimethylsilyl ether glycerol 308
2 1949 82 Not identied 1681 147 Propanoic acid 322
3 2085 147 Acid malic 350 1843 239 Hydroquinone 254
4 2644 204 Arabitol 512 2676 75 D-Fructose 569
5 2838 217 D-Ribofuranose 438 2770 217 Arabinofuranose 438
6 2823 217 D-Ribofuranose 438 2835 217 b-D-Galactofuranose 540
7 2939 345 Not identied 2850 217 D-Ribofuranose 438
8 2995 103 Sorbitol 614 2909 147 D-Erythrotetrofuranose 336
9 3216 147 D-Gluconic acid 628 3007 204 Arabitol 512
10 3365 147 Myo-inositol 612 3189 204 Glucopyranose 540
11 3375 147 Myo-inositol 612
12 3418 396 Cinnamic acid 396
13 4196 182 Glycosylated hydroquinone 560
14 4979 396 Cinnamic acid 396
15 5131 129 b-sitosterol 486
* Ion fragment attributed to the main peak of trimethylsilyl ether derivatives.
** Molecular weight of silanized compounds as proposed by the NIST library.
2 Letters in Applied Microbiology 2013 The Society for Applied Microbiology
Inhibition and oxidative stress in wood decay fungi T.S. Bento et al.
01 mg ml
1
(Fig. 1). However, P. sanguineus showed
more susceptibility to the plant extracts as their biomass
was reduced around 87% at the end of 16 days of culture,
whereas the biomass production by T. villosa was reduced
by 70% in this same period.
Extracts of ve Casearia species showed antimicrobial
activity against the human pathogens Staphylococcus
aureus, Escherichia coli, Pseudomonas aeruginosa and Can-
dida albicans (Mosaddik et al. 2004). According to the
authors, the Casearia sp and Casearia grewiifolia extracts
had the highest antimicrobial activity with minimal inhib-
itory concentration (MIC) corresponding to >5 mg ml
1
,
concentration 50-folds higher than that employed in this
study to control the basidiomycetes.
Clerodane diterpenoids identied in the C. sylvestris
extract is a class of secondary metabolites widespread in
some Casearia species and has been associated with several
medicinal properties (Ferreira et al. 2011). Oberlies et al.
(2002) isolated from leaves and twigs of C. sylvestris three
clerodane diterpenoids denominated casearvestrins with
cytotoxicity activity on tumour cell lines and antifungal
activity on Aspergillus niger. The antimicrobial activity of
cinnamic acid, hydroquinone and b-sitosterol, compounds
identied in the C. decandra extract, has also been demon-
strated in some studies (Amoroso et al. 2009; Sova 2012;
Wong et al. 2012). A wood preservative formulation based
on cinnamic acid at 15% protected wood samples against
Trametes versicolor for 3 months (Kartal et al. 2006).
The lesser susceptibility of T. villosa to the Casearia
extracts could be explained at least in part by their higher
ligninolytic potential compared with P. sanguineus. More-
ira-Neto et al. (2013) veried that T. villosa produced
high levels of ligninolytic enzymes while P. sanguineus
produced only low levels of laccase. The presence of aro-
matic compounds such as hydroquinone in C. decandra
extract could have increased the expression of ligninolytic
enzymes by T. villosa, which oxidized these compounds.
Elisashvili et al. (2010) observed increased production of
laccase by T. versicolor in hydroquinone-supplemented
medium. According to Voda et al. (2003), T. versicolor
was less susceptible to antifungal phenols and aromatic
compounds than Coniophora puteana. As a brown wood
4
3
B
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o
m
a
s
s

(
g
)
B
i
o
m
a
s
s

(
g
)
2
1
0
ns
ns
ns
ns
4
3
2
1
0
0 4
Time (days)
8 12 16 0 4
Time (days)
8 12 16
(a) (b)
(c) (d)
Figure 1 Biomass production by the fungi (a) Trametes villosa and (b) Pycnoporus sanguineus exposed to Casearia sylvestris, and by (c) Trametes
villosa and (d) Pycnoporus sanguineus exposed to Casearia decandra. The time 0 indicates the moment when the 4-day-old cultures were trans-
ferred to the plant extract-supplemented PDA. Values are means of three replicates (SD). ns = nonsignicant difference at P < 005, Tukeys
test. ( ) Control and ( ) 01 mg ml
1
.
Letters in Applied Microbiology 2013 The Society for Applied Microbiology 3
T.S. Bento et al. Inhibition and oxidative stress in wood decay fungi
decay fungus, C. puteana produces less extracellular lac-
case, reducing their capacity to oxidize phenolic and other
aromatic antimicrobial compounds. Induction of laccase
by aromatic compounds has been considered a protective
response in fungi against toxic compounds produced
during the lignin degradation (Souza et al. 2004).
The Fig. 2 shows the light microscopy analysis of the
hyphae of T. villosa and P. sanguineus exposed for 4 days
to the Casearia extracts. The C. sylvestris extract caused
the major changes in the hyphal morphology of both
fungi. The exposure to the extract resulted in hyphae
more branched and segmented and the formation of
defective clamp connections. The C. decandra extract does
not cause perceptible modications in T. villosa; however,
the plant extract induced the formation of clamp connec-
tions and decreased the hyphal winding in P. sanguineus.
Signicant changes were not observed in the average
thickness of the hyphae (375 lm) in the fungi exposed
to the extracts. Changes in the hyphal morphology may
suggest the cell wall as a target of antifungal compounds.
According to Amoroso et al. (2009), hydroquinone
caused damage in up to 55% of the cells of the basidio-
mycete Ceriporiopsis subvermispora. The transmission elec-
tron microscopy showed the formation of microvesicles
and extensive cytoplasm degeneration. The Echinacea sp
extract inhibited 10 genes involved in cell wall integrity
and structure of Saccharomyces cerevisiae and the uores-
cence microscopy showed more susceptibility to cell wall
damage (Mir-Rashed et al. 2010).
As the knowledge on antifungal action mechanisms of
plant extracts is relatively limited, the present work inves-
tigated the oxidative stress in the fungi exposed to the
Casearia extracts employing the enzymes CAT and GR as
oxidative stress markers. Antioxidative response to several
chemical and physical factors had been studied in bacteria,
animal and plant cells and yeasts. However, information
(a) (d)
(b) (e)
(c) (f)
Figure 2 Light microscopy analysis of the fungi exposed for 4 days to the plant extracts (400 9 magnication, scale bar = 20 lm). (a) Trametes
villosa control, (b) Trametes villosa exposed to Casearia sylvestris extract, (c) Trametes villosa exposed to Casearia decandra extract, (d) Pycnopo-
rus sanguineus control, (e) Pycnoporus sanguineus exposed to Casearia sylvestris extract and (f) Pycnoporus sanguineus exposed to Casearia dec-
andra extract.
4 Letters in Applied Microbiology 2013 The Society for Applied Microbiology
Inhibition and oxidative stress in wood decay fungi T.S. Bento et al.
on lamentous fungi is still scarce with most of the studies
developed in Neurospora crassa and A. niger.
The CAT activity in T. villosa and P. sanguineus
increased after exposure to the C. sylvestris extract
(Fig. 3ab). The extract increased the CAT activity in
T. villosa after 4 and 8 days; however, the enzyme
activity decreased considerably after 12 and 16 days
reaching values compatible to the fungi in normal con-
ditions. The CAT activity in P. sanguineus increased
after 8 and 12 days, but decreased thereafter. The effect
of the C. decandra extract on CAT activity was less evi-
dent (Fig. 3cd). The extract inhibited signicantly the
CAT activity in T. villosa after 4 and 8 days, whereas in
P. sanguineus the enzyme activity increased after 8 days,
but thereafter the enzyme activity was not signicantly
affected.
The GR activity in T. villosa was inhibited by C. sylves-
tris extract; however, the activity increased in P. sanguin-
eus after 8 and 12 days (Fig. 4ab). The C. decandra
extract increased the GR activity in T. villosa, while in
P. sanguineus the enzymatic levels increased signicantly
only after 12 days and decreased thereafter to normal
levels (Fig. 4cd).
The CAT in wood decay fungi is important to avoid
harmful ROS levels during the lignin breakdown, a highly
oxidative process due to production of aromatic
compounds and H
2
O
2
(Rabinovich et al. 2004). The over-
all increased CAT activity after exposure to the Casearia
extracts indicates oxidative stress process due to H
2
O
2
for-
mation as CAT is directly responsible for their detoxica-
tion. In addition, GSH-dependent enzymes have also been
associated with peroxides detoxication and in the resto-
ration of the cellular GSH/GSSG balance. However, studies
on the GSH pathway in lamentous fungi are scarce, and
the results have been unclear or contradictory (Li et al.
2008). Aromatic compounds such as hydroquinone in the
C. decandra extract could have caused the oxidative stress
in the fungi. The exposure of C. subvermispora to hydro-
quinone resulted in decreased GSH/GSSG ratio and
increased glutathione-S-transferase mRNA levels, indicat-
ing oxidative stress (Amoroso et al. 2009).
Therefore, the extracts were able to reduce the develop-
ment of T. villosa and P. sanguineus and triggered a pro-
cess of oxidative stress. Although the accumulation of
toxic levels of ROS could be a possible mechanism of
action, other mechanisms must be investigated to better
1600
1200
800
400
S
p
e
c
i
f
i
c

a
c
t
i
v
i
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y

(
U

m
g

1

p
r
o
t
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)
S
p
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(
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)
0
1600
1200
800
400
0
4
Time (days)
8 12
ns
ns
ns
ns ns
ns
ns
ns
16 4
Time (days)
8 12 16
(a) (b)
(c) (d)
Figure 3 Catalase activity in the fungi (a) Trametes villosa and (b) Pycnoporus sanguineus exposed to Casearia sylvestris and in (c) Trametes villosa
and (d) Pycnoporus sanguineus exposed to Casearia decandra. Values are means of three replicates (SD). ns = nonsignicant difference at
P < 005, Tukeys test. ( ) Control and ( ) 01 mg ml
1
.
Letters in Applied Microbiology 2013 The Society for Applied Microbiology 5
T.S. Bento et al. Inhibition and oxidative stress in wood decay fungi
comprehend the inhibitory activity of plant extracts and
to develop environmentally safer products to control
wood decay fungi.
Materials and methods
Plant extracts and chemical analysis
The ethanolic extracts of leaves of Casearia sylvestris
Swartz (RM17) and C. decandra Jacq (M742) were
obtained from the plant extract bank located in the
Center of Plant Physiology and Biochemistry of the
Institute of Botany of S~ao Paulo, Brazil.
After derivatization with methoxyamine hydrochloride
in pyridine, and N,O-bis(trimethylsilyl)triuoroacetamide
(BSTFA) with 1% trimethylchlorosilane (TMCS), as
described by Roessner et al. (2001), the samples were analy-
sed by GC-MS employing a gas chromatograph Agilent
6890 Series GC equipped with a quadrupole mass spec-
trometer Agilent 5973N MSD (Agilent Technologies, Santa
Clara, CA, USA). The total ion chromatograms and mass
spectra were evaluated using the program Chem Station
(Agilent Technologies). The detected peaks were compared
with data from the library NIST 08 Mass Spectral Library.
Antifungal activity
The basidiomycetes Trametes villosa CCIBT2628 and
Pycnoporus sanguineus CCIBT3732 were obtained from
the Culture Collection of the Institute of Botany of S~ao
Paulo, Brazil. To obtain the inoculum, the fungi were cul-
tured in Potato Dextrose Agar (PDA) for 7 days at
28 2C. An agar plug (4 mm diameter) containing the
mycelium was transferred to a 9-cm-diameter cellophane
membrane-covered PDA and kept at 28 2C until the
fungal colony reach about 3/4 of the plate (4 days).
Thereafter, the cellophane membranes containing the fun-
gal colony were aseptically removed and transferred to
new plates containing PDA supplemented with the etha-
nol extracts at nal concentration of 01 mg ml
1
and
maintained at 28 2C. Every 4 days for 16 days, sample
plates were collected and the mycelium harvested to bio-
mass determination (fresh weight) and enzyme extraction.
The experiment was carried out in triplicate.
Morphological analysis
Samples were collected from the edge of the fungal col-
ony with a dissecting needle and placed on a microscope
100
80
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40
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80
60
40
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4
Time (days)
8
ns
ns
ns
ns
ns
ns
ns
ns
12 16 4
Time (days)
8 12 16
(a) (b)
(c) (d)
Figure 4 Glutathione reductase activity in the fungi (a) Trametes villosa and (b) Pycnoporus sanguineus exposed to Casearia sylvestris, and in (c)
Trametes villosa and (d) Pycnoporus sanguineus exposed to Casearia decandra. Values are means of three replicates (SD). ns = nonsignicant
difference at P < 005, Tukeys test. ( ) Control and ( ) 01 mg ml
1
.
6 Letters in Applied Microbiology 2013 The Society for Applied Microbiology
Inhibition and oxidative stress in wood decay fungi T.S. Bento et al.
slide containing a drop of 3% (w/v) KOH combined
with a drop of 1% (w/v) phloxine. The material was
examined in an optical microscope Leica DM 1000,
using the program EZ-LAS. The microscopic structures
of the mycelium were observed according to Nobles
(1948).
Protein extraction and enzymatic assays
The frozen mycelia were grounded in mortar with liquid
nitrogen and added 100 mmol l
1
potassium phosphate
buffer (pH 75) containing 1 mmol l
1
EDTA and
3 mmol l
1
dithiothreitol (5 ml g
1
mycelium). The
homogenates were centrifuged at 15 000 g (20 min at 4C),
and the supernatant was collected and stored at 20C.
The protein concentration was quantied according to
Bradford (1976).
The CAT activity was determined using a reaction mix-
ture containing 29 ml 0036% (v/v) H
2
O
2
in
100 mmol l
1
sodium phosphate buffer (pH 68). After
addition of 01 ml of protein extract, the H
2
O
2
decompo-
sition was monitored at 240 nm for 1 min at 25C. The
specic enzyme activity was expressed in U mg
1
protein,
where one unit (U) corresponds to the decomposition of
1 lmol of H
2
O
2
per min under the assay conditions
(Beers and Sizer 1952).
The GR activity was determined using a reaction mix-
ture consisting of 1 ml of 100 mmol l
1
potassium
phosphate buffer (pH 75), 05 ml of 3 mmol l
1
DTNB
[5,5-Dithiobis (2-nitrobenzoic acid)], 01 ml of
2 mmol l
1
GSSG and 01 ml of 2 mmol l
1
NADPH.
After addition of 01 ml of protein extract, the GSSG
reduction was monitored at 412 nm for 1 min at 30C.
The specic enzyme activity was expressed in U mg
1
protein, where one unit (U) corresponds to 1 lmol of
reduced GSSG per min under the assay conditions
(Azevedo et al. 1998).
Statistical analysis
Data were analysed by analysis of variance (ANOVA) using
Tukeys test at P < 005 to examine signicant differences
between treatments. All results were expressed as
means standard deviation (SD).
Acknowledgements
This research was supported by CAPES (Coordination for
Enhancement of Higher Education Personnel).
Conict of Interest
The authors declare no conict of interest.
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