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BIOTROPIA NO. 21, 2003 : 19 - 31

ANTAGONISTIC EFFECT OF THREE FUNGAL ISOLATES TO
AFLATOXIN-PRODUCING^spergiY/HS/JavHS

OKKY SETYAWATI DHARMAPUTRA

SEAMED BIOTROP, P.O. BOX 116, Bogor, Indonesia and Faculty of Mathematics and
Natural Sciences, Bogor Agricultural University, Bogor, Indonesia

ASMARINA S.R. PUTRI, INA RETNOWATI and SANTIAMBARWATI
SEAMEO BIOTROP, P.O. BOX 116, Bogor, Indonesia

ABSTRACT

Aflatoxin contamination in preharvest peanuts can be controlled among others by using antagonistic fimgi
to aflatoxin-producing fungi. Aspergillus flavus is one of the fungal species where certain strains can produce
aflatoxin. Informations regarding the type of interactions between antagonistic fungi and toxigenic A. flavus, and the
effects of culture filtrates of the test fungi on the growth and aflatoxin production of toxigenic A. flavus are
necessary, before antagonistic fungi could be used as biocontrol agent. Three fungal isolates (nontoxigenic A.
flavus BIO 2127, A. niger BIO 2129 and Trichoderma harzianum BIO 19130) were tested for their antagonistic
properties against toxigenic A. flavus BIO 2132 using direct and indirect confrontation methods.

On direct confrontation method, four kinds of agar media were used, i.e PDA (Potato Dextrose Agar), MEA
1% (Malt Extract Agar 1%), SMKYA (Sucrose 200 g, MgSO47H2O 0.5 g, KNO3 3 g, yeast extract 7 g, and block agar
AA 20 g), and the mixture of MEA 1 % + SMKYA (1:1). The results indicated that the type of interactions between
toxigenic A. flavus either with nontoxigenic A. flavus or with T. harzianum was B type. In this type of interaction,
the growth of both toxigenic A. flavus and the test fungi inhibited each other (mutual inhibition) with the zone of
inhibition < 2 mm. Type of interaction between toxigenic A. flavus and A. niger depended on the kind of media. On
SMKYA and MEA 1% + SMKYA media, the interaction was B type, while on PDA and MEA 1% media it was
D type. In this D type of interaction, toxigenic A. flavus and A. niger inhibited each other (mutual inhibition) at a
distance > 2 mm.

Culture filtrates derived from nontoxigenic A. flavus and A. niger grown on ME 1%, SMKY and ME 1% +
SMKY inhibited the growth (based on dry weight) of toxigenic A. flavus, except culture filtrates derived from T.
harzianum grown on SMKY and ME 1% + SMKY media stimulated the growth of toxigenic A. flavus.

Culture filtrates of nontoxigenic A. flavus, A. niger and T. harzianum inhibited aflatoxin B\ production
of toxigenic A. flavus. Culture filtrates of A. niger and T. harzianum with conidial concentrations of IxlO6,
2xl06 and 3xl06 per ml inhibited aflatoxin B, production up to 100%. The percentage of inhibition of aflatoxin Bi
production increased with the increase of conidial concentrations of nontoxigenic A. flavus. The highest percentage
of inhibition of aflatoxin BI production (62.5%) was obtained from conidial concentration of 3xl06 per ml.

Aspergillus niger was the most potential fungus in inhibiting the growth of toxigenic A. flavus, either on
agar media or on culture filtrates of test fungi. Culture filtrate of A. niger was also the most potential filtrate in
inhibiting aflatoxin BI production of toxigenic A. flavus.

Keywords: Antagonistic effect / Aspergillus flavus I Aspergillus niger I Trichoderma harzianum I aflatoxin

BIOTROPIA NO. 21,2003

INTRODUCTION

Aflatoxin contamination in peanuts occurs when kernels become infected by Aspergillus
flavus, A. parasiticus and A. nomius, under drought stress before harvest, during the drying phase in
the field, or under unsuitable storage conditions. Pitt et al. (1991) reported that A. flavus (and A.
parasiticus) are able to grow as commensals in developing peanut plants, and from there the fungi can
invade developing peanuts.

Levels of 1000 ppb of aflatoxin could cause acute liver damage in man and animals. Lower
levels of aflatoxin in peanut products if consumed could cause liver cancer and premature death in
humans, as well as reducing productivity of livestock (Pitt and Hocking 1996).

According to Pitt and Hocking (1996), 45% of 215 peanut samples collected from retailers
in Bogor and Yogyakarta contained more than 50 ppb of aflatoxin, 33% more than 300 ppb, and
22% exceeding 1000 ppb. In foodstuff, the limit of aflatoxin content determined by U.S. Food
and Drug Administration was 20 ppb (Park 1993). Codex Alimentarius Commision adopted the
maximum level of total aflatoxin content in peanuts intended for further processing at 50 ppb
(FAO and WHO 1999).

During the past 20 years, a number of approaches have been advocated and tested for
reduction of aflatoxins in peanuts: resistant cultivars, thickened shells, waxy testa, improved
farm management techniques and postharvest procedures involving drying and storage. Some of
these approaches have merit, but despite the expenditure of large sums of research funding aflatoxin
in peanuts remains a serious commercial problem. Other approaches such as using antagonistic fungi
to aflatoxin-producing fungi are still needed (Pitt 1999).

One of the more promising method is the concept of biocontrol by competitive exclusion (Pitt
1999). This involves the use of competitive fungi to reduce the possibility of toxigenic fungi
present in the soil entering developing peanuts and then producing aflatoxins in them. According
to Dorner et al. (1992), nontoxigenic A. parasiticus can be used as biocompetitive agent to control
aflatoxin contamination in peanuts before harvest. Dharmaputra et al. (2001) reported that among
fungi isolated from the soil of peanut farms at Wonogiri regency (Central Java), nontoxigenic
A. flavus BIO 2127, A. niger BIO 2129 and Trichoderma harzianum BIO 19130 were antagonistic
to toxigenic A. flavus BIO 2128.

The objectives of this study were to get information on the type of interactions between each
of the three fungal isolates (nontoxigenic A. flavus, A. niger and T. harzianum) and toxigenic A.
flavus; and to investigate the effects of culture filtrates of the test fungi on the growth and aflatoxin
production of toxigenic A. flavus.

MATERIALS AND METHODS

Fungal isolates

Fungal isolates used were toxigenic A. flavus BIO 2132, isolated from peanut farms in Pati
regency, Central Java, in January 2002; nontoxigenic A. flavus BIO

Antagonistic effect of three fungal isolates - Okky S. Dharmaputra et al.

2127, A. niger BIO 2129 and T. harzianum BIO 19130, isolated from soils of peanut farms at
Wonogiri regency, Central Java, in May 2000 (Dharmaputra et al 2001). The four fungal isolates
belong to the Plant Pathology Laboratory Culture Collection, SEAMEO BIOTROP. Each fungal
isolate was subcultured on Potato Dextrose Agar (PDA) medium and incubated at room temperature
(28 ± 2°C) for 7 days.

Test of antagonism between three fungal isolates and toxigenic A. flavus

1. Direct confrontation between fungal colonies

Nontoxigenic A. flavus, A. niger and T. harzianum isolates were tested for their antagonistic
property against toxigenic A. flavus using direct opposition method (Dennis and Webster 1971) on
four agar media, i.e. PDA, ME A 1% (Malt Extract Agar 1%), SMKYA (Sucrose 200 g, MgSO4.7H
O 0.5 g, KNO3 3 g, yeast extract 7 g, block agar AA 20g) and the mixture of MEA 1% + SMKYA
(1:1). Toxigenic A. flavus (4 mm in diam) was placed on the four media in a Petri dish (diam 9
cm). At the same time at a distance of 3 cm from toxigenic A. flavus inoculum, each test fungus
was inoculated on the same dish. The plates were incubated at room temperature. Three
replications were used for each treatment. Observations were carried out seven days after
incubation on the inhibition of mycelial growth of toxigenic A. flavus by the test fungi using the
formula of Fokkema (1973):

r i - r 2
1= x l O O %
R1

where I = percentage of inhibition
f] = radius of toxigenic A. flavus away from the test fungi
r2 = radius of toxigenic A. flavus towards the test fungi

The type of interactions between toxigenic A. flavus and each of the test fungus was
determined based on Wheeler and Hocking (1993), adapted from Magan and Lacey (1984), as
presented in Table 1.

2. Indirect confrontation

2.1. Preparation of cultural filtrates of three test fungal isolates

Isolates of nontoxigenic A. flavus, A. niger and 7". harzianum were grown respectively on
PDA medium in Petri dishes (9 cm in diam) and incubated at room temperature for 7 days.
Fungal conidia were suspended in sterile distilled water. Two ml of each test fungal isolate
suspension with three conidial concentrations (IxlO6, 2xl06 and 3xl06 conidia/ml) were'grown
on 100 ml ME 1%, 100 ml SMKY, and 100 ml of the mixture of ME 1% + SMKY (1:1) liquid
media in 250 ml Erlenmeyer flask, respectively, and then incubated at room

BIOTROPIA NO .21,2003

Antagonistic effect of three fungal isolates - Okky S. Dharmaputra et al.

temperature for 14 days. The concentrations of conidia were determined by microscopically
counting the number of conidia using a haemacytometer. After incubation, the culture filtrates of
the fungi were separated from their colonies using sterile filter paper No. 1, followed by
sterilization of the culture filtrates using Millipore (0.45 jam pore size).

2.2. Effect of culture filtrates of test fungi on the growth and aflatoxin production of
toxigenic A. flavus

One ml of conidial suspension of toxigenic A. flavus ( I x l O 6 conidia/ml) was inoculated into 50
ml of sterilized culture filtrates derived from each treatment combination in a glass bottle volume
100 ml. They were then incubated at room temperature for 10 days. As control, toxigenic A. flavus
was grown on ME 1%, SMKY and in the mixture of ME 1% + SMKY liquid media. Three
replications were used for each treatment (including the control). Observations were made on the dry
weight of mycelia of toxigenic A. flavus and aflatoxin production. The dry weight of mycelia was
determined by drying the fungal colonies in an oven at 95°C until a constant weight was attained
(Gourama and Bullerman 1995). Aflatoxins were analyzed using a Thin Layer Chromatography
method (Bainton et al. 1980). In this case, aflatoxin in culture filtrates was extracted using
chloroform and identified by two dimensional TLC using standard comparison. Aflatoxin content
produced by the most potential test fungal isolate was confirmed using a High Performance Liquid
Chromatography method (Rodriguez and Mahoney 1994).

Statistical analysis

The data were analyzed using a completely randomized factorial design with 2 factors. The first
and the second factors were the isolates of test fungi (nontoxigenic A. flavus, A. niger, and T.
harzianum) and conidial concentration of test fungi (1x106, 2x106 and 3xl06 conidia/ml), respectively.

RESULTS AND DISCUSSION

Type of interaction and percentage of growth inhibition between toxigenic A. flavus and test
fungi on various agar media

Two types of interactions (B and D types) were found between each of the three test fungi and
toxigenic A. flavus after 7 days of incubation (Table 2). On the type of interaction B, the test fungi
and the toxigenic A. flavus inhibited each other's growth with a zone of inhibition < 2mm. This type
of interaction was found on the interaction between the toxigenic and the nontoxigenic A.flavus on
PDA, MEA 1%, SMKY A, and MEA 1% + SMKYA media; between the toxigenic A. flavus and

BIOTROPIA NO. 21,2003

A.niger on SMKYA and MEA 1% + SMKYA media; between the toxigenic A. flavus and T.
harzianum on PDA, MEA 1%, SMKYA, and MEA 1% + SMKYA media. On the D type of
interaction, the test fungi and the toxigenic A. flavus inhibited each other's growth with a zone of
inhibition > 2 mm. This type of interaction was found on the interaction between the toxigenic A.
flavus and A. niger on PDA and MEA 1% media. On the two media a zone of inhibition was
found between the colony of A. niger and toxigenic A. flavus. It was assumed that A. niger produced
antibiotic. According to Jeffries and Young (1994), production of extracellular metabolites (such
as antibiotics and lytic enzymes) was one of the mechanisms of antagonism between two fungal
isolates. Dharmaputra et al. (2001) reported that the type of interaction between toxigenic A. flavus
isolate 557 and T. harzianum, and between A. flavus isolate 102 and T. harzianum were A and B
types, respectively; between toxigenic A. flavus isolate 23j and nontoxigenic A. flavus isolate
18t was B type; between toxigenic A. flavus isolate 102 and A. niger was D type.

The kind of media did not give significant differences on the percentage of growth
inhibition (based on mycelial growth) of toxigenic A. flavus by the three test fungal isolates.
Nevertheless, the highest percentage of growth inhibition was found on SMKYA medium (43.77%)
(Table 3).

The test fungal isolates gave very significant differences on the percentage of growth
inhibition of toxigenic A. flavus on SMKYA medium. Trichoderma harzianum caused the highest
growth inhibition (49.09%) compared to toxigenic A. flavus on SMKYA medium (Table 4).

Antagonistic effect of three fungal isolates - Okky S. Dharmaputra et al

The effect of culture filtrate of test fungi on the growth and aflatoxin production of
toxigenic A. flavus

The effect of culture filtrates of nontoxigenic A. flavus and A. niger on the percentage of
growth inhibition of toxigenic A. flavus (based on the dry weight of mycelia) was not significantly
different. Nevertheless, the highest percentage of growth inhibition (69.44%) of toxigenic A.
flavus caused by the culture filtrate of nontoxigenic A. flavus was on ME 1%, while that of the
culture filtrate of A. niger (72.51%) was on SMKY (Table 5). It indicated that nontoxigenic A. flavus
was more competitive to toxigenic A. flavus on ME 1% medium compared with those of SMKY
and ME 1% + SMKY media, while A. niger was more competitive on SMKY medium compared
with those on ME 1% and ME 1% + SMKY media.

Culture filtrate derived from T. harzianum grown only on ME 1% medium inhibited the
growth of toxigenic A. flavus (63.64%), while on SMKY and ME 1% + SMKY media the filtrate
stimulated the growth of toxigenic A. flavus (38.0 and 31.58%, respectively) (Table 6). It indicated
that culture filtrate of T. harzianum was only effective in inhibiting the growth of toxigenic A. flavus
on ME 1% medium.

Using TLC method, only aflatoxin B, was detected by toxigenic A. flavus. As aflatoxin was
not produced on ME 1 % medium, consequently only SMKY and ME

BIOTROPIA NO. 21, 2003

1% + SMKY media were used to study their effects on the growth (based on dry weight of
mycelia) and aflatoxin production of toxigenic A. flavus.

The kind of culture filtrates of test fungi on the growth of toxigenic A. flavus on SMKY
media was significantly different. Conidial concentration of test fungi and the interaction between the
kind of the culture filtrates and conidial concentrations were not significantly different. The
percentage of growth inhibition of toxigenic A. flavus caused by culture filtrate of A. niger
(72.51%) was higher than that of nontoxigenic A. flavus (45.62%). On ME 1% + SMKY medium,
culture filtrates and conidial concentration of test fungi, and their interaction were not
significantly different. Nevertheless, the percentage of growth inhibition of toxigenic A. flavus
caused by culture filtrate of A. niger (68.68%) was higher than that of nontoxigenic A. flavus
(64.0%).

Antagonistic effect of three fungal isolates - Okky S. Dharmaputra et al.

The inhibition percentage of aflatoxin B, production caused by culture filtrate derived from
nontoxigenic A, flavus grown on SMKY medium (41.67%) was not significantly different with that
of ME 1% + SMKY medium (58.33%).

Culture filtrate derived from A. niger either grown on SMKY or ME 1% + SMKY media
completely inhibited aflatoxin production effectively (100%) (Table 7). Culture filtrate of T.
harzianum grown on the two media also completely inhibited aflatoxin BI production up to 100%
(Table 7), but also stimulated the growth of toxigenic A. flavus (Table 6).

The kind of culture filtrates derived from test fungi and conidial concentration of test fungi and
their interaction gave significantly different effects on the percent inhibition of aflatoxin
production using SMKY medium. On ME 1% + SMKY medium, the kind of culture filtrates of
the test fungi gave very significantly different effect on aflatoxin B) production, while conidial
concentrations and the interaction between kind of culture filtrates and conidial concentrations
were not significantly different.

Culture filtrates derived from A. niger with the concentrations of 1x106, 2xl06 and 3xl06
conidia/ml grown on SMKY medium inibited aflatoxin Bt production up to 100% (Table 8). The
percent inhibition of aflatoxin B, production increased with the increase of conidial concentrations
of nontoxigenic A. flavus. The culture filtrate of nontoxigenic A. flavus with the concentration of
1x106 conidia/ml was not significantly different with that of 2x106 conidia/ml against the inhibition
percentage of aflatoxin Bj production, while that of 3xl06 conidia/ml was significantly different.

The culture filtrate derived from A niger grown on ME 1% + SMKY medium inhibited
aflatoxin B] production higher (100.00%) than that of nontoxigenic A. flavus (56.25%).

Aflatoxins produced by the most potential test fungal isolate were confirmed by using High
Performance Liquid Chromatography (HPLC). Two kinds of aflatoxins (Bi and G|) were found
using this method.

BIOTROPIA NO. 21,2003

The results of test of antagonism using direct confrontation showed that SMKYA
medium was the best medium for antagonism study between toxigenic A. flavus either with
nontoxigenic A. flavus, A. niger or T. harzianum. This is because the highest percentage of growth
inhibition of toxigenic 'A. flavus caused by the three test fungal isolates occurred on SMKYA
medium (Table 3). On SMKYA medium the highest percentage of growth inhibition of toxigenic
A. flavus was caused by T. harzianum followed by A. niger, while the lowest was caused by
nontoxigenic A. flavus (Table 4). However, the results of test of antagonism study using
indirect confrontation showed that the culture filtrate derived from T. harzianum grown on SMKY
medium stimulated the growth of toxigenic A. flavus (Table 6). Therefore, A. niger was the most
potential test fungus in inhibiting the growth and aflatoxin B, production of toxigenic A. flavus,
either on agar media or on culture filtrates of the test fungi.

Confirmation of aflatoxin contents produced by toxigenic A. flavus grown on culture filtrate
of A. niger cultivated on SMKY and ME 1% + SMKY is presented in Table 9. The results showed
that there were differences in aflatoxin BI contents analyzed using TLC and HPLC methods,
because different methods could have different results. Nevertheless, based on the two
methods used, culture filtrates derived from A. niger cultivated either on SMKY or ME 1% +
SMKY liquid media with different conidial concentrations completely inhibited aflatoxin B]
production of toxigenic A. flavus.

Antagonistic effect of three fungal isolates - Okky S. Dharmaputra et al.

Table 9. Aflatoxin Bj contents produced by toxigenic A. flavus grown on culture filtrate derived from A. niger cultivated on
SMKY and ME 1% + SMKY liquid media with various conidial concentrations.

Aflatoxin Bl

content (ppb)

Conidial concen
tration of A. niger
(conidia/ml)

Media
TLC method HPLC method

1 x 106

Culture filtrate (using SMKY) 0 0

Control (SMKY) 160 97.57
Culture filtrate (using ME 1% + SMKY)

Control (ME 1% + SMKY) 120 60.79
2x10" Culture filtrate (using SMKY) 0 0

Control (SMKY) 160 98.89
Culture filtrate (using ME 1% + SMKY)

Control (ME 1% + SMKY) 120 69.37
3x 10' Culture filtrate (using SMKY) 0 0

Control (SMKY) 160 103.58
Culture filtrate (using ME 1 % + SMKY)

Control (ME 1% + SMKY) 120 73.68

TLC = Thin Layer Chromatography; HPLC = High Performance Liquid Chromatography

CONCLUSIONS

The type of interactions between toxigenic A. flavus either with nontoxigenic A. flavus or
with T. harzianum was B type, respectively. In this type of interaction, mutual inhibition on
contact or space between the test fungi and toxigenic A. flavus was small (< 2 mm). The type of
interactions between toxigenic A. flavus and A. niger depended on the kind of media. On SMKYA
and MEA 1% + SMKYA media, the interactions were B type, while on PDA and MEA 1% media,
it was D type. In this D type of interaction, toxigenic A. flavus and A. niger inhibited each other
(mutual inhibition) at a distance > 2mm.

Culture filtrates derived from nontoxigenic A. flavus and A. niger grown on ME 1%, SMKY
and ME 1% + SMKY inhibited mycelial growth (based on dry weight) of toxigenic A. flavus,
except those derived from T. harzianum grown on SMKY and ME 1% + SMKY media which
stimulated the growth of toxigenic A. flavus.

BIOTROPIA NO. 21,2003

The culture filtrates of nontoxigenic A. flavus, A. niger and T. harzianum inhibited aflatoxin
BI production of toxigenic A. flavus. The culture filtrates of A. niger and T. harzianum with the
conidial concentrations of 1x106, 2xl06 and 3xl06 per ml could inhibit aflatoxin B] production up to
100%. The percent inhibition of aflatoxin B, production increased with an increase of conidial
concentrations of nontoxigenic A. flavus. The highest percent inhibition of aflatoxin B\ production
(62.5%) was obtained from the conidial concentration of 3x106 per ml.

Aspergillus niger was the most potential fungus in inhibiting the growth of toxigenic A.
flavus, either on agar media or on culture filtrates of the test fungi. The culture filtrate of A. niger
was also the most potential filtrate in inhibiting aflatoxin BI production of toxigenic A. flavus.

The results of this study gave important informations on antagonistic effect of three fungal
isolates (nontoxigenic A. flavus, A. niger and T. harzianum) before they could be used to control
aflatoxin-producing A. flavus in peanuts grown under green-house and field conditions.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the financial support of the Government of Indonesia. We
also thank the technicians of the Plant Pathology Laboratory, SEAMEO BIOTROP, who have in
one way or another contributed to this research.

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