J[ Phytopathology 037\ 486488 "1999#

1999 Blackwell Wissenschafts!Verlag\ Berlin

ISSN 9820!0674
Biomed RRC\ Pai Chai University\ Taejon\ Korea
Comparative In~uence of Flavonoids on the Toxicity of Copper to Alternaria
alternata
S[ H[ PARK
0\1
\ O[ S[ KWON
1
\ C[ W[ CHOI
2
and C[!J[ KIM
1
Authors| addresses]
0
Research Center for Bio!medicinal Resources "Biomed RRC# and
2
Department of Biology\ Pai Chai
University\ Doma!1 dong\ Taejon 291624\ Korea^
1
Korea Research Institute of Bioscience and Biotechnology\ PO Box 004\
Yusong\ Taejon\ 294599\ Korea "correspondence to S[ H[ Park#
With one _gure
Received October 14\ 0888^ accepted April 19\ 1999
Keywords] Alternaria alternata\ copper toxicity\ ~avonoids\ protection
Abstract
Three ~avonols\ morin\ rutin and quercetin\ and a ~a!
vonone\ naringenin\ were in vitro tested for their in~uence
on the toxicity of copper to Alternaria alternata[ Copper
sulphate "CuSO
3
# displayed dose!dependent inhibition of
fungal growth\ resulting in a reduction in growth by one!
half at 9[96 mM[ The addition of quercetin and morin
conferred substantial protection against the inhibition of
fungal growth by copper\ whereas naringenin and rutin
o}ered protection less e}ectively[ Higher concentrations
of copper could overcome the ~avonoid e}ect\ but the
reduction of copper toxicity was more apparent in the
presence of quercetin than morin[ The metal chelating
ability of quercetin was highly speci_c for copper among
toxic metals such as Co\ Ni\ Zn\ and Cd[ The results show
that the protection e}ect of the ~avonoids against copper
toxicity is highly dependent on the keto function and
hydroxyl group substitution of the ~avonoid skeleton[
Zusammenfassung
Vergleich der Wirkung verschiedener Flavonoide auf die Toxizitat
von Kupfer gegenuber Alternaria alternata
Die drei Flavonole Morin\ Rutin und Quercetin sowie
das Flavonon Naringenin wurden in vitro hinsichtlich
ihres Ein~usses auf die Toxizitat von Kupfer gegenu ber
Alternaria alternata gepru ft[ Kupfersulfat "CuSO
3
#
bewirkte eine dosisabhangige Hemmung des
Pilzwachstums\ bei 9\96 mM kam es zu einer Halbierung
des Wachstums[ Der Zusatz von Quercetin und Morin
verlieh einen deutlichen Schutz gegen die Hemmung des
Pilzwachstums durch Kupfer\ Naringenin und Rutin
zeigten eine weniger ausgepragte schu tzende Wirkung[
Bei ho heren Kupferkonzentrationen war die Wirkung
der Flavonoide nicht so o}ensichtlich\ doch Quercetin
minderte die Toxizitat des Kupfers starker als Morin[
Wurden au)er Kupfer auch verschiedene andere toxische
Metalle angeboten\ zum Beispiel Co\ Ni\ Zn und Cd\
U[ S[ Copyright Clearance Center Code Statement] 98200674:1999:37019486 , 04[99:9
bildete Quercetin hochspezi_sch mit Cu Chelate[ Die
Ergebnisse zeigen\ da) die schu tzende Wirkung der Fla!
vonoide vor Kupfertoxizitat in hohem Ma)e von der
Substitution der Ketofunktion und der Hydroxylgruppe
des Flavonoidgeru sts abhangt[
Introduction
Copper has a wide spectrum of e}ectiveness against a
variety of micro!organisms and thus copper!containing
compounds such as Bordeaux mixture\ copper sulphate\
etc[\ have been used for the control of plant pathogenic
fungi and bacteria on most agricultural crops "Horsfall\
0845#[
Natural chelating agents can reduce copper toxicity\
by binding part of the available copper\ and protect
against the toxicity of copper towards bacteria and fungi
"Arman and Wain\ 0847^ Menkissoglu and Lindow\ 0880^
Gadd\ 0882^ Cabral\ 0883#[ Flavonoids are phenolics that
occur widely in the plant kingdom\ and many ~avonoids
are known to chelate metal ions "Thompson et al[\ 0865^
Harborne\ 0883#[ Recently\ it was demonstrated in fungal
culture systems that quercetin\ a bio~avonoid\ is able to
protect Fusarium culmorum from the toxicity of copper
"Park et al[\ 0887#[
To better understand the protection of copper toxicity
by ~avonoids\ four ~avonoids\ which are in common
in plants\ quercetin\ rutin\ naringenin and morin\ were
chosen and studied their in~uence on the toxicity of cop!
per to Alternaria alternata[ This fungus commonly occurs
on plant surfaces or in dying or dead tissues of plants
and various formulations containing copper are currently
applied to control the diseases caused by this and related
fungi[
Materials and Methods
Alternaria alternata KCTC 5994 was obtained from the
Korean Collection for Type Cultures\ Taejon\ Korea[
487 PARK et al[
Fig[ 0 E}ect of copper on mycelial growth of Alternaria alternata in
solid culture[ The medium contained 0[9) malt extract with copper at
9 "#\ 9[91 "#\ 9[92 "T#\ 9[94 "t#\ 9[0 "#\ and 9[04 mM "#
The ~avonoids "morin\ rutin\ naringenin\ and quercetin#\
and metals were obtained from Sigma "St[ Louis\ MO\
USA#[
The sensitivity of the fungus to copper was investigated
by assessing growth at 14>C on 0[9) malt extract agar[
Cupric sulfate "CuSO
3
4H
1
O# was sterilized separately in
a small volume of distilled water and a sterile stock solu!
tion of each ~avonoid was prepared by dissolving in
ethanol[ Rutin was aseptically dissolved in distilled water[
Equal volumes of ~avonoid and copper solution were
added to sterile warm "44>C# agar to give various con!
centrations[ The control contained the mediumwith etha!
nol alone[ Compound!amended agar media were
dispersed into 7[4 cm diameter plastic plates "19 ml per
dish#[ The inocula consisted of plugs "6 mm diameter#
taken from the edge of actively growing colonies and
inverted on the agar[ Each experiment was carried out in
three to four replicates[ The plates were examined daily
up to 4 days and the diameter of the growing colonies
was measured in two directions at right angles to each
other[ The inhibition of fungal growth was expressed as
the mycelial growth or the percentage of the mycelial
growth with the control colony[
Results and Discussion
When copper sulphate was added to the medium\ fungal
growth was slowed\ with increasing concentrations of
copper leading to increased inhibition of growth "Fig[ 0#[
From these data it is possible to estimate the dose of
copper that results in a 49)inhibition of the growth "See
the IC
49
value in Table 1#[
Table 0 shows the speci_c in~uence of each ~avonoid
on the toxicity of copper to the fungus[ For this particular
Table 0
E.cacy of ~avonoids on the protection of copper toxicity to Alternaria
alternata
*
Mycelial growth ") of control#
Compound added *
"9[1 mM# Without copper With copper
*
Quercetin 88
a
099
Morin 091 87
Naringenin 71 54
Rutin 093 50
Water 099 42
*
The agar medium contained 9[96 mM copper in the presence of 0[9)
malt extract
a
Each value represents an average of four replicates
purpose\ the level of copper was adjusted to a con!
centration to observe the recovery of the inhibition of
fungal growth by copper[ The addition of quercetin and
morin conferred substantial protection against the inhi!
bition of fungal growth by copper[ Rutin\ a rutinoside of
quercetin\ o}ered less e}ective protection[ Naringenin by
itself was an inhibitor of A[ alternata\ but could only
protect the fungus slightly[ Thus\ the reduced copper
toxicity on fungal growth in the presence of each ~a!
vonoid may be a direct consequence of the depletion of
free copper "Cu

# in the medium[
Because morin and quercetin acted as e.cient copper
antagonists\ the in~uence of these compounds at 9[1 mM
was further compared with increasing concentrations of
copper[ Both compounds protected the fungus com!
pletely at a lowlevel of copper[ Ahigher concentration of
copper overcame the ~avonoid e}ect\ but the protective
e}ect could still be observed[ Thus\ the percentage of
mycelial growth at 9[1 mM copper was 46\ 16\ and 07)
of the control in the presence of quercetin\ morin and
water alone\ respectively[
Morin is structurally similar to quercetin\ but has 1?\3?!
dihydroxy systemin the B!ring instead of 2?\3?!dihydroxy
system in quercetin[ The fact that rutin was not e}ective
also indicates that conjugation of a sugar moiety to the
2!hydroxy group in the C!ring of queretin reduces the
copper!chelation ability[ Thus\ quercetin that had the
greatest propensity for copper ion chelation is well
accorded with that and the 2!hydroxy!3!keto system in
the C!ring and 4!hydroxy!3!keto group of the ~avonoid
which form strong complexes with copper\ and that an
additional 2?\3?!dihydroxy systemin the B!ring also func!
tions as a chelation site "Thompson et al[\ 0865#[
Since divalent metals are toxic to fungi and some of
them are among the ingredients for fungicide formu!
lation\ quercetin was tested as a metal chelator "Table 1#[
All metals inhibited mycelial growth of A[ alternata at
low level and their toxicity was in the order of Cu
Ni CoCdZn[ However\ the addition of quercetin
at 9[4 mMcould protect the fungus and caused an increase
of the IC
49
value of copper\ but not a signi_cant increase
in the IC
49
value of the other metals\ indicating that
quercetin is more speci_c for copper than the other metals
tested[
488 In~uence of Flavonoids on Toxicity of Copper to Alternaria alternata
Table 1
In~uence of quercetin on the toxicity of metals to Alternaria alternata
*
IC
49
"mM#
*
Metal Without quercetin With quercetin
*
Cu 62 219
a
177 204
Zn 339 212 364 225
Cd 119 201 144 212
Ni 89 200 004 206
Co 034 209 078 202
*
IC
49
represents concentration of each metal in media giving 49)
reduction of mycelial growth of the fungus[ The concentration of quer!
cetin was 499 mM[
a
Each value represents a mean 2SD of four replicates[
As the presence of copper at higher levels in its free
ionic form "Cu

# is detrimental to living organisms\

binding of copper to organic materials can reduce copper
toxicity "Arman and Wain\ 0847^ Gadd\ 0882^ Cabral\
0883#[ Previously\ Menkissoglu and Lindow"0880# stated
that some or all of the organic compounds formed on
leaves greatly a}ect the toxicity of copper towards
Pseudomonas syrin`ae and other phytopathogens[ Fla!
vonoids are synthesized in substantial amounts "9[4
0[4)# in plants and various ~avonoids are accumulated
on leaf surfaces and exuded from the leaves "Valant!
Vetschera and Wollenweber\ 0877^ Bohm and Constant\
0889#[ Therefore\ some or all of the ~avonoids together
with other organic compounds in plants might a}ect the
apparent toxicity of copper[ More detailed studies are
needed to clarify under what conditions and to what
extent the ~avonoids are released\ and how copper actu!
ally interacts with ~avonoids in plants[
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