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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
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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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8 Letters in Applied Microbiology 2013 The Society for Applied Microbiology
Inhibition and oxidative stress in wood decay fungi T.S. Bento et al.