Letters in Applied Microbiology 1998, 26, 363366

Inuence of quercetin, a bioavonoid, on the toxicity of

copper to Fusarium culmorum
S.H. Park, C.W. Choi
1
, K.-S. Lee
1
and C.-J. Kim
Korea Research Institute of Bioscience and Biotechnology, Yusong, and
1
Department of Biology, PaiChai University,
Taejon, Korea
1680/97: received 24 October 1997 and accepted 2 February 1998
S. H. PARK, C. W. CHOI , K. - S. LEE AND C. -J. KI M. 1998. The effect of quercetin on copper
toxicity to the mycelial growth of Fusarium culmorum was investigated. Increasing
concentrations of copper produced dose-dependent inhibition in yeast extract and malt
extract agar. However, the toxic level of copper against fungal growth was signicantly
affected by the concentration of yeast extract in the medium, compared to that of malt
extract. Apart from the difference in toxic level of copper, the addition of quercetin
antagonized copper toxicity to hyphae morphology and resulted in the reversal of fungal
growth inhibition. Quercetin showed a protective ability similar to citrate, and was more
effective than acetate and proline.
I NTRODUCTI ON
Copper is an essential mineral nutrient which is required at
low concentrations for normal growth and development of
living organisms. However, copper present at higher levels
in its free ionic form (Cu
2
) is detrimental to all types of
cells. In a wide range of micro-organisms, copper shows, in
vitro, very high antimicrobial activity (Gadd and Grifth
1978 ; Gadd 1993). Therefore, copper-containing com-
pounds, including cupric hydroxide, Bordeaux mixture, and
various formulations of basic copper sulphate, were among
the rst biocides and have been used for the control of phyto-
pathogenic fungi and bacteria.
On the other hand, organically complexed copper is rela-
tively non-toxic. Strong chelating agents can markedly reduce
copper toxicity, by binding part of the available copper, and
eventually, protect against the cytotoxicity of copper in the
living organisms (Cervantes and Gutierrez-Corona 1994).
Many reports have shown that binding of copper to environ-
mental constituents such as organic materials has considerable
effects on the biological availability of copper and reduces
the toxicity of copper towards micro-organisms (Gadd and
Grifth 1978 ; Gadd 1993). A large number of organic sub-
stances have been described from plants, including sugars,
organic and amino acids, phenolics and vitamins. Thus, some
or all of the organic compounds found in plants could greatly
Correspondence to: Dr C. W. Choi, Department of Biology, Pai Chai
University, 439-6 Doma 2-dong, Seo-gu, Taejon 302-735, Korea
(e-mail : choicw@woonam.paichai.ac.kr).
1998 The Society for Applied Microbiology
affect the apparent toxicity of copper compounds toward
micro-organisms associated with living plants or their debris
in nature.
Flavonoids are phenolics which occur widely in the plant
kingdom. A variety of avonoids has been reported to have
antioxidant properties, to inhibit several enzymes, and to
possess antimicrobial activities (Harborn 1994). Besides other
biological activities, they have also been shown to chelate
metal ions (Thompson et al. 1976 ; Harborn 1994). There are
many reports of their chemical and physical properties for
metal chelating (Thompson et al. 1976 ; Bors et al. 1993).
However, no information is available on the effect of avo-
noids on the toxicity of copper to growth of fungal species in
the ecological niche.
The objective of this study was to conrm that quercetin
(3,5,7,3?,4?-penta hydroxyavonone), one of the most abun-
dant and naturally occurring avonoids, protects a plant
pathogenic fungus, Fusarium culmorum, from toxic copper in
vitro.
MATERI ALS AND METHODS
Fusarium culmorum, a plant pathogenic fungus, was grown
and maintained on potato dextrose agar at 25 C. Potato,
malt and yeast extract were obtained from Difco. Quercetin,
sodium citrate, sodium acetate, proline and cupric sulphate
(CuSO
4
.5H
2
O) were from Sigma Chemical Co.
The actual level of metals that allows optimal fungal growth
depends not only on the fungal species but also on the med-
364 S. H. PARK ET AL.
ium used because metals bind to organic and inorganic con-
stituents of the medium (Gadd 1993). Therefore, in order to
investigate which concentration of copper will produce an
optimum level of inuence on fungal growth, the sensitivity
of the fungus to copper was tested by assessing growth at
25 C on yeast extract and malt extract agar amended with
different concentrations of copper. The cupric sulphate was
sterilized separately in a small volume of distilled water added
to each medium autoclaved at 120 C for 15 min. A sterile
stock solution of quercetin was prepared by dissolving the
appropriate amounts of compound in 2 ml of 100% ethanol.
Sonication and heating (Rivera-Vargas et al. 1993) were often
used to dissolve quercetin completely. Equal volumes of
diluted compounds were added to sterile warm (55 C) agar
to give various concentrations. The control contained the
medium with ethanol alone. Compound amended agar media
were dispersed into 85 cm diameter plastic Petri plates (20
ml dish
1
). Inocula consisted of plugs (7 mm diameter) taken
from the edge of actively growing colonies and inverted on
the agar. Each experiment was carried out with three to ve
replicates, and mycelial growth was determined by measuring
the colony diameter. The plates were examined micro-
scopically using a light microscope to investigate the morph-
ological alteration of hyphae.
RESULTS AND DI SCUSSI ON
Four and ve days after treatment, culture of the fungus with
increasing concentrations of copper indicated that copper
produced dose-dependent inhibition in yeast extract and malt
extract medium, respectively. However, the toxic levels of
copper to fungal growth were signicantly affected by the
amounts of yeast extract. Copper at 03 mmol l
1
was suf-
cient to inhibit half the mycelial growth compared to the
control in the medium containing 01% extract, whereas a
much higher level (10 mmol l
1
) of copper was required to
inhibit that of fungal growth in the mediumcontaining 025%
extract (Fig. 1). Copper at 20 mmol l
1
was therefore not
inhibitory to fungal growth in the medium containing 05%
or more of yeast extract (data not shown). In contrast, the
toxicity of copper to fungus on malt extract agar was slightly
affected by the amounts of extract added. The inhibitory
doses of copper to F. culmorum grown on malt agar were
similar to those of copper-sensitive fungi reported by Baath
(1991). The difference between inhibitory doses on malt and
yeast extract could be attributed to the fact that yeast extract
contains more diverse organic constituents, such as amino
acids, organic acids and vitamins, than malt extract. There-
fore, the inhibitory doses of copper are only valid for two of
the media used in this experiment.
Copper is considerably less toxic to living cells when pre-
sent as complexes. If the inhibition of growth induced by
copper is due to its interference with normal metabolism
1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 363366
Fig. 1 Effect of copper on mycelial growth of Fusarium culmorum
in solid culture. The medium contained 01% yeast extract (R),
025% yeast extract (E), 05% malt extract () and 15% malt
extract ()
of the fungus, it would be possible to compensate for its
detrimental effect by the addition of exogenous copper chel-
ators. A variety of avonoids has been shown to chelate metal
ions, and there have been several reports on the mechanism
of transition metal chelating by the polyhydroxyl group of
avonoids (Harborn 1994). Discussion has mainly focused on
the fact that the 3-hydroxy-4-keto system in the C-ring and
the 5-hydroxy-4-keto group of quercetin form strong com-
plexes with copper, and an additional 3?,4?-dihydroxy system
in the B-ring functions as a chelation site (Thompson et al.
1976 ; Bors et al. 1993). Previously, Gadd and de Rome (1988)
reported that melanin could bind metals and determined a
binding afnity of L-Dopa (3,4 dihydroxy-phenylalanine) for
copper. Therefore, quercetin was used to model whether a
avonoid could protect the fungus from the toxicity of
copper. The level of copper was adjusted to an appropriate
concentration to show the inhibition of fungal growth in the
given medium because the actual level of free copper (Cu
2
)
was not determined. As anticipated, the reversal of inhibition
by quercetin could already be seen the rst day after addition.
The effect of copper on mycelial growth was reversed by the
addition of an increasing amount of quercetin in both malt
and yeast extract media (Fig. 2b). However, quercetin by
itself did not affect the growth of the fungus at 1 mmol l
1
concentration in both media.
Qualitative light microscope observation revealed that the
hyphae of fungus grown on media containing copper were
EFFECT OF QUERCETI N ON COPPER TOXI CI TY 365
Fig. 2 Inuence of quercetin on the toxicity of copper to mycelial growth of Fusarium culmorum. (a) The medium contained 01%
yeast extract with copper (05 mmol l
1
) ; water-treated control (), quercetin at 0 (), 025 (T), 05 (t) and 075 mmol l
1
(), and
quercetin (1 mmol l
1
) alone (). (b) The medium contained 15% malt extract with copper (025% mmol l
1
) ; water-treated control
(), quercetin at 0 (), 01 (T), 025 (t) and 05 mmol l
1
(), and quercetin (1 mmol l
1
) alone ()
usually swollen and twisted. The mycelium was scant and
chlamydospores were also observed. However, the hyphae of
fungus grown on the mixture of copper and quercetin, and
quercetin alone, showed the same type of mycelium and
hyphae as the normal control (data not shown).
The results suggest that the negative effect on growth
induced by quercetin is a direct consequence of the depletion
of free copper (Cu
2
) in the medium. To conrm this obser-
vation, the efcacy of quercetin and some organic compounds
on the reversal of copper toxicity was compared. Acetate,
proline and citrate were chosen as examples of weak, moderate
and strong copper chelating ligands, respectively (Men-
kissoglu and Lindow 1991 ; Cabral 1994). As shown in Table
1, the addition of each compound conferred substantial pro-
tection against the inhibition of mycelial growth by copper.
It should be noted that the levels of quercetin and organic
compounds added to detoxify copper are not actual molar
concentrations because these compounds can form complexes
with the constituents of the medium. Nevertheless, quercetin
showed a similar protecting ability to citrate. Proline and
acetate did not reduce the toxicity of added copper as much
as the same concentrations of quercetin and citrate.
The results of this study indicate that quercetin anta-
gonizes copper toxicity in vitro regardless of the types of
medium on which the fungus is grown. Flavonoids are phenol
1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 363366
Table 1 Efcacy of quercetin and organic compounds on the
protection of copper toxicity to Fusarium culmorum

Mycelial growth (cm)

Compound added
(mmol l
1
) 15% ME 025% YE

Quercetin 025 462026

a
nd
b
05 522012 592015
10 nd 632016
Citrate 05 472023 552009
10 492014 632023
Proline 05 272015 422017
10 382024 542015
Acetate 05 272015 452021
10 292036 502013
Water 192031 382010

The agar medium contained 03 mmol l

1
and 125 mmol l
1
copper in the presence of malt extract (ME) and yeast extract
(YE), respectively.
a
Each value represents a mean2S.D. of four replicates.
b
Not determined.
366 S. H. PARK ET AL.
derivatives synthesized in substantial amounts (0515%)
and widely distributed in plants. More than 4000 individual
avonoids have been identied in higher and lower plants
(Harborn 1994). As less attention has been given to the poss-
ible interactions of avonoids with copper and the role in the
plant ecosystemof the complexes they form, it will be valuable
to compare the protection effects of several types of avonoids
on the toxicity of copper to various fungi. Also, a knowledge
of this type of detoxication mechanism may be useful when
considering the reason for failure of copper-based agro-
chemicals.
ACKNOWLEDGEMENTS
This work was nancially supported by the Research Grant
#295138-3, Technical Development Project of Agriculture
and Forestry from Ministry of Agriculture and Forestry.
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