J.

Phytopathology 146, 421-425 (1998)

1998 Blackwell Wissenschafts-Verlag, Berlin
ISSN 0931-1785

Unipersidade Federal de Vii;osa, Vi(;osa, MG, Brazil

In Vitro Effect of Plant Leaf Extracts on Mycelial Growth and Sclerotial

Germination of Sclerotium cepivorum
C. M. F. PINTO', L . A. MAFFIA-, V. W. D. CASALI' and A. A. CARDOSO'

Authors' addresses: 'EMBRAPA EPAMIG, Centro Teenologico da Zona da Mata de Minas Gerais, 36570-000, Vifosa,
MG, Brazil; "Universidade Federal de Vifosa, Departamento de Fitopatologia, 36571-000. Vi9osa, MG, Brazil;
'Universidade Federal de Vifosa, Departamento de Fitotecnia, 36571-000. Vifosa, MG, Brazil
With 2 figures
Received May 21. 1996; accepted January 27. 1998

Abstract
The effects of leaf extracts of Brachiaria hurtiidicola, Crofalaria paulina. Eucalyptus citriodora. and Brassica oleracea var. capitata on mycelial growth and sclerotial
germination of Sclerolium cepivorum were evaluated in
vitro. Water extracts of leaves had little effect on either
growth or germination. However, acetone-water leaf
extracts usually had the greatest inhibitory effects. Acetone-water extracts reduced mycelial growth more than
sclerotial germination. Leaf extracts of E. citriodora at
1000 and 10000p.p.m. in acetone-water completely
inhibited mycelial growth and sclerotial germination of
S. cepivorum. Thus, this extract is potentially useful for
the control of garlic white rot. and its effect will be evaluated under field conditions.

Zusammenfassung
Jn vitro EinfluB von Pfianzenextrakten auf das Myzelwachstum
und die Sklerotienbiidung von Sclerotium cepivorum

Untersucht wurden die Einflllsse von Blattextrakten von

Brachiaria humidicola, Crntalaria paulina.. Eucalyptus
citridora und Brassica oleracea var. capitata auf das
Myzelwachstum und die Sklerotienbiidung von Sclerotium cepivorum in vitro. Wasserextrakte der Blatter hatten
kaum EinfluB, weder auf das Wachstum noch auf die
Keimung. Am haufigsten hatten jedoch Aceton-Wasser
Blattextrakte die groflten hemmenden Effekte. Die Aceton-Wasser-Extrakte reduzierten das Myzelwachstum
starker als die Sklerotienkeimung. Die Blattextrakte von
E. citriodora verursachten eine voUige Hemmung des
Myzelwachstums und der Sklerotienkeimung von S. cepivorum bei einer Konzentration von 1000 bzw. 10000
ppm in Aceton-Wasser. Von daher hat dieses Extrakt
das Potential als BekampfungsmaBnahme gegen die
Mehlkrankheit in Knoblauch, und dessen Effekt wird
unter Freilandbedingungen beurteilt.

Introduction
Development of synthetic products to control plant diseases has become difficult because of strict requirements
of their efficacy, selectivity, toxicology, and general
impact on the environment (McLaren, 1986). Consequently, there is an increasing interest in evaluating other
mechanisms of control, including the effect of plant
metabolites on plant pathogens.
Secondary compounds, considered as final products of
plant metabolism or metabolite refuses, have important
ecological functions for the plants which synthesize them.
One of these functions is to protect the plants against
infection by pathogens (Whittaker and Fenny, 1971;
Swain, 1977; McLaren, 1986; Wink, 1988; Taiz and
Zeiger, 1991). Several authors including Pordesimo and
Hag (1976), Moore and Atkins (1977), Alfenas etal.
(1982), Rai and Tripathi (1984), Tewari and Dath (1984),
Ismail etal. (1988, 1989), and Ferracine etal. (1990) verified that most plant extracts have antifungal properties.
These properties depend on the plant organ used, fungal
species tested, solvent used for extraction, and compound
dose and structure. The mycelia! growth of Cylindrocladium clavatum, Fusarium moniliforme var. subgiutinans. Rhizoctonia solani, Giberella zeae, Alternaria
ziniae. Macrophomina pha.seolina, Fusarium oxrsporumf.
sp. ciceri and Sclerotinia sclerotiorum was considerably
reduced in vitro by extracts of garlic bulb and leaves
(Singh etal., 1979; Bolkan and Ribeiro, 1981; Chalfoun
and Carvalho, 1987a,b). According to Bastos (1992),
garlic-bulb extract at lOOOOp.p.m. in Potato Dextrose
Agar (PDA) completely inhibited mycelial growth, and
at 1000 p.p.m. inhibited the germination of basidiospores
and zoosporangia of Crinipellis pemiciosa and Phytophthora palmivora, respectively. The growth inhibition
of certain soil borne plant pathogens was attributed to
alicin, a volatile sulphur product found in garlic bulbs
(Ark and Thompson, 1959). Mycelial growth and scler-

L.S.Copyngh, ClearanceCemcrcode S,.,e,ne: 0931-1785/98/4609-0421 $ 14.00/0

422

otial germination of Sclerotium rolfsii were completely

inhibited by ethanolic extract of Chenopodium amhrozioides (Ferracini etal., 1990). Phenolic substances produced by Eucalyptus spp. and Laccaria laccaia inhibited
mycelial growth and spore germination of Cryphonectria
cubensis and Fusarium oxysporum (Alfenas etal., 1982;
Sylvia and Sinclair, 1983). Ismail etal. (1989) observed
total remission of sclerotial germination of Sclerotium
cepivorum by 10 p.p.m. of acetone-water leaf extract of
Eucalyptus rostrata.
White rot, caused by Sclerotium cepivorum, is an
important disease of garlic in Brazil. The usual practices
for the control of plant diseases have no effect on white
rot. There are limitations with the use of crop rotation
(Croweetal., 1980; CoJey-Smith etal., 1990), solarization
(Entwistle, 1990a), biological control (Oliveira etal.,
1984; Entwistle, 1990b; Brix and Zinkemagel, 1992).
resistance (Coley-Smith and Entwistle, 1988), or fungicides (Utkhede and Rahe, 1983; Coley-Smith, 1990; Stewart and FuUerton, 1990; Slade etal., 1992) to control
the disease.
Therefore, in the quest for alternative practices for
white rot management, it would be valuable to select
plants which produce active compounds against S. cepivorum. The first step is to test plant extracts against the
target organisms. Thus, the objective of this research
was to evaluate the effect of leaf extracts of Brachiaria
humidicota, Crotaiaria paulina. Eucalyptus citriodora and
Brassica oleracea var. capitata on mycelial growth and
sclerotial germination of S. cepivorum, in vitro.

PINTO et al.

Preparation of leaf extracts

Green leaves of brachiaria (B. himidicola), crotaiaria (C.
paulina), eucalyptus {E. citriodora), and cabbage (B. oleracea var. capitata), lOOg of each, were rinsed under tap
water and blended with 1000 ml of either distilled water
or acetone-water (9; 1, v/v). The extracts were filtered
through coarse filter paper. The filtrates from the acetone-water extracts were subjected to a Rotavapor (TECNAL, Piracicaba, SP. Brazil), to eliminate acetone
residues. The extracts were stored in dark bottles under
an Ni atmosphere in a refrigerator until used. The
extracts were sterilized by filtering through a Millipore*
membrane (0.45 fim) and then were mixed with PDA at
48"C to obtain concentrations of 100, 1000 and
10 000 p. p.m.
Evaluation of mycelial growth
The amended media were poured into 9-cm plates (20 ml
per plate). One 5-mm mycelial disc cut from a 7-dayold colony was seeded in the centre of each plate and
incubated at 2 0 C The experiment was conducted in a
completely randomized design, with six replications.
Each replication was represented by two culture plates.
The colony diameter was measured daily until the fourth
day of incubation, when mycelial growth covered the
surface of all cultures in the control treatment. Inhibition
of growth was calculated in relation to the growth in the
control, according to the equation proposed by
Sztejnbergetal. (1983):
% inhibition =

Materials and Methods

Sclerotia used for bioassay
Soil samples were collected to a depth of 20 cm in a
garlic-producing area infested with 5. cepivorum near
the community of Santo Antonio, Vif osa-MG. Sclerotia
were extracted from these samples using a modification
to the technique of Crowe etal. (1980), Resende and
Zambolim (1986), and Vimard etal. (1986). In the laboratory, a lOOg sample of soil was blended in 1000 ml of
water at a slow speed. The suspension was passed through
a 20-mesh sieve in tandem with a 60-mesh sieve. The
material retained on the 60-mesh sieve was rinsed with
tap water, and suspended in 300 ml of 2.5 M sucrose.
The suspension was agitated for 1 min and the floating
material was poured through a 60-mesh sieve, rinsed
under tap water, collected on a filter paper, and left to
dry under laboratory conditions. After 48 h, the material
on the filter paper was passed through a 60-mesh sieve
and the scierotia were separated from the plant and soil
residues with the aid of a stereoscopic microscope. The
sclerotia were surface disinfested (1.5% sodium hypochlorite, for 3 min) and transferred to a Petri dish containing PDA. After 15 days incubation at 20C, the fungal
colonies were removed and rinsed under tap water over
a 60-mesh sieve to leach most nutrients from the medium.
The sclerotia on the sieve were left to dry under laboratory conditions.

Diameter of treated colonv\

1
X 100.
Diameter of control colony j

Evaluation of sclerotial germination

Surface-sterilized sclerotia (1.5yo sodium hypoclorite for
3 min) were transferred to sterile filter paper to remove
excess moisture, inside a laminar flux chamber, and were
plated on the experimental media described above. Five
sclerotia, equally spaced, were placed in each plate containing medium with the respective extract and incubated
at 20 C. The experiment was conducted using a completely randomized design, with five replications (each
replication consisted of two plates). Sclerotial germination was evaluated daily until the fifth day, when
germination was 100% in the controls. The inhibition of
sclerotial germination was calculated according to the
equation proposed by Sztejnberg etal. (1983);
% inhibition =
Germination of treated sclerotia\
Germination of control sclerotia j

Results and Discussion

Water leaf-extract of crotaiaria inhibited mycelial growth
by 67, 53 and 27%, at 100,1000 and 10000p.p.m., respectively. Brachiaria extracts reduced growth by 64% at
10 000p.p.m., but at other concentrations the growth was
not affected. Aqueous leaf-extracts of eucalyptus and
cabbage did not inhibit mycelial growth of the fungus at

Effect of Plant Leaf Extracts on Mycelial Growth and Germination of Scleroiium cepivorum

423

100

g,
c

80-

.^

60

40

I
100

1000

10000

100

Concentration (PPM)
100-

minat Ion Inhlibition (%)

10080604020-

0100

1000

10000

BO'
60
40'
20'

B Eucalyptus M Cabbage [

11
1"

ffa.til.A

10000

Concentration (PPM)
I B Brachiaria B Crotalaria

1000

Concentration (PPM)

Concentration
100
1000(PPM) 10000

! 1 Brachiaria Q Crotalaria

Eucalyptus Cabbage

Fig. 1 Inhibition of mycelial growth o( Stierotium cepivorum. in culture

medium amended with water (A) or acetone-water (B) leaf extracts of
brachiaria, crotalaria, eucalypttis. and cabbage. Same letters over the
bars indicate no difference in the values for plant species at individual
concentrations (Tukey, P = 0.05)

Fig. 2 Inhibition of sclerotial germination of Sclerotium cepivorum in

cuiture medium amended with water (A) or acetone-water (B) leaf
extracts of brachiaria, crotalaria, eucalyptus, and cabbage. Same letters
over the bars indicate no difference in the valties for plant species at
indi\'idual concetrations (Tukey, P = 0.05)

any of the concentrations tested (Fig. lA). Acetonewater leaf-extract of eucalyptus inhibited mycelia! growth
by 36% at 1000 and 100% at lOOOOp.p.m., but no inhibition occurred at 100 p.p.m. Acetone-water leaf-extracts
of crotalaria inhibited growth by 58, 37, and 32%, at 100,
1000, and lOOOOp.p.m., respectively. The lOOOOp.p.m.
extracts of brachiaria and cabbage inhibited growth by
20 and 32%, respectively, but there were no effects at 100
and 1000p.p.m. with these latter two extracts (Fig. IB).
All water leaf-extracts had httle effect on sclerotial
germination. Even at 10 000 p.p.m., germination was only
reduced by 6-10% with any of the extracts (Fig.2A).
The acetone-water leaf-extract of eucalyptus completely
inhibited germination at 1000 and lOOOOp.p.m., whereas
those of other plant species had little effect on sclerotial
germination (Fig. 2B).
Acetone-water leaf-extracts of E. citriodora at 1000
and lOOOOp.p.m, completely inhibited mycehal growth
and sclerotial germination of S. cepivorum. Thus this
extract is potentially useful for the control of garlic white
rot. Salama etal, (1985) reported inhibitory effects of
phenolic compounds, such as salicylic and galic acids,
extracted from leaves of E. rostrata, on several fungi. Soil

amendment with eucalyptus leaves completely inhibited

sclerotial germination of S. cepivorum (Salama etal.,
1988). Ismail etal, (1989) concluded that three phenolic
compounds in the acetone-water extract of E. rostrata
were responsible for the inhibition of fungal growth. Citronellal, cineole, and limonene (Wink, 1988; Boland
et al,, 1991; Taiz and Zeiger, 1991) may have been responsible for the inhibitory effect of S. cepivorum in the present
research.
Aqueous leaf-extracts of E. citriodora had no effect
on mycehal growth nor on sclerotial germination of S.
cepivorum. Ismail etal. (1989) also observed no effect of
aqueous leaf-extract of E. rostrata on the growth of S.
cepivorum, but high inhibition was found with acetotiewater extract, which had higher concentrations of phenohc compounds.
Inhibition of mycelial growth of S, cepivorum with
brachiaria leaf extracts at lOOOOp.p.m. and negligible
inhibition of sclerotial germination probably were due to
saponins, Sonoda (1978) analysed aqueous extracts of
this platit and attributed the growth inhibition of S. rolfsii
to saponin. According to Bossahard (1992), saponin from
the aqueous leaf extract of Hedera helix was responsible

424

for inhibition of conidial germination and tnycelial

growth of Venturia inaequalis and Podosphaeria leucotricha.
Lewis and Papavizas (1971) concluded that mercaptans
in the cabbage leaf-extracts were responsible for the
inhibitory eflFects on sclerotial germitiation of S. cepivorum. Possibly these compounds also inhibited the
myceliai growth of S. cepivorum at 10000 p.p.m. with
cabbage leaf extract in this assay. However, it is important to consider that more than 20 sulphur-containing
volatile compounds are liberated in the soil when cabbage
residues are decomposing (Zavaleta-Mejia and Reyna.
1990).
The results of this study are promising. Considering
both the inhibitory effect of soil amendment with leaves
of E. rostrata (Salama etal., 1988) and toxicity of leaf
extracts of E. citriodora on the growth and sclerotia!
germination of S. cepivorum, the addition of leaves of E.
citriodora to soil may reduce the incidence of white rot in
garlic growing areas infested with 5*. cepivorum. However,
further studies are required to evaluate the effect under
field conditions. Chemical analysis of the leaf extracts of
E. citriodora is also needed to identify and purify the
active principals that are inhibitory to the myceliai
growth and sclerotial germination of S. cepivorum.

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