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New Biotechnology �Volume 28, Number 6 �October 2011 RESEARCH PAPER

Characterization of a fungistaticsubstance produced by Aspergillus flavusisolated from soil and its significance innatureYen-Ting Chen, Mei-Ju Lin, Ching-Hui Yang and Wen-Hsiung Ko

Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan

A fungus capable of using vegetable tissues for multiplication in soil was isolated and identified as

Aspergillus flavus based on morphological characteristics and sequence similarity of ITS and 28S. When

grown in liquid medium prepared from the same vegetable tissues used in soil amendment, the isolate of

A. flavus produced a substance capable of preventing disease development of black leaf spot of mustard

cabbage caused by Alternaria brassicicola and inhibiting the germination of A. brassicicola conidia. The

inhibitory substance was fungistatic, and was very stable under high temperature and high or low pH

value. It was soluble in ethanol or methanol, moderately soluble in water, and insoluble in acetone, ethyl

acetate or ether. The inhibitor is not a protein and has no charges on its molecule. This is the first

discovery of the production of a fungistatic substance by this deleterious fungus. Results from this study

suggest the possession of a strong competitive saprophytic ability by A. flavus, which in turn may

explain the widespread occurrence of this fungus in soils. Production of a fungistatic substance when A.

flavus was grown in medium prepared from vegetable tissues suggests the importance of antibiotic

production in its competitive saprophytic colonization of organic matters in soils.

IntroductionAspergillus flavus is an important deleterious fungus due to its

production of aflatoxins especially aflatoxin B1 which is the most

toxic and most potent naturally occurring carcinogen character-

ized [1,2]. It is also a common cause of invasive and non-invasive

aspergillosis in human and animals, and an opportunistic patho-

gen of many agricultural crops such as corn, cotton, peanuts as

well as tree nuts [3–5]. The fungus is found throughout the world

in soils including agricultural, forest, orchard, grassland and wet-

land soils [6,7]. It was also isolated from virgin as well as cultivated

desert soils [8–10]. The widespread occurrence of A. flavus in soil

has been associated with its ability to colonize plant debris in soil

[11–13]. However, little is known about the competitive sapro-

phytic ability [14] of this fungus in soils.

During our recent research, microorganisms capable of utilizing

vegetable tissues in soil for multiplication were selectively isolated.

Broth prepared from the same vegetable tissues was used to culti-

Corresponding author: Ko, W.-H. (kowh@dragon.nchu.edu.tw)

1871-6784/$ - see front matter � 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2011.02.005

vate these organisms. Triturated cultures were screened for ability

to control black leaf spot of mustard cabbage caused by Alternaria

brassicicola. The culture of a fungus, which was subsequently

identified as A. flavus, was found to be very effective in controlling

the disease and its extract was strongly inhibitory to the pathogen.

This is the first discovery of the production of a fungistatic sub-

stance by this important fungus.

In this report, we describe the isolation and identification of A.

flavus from soil, the production and characterization of the fungi-

static substance produced by this fungus and the significance of

the discovery in the explanation of its strong competitive sapro-

phytic ability in soil.

Materials and methodsIsolation of soil microorganismsSoil samples collected from six locations in central Taiwan were

taken from a depth of 0–10 cm, sifted and moistened to about 65%

water-holding capacity. Media for selective isolation of fungi,

bacteria and actinomycetes were prepared as previously described

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[15]. Approximately 1.3 g soil was mixed with 100 ml sterile dis-

tilled water in an Omni mixer chamber at 5000 rpm for 30 s. The

suspension was diluted to 10�4, 10�5 and 10�6 for fungi, 10�5, 10�6

and 10�7 for actinomycetes and 10�6, 10�7 and 10�8 for bacteria to

determine the dilution needed to obtain soil suspension essen-

tially free of each group of microorganisms. A 1-ml aliquot of

diluted soil suspension was mixed with 20 ml of a molten selective

medium at 458C in a Petri plate. Five plates were used for each

treatment.

Vegetables including fruit of tomato (Lycopersicon esculentum),

tubers of sweet potato (Ipomoea batatas), and leaves and stems of

spinach (Spinacia oleracea), ong choy (Ipomoea aquatica) and com-

mon purslane (Portulaca oleracea) were purchased from local mar-

ket and chopped into small pieces. For soil amendment, about

500 g soil was mixed with 4% of each chopped vegetable in a 1000-

ml bottle and incubated at 248C for at least two weeks before use.

To isolate microorganisms with ability to utilize amended nutri-

ents for multiplication, suspension of amended soil was diluted to

the concentration pre-determined for each group of microorgan-

isms and plated on the selective medium as described above. After

incubation at 248C for seven days, colonies appeared were indi-

vidually transferred to 10% V-8 agar (10% V-8 juice, 0.02% CaCO3

and 2% agar) plates.

Culture of isolated microorganisms in liquid mediumLiquid medium was prepared by triturating 4 g each of the five

chopped vegetables in 100 ml water in an Omni mixer at 4000 rpm

for 3 min and dispensing 50 ml broth in a 250-ml flask. After

autoclaving, each flask was inoculated with two loopfuls of bac-

terium, or a piece (ca. 4 � 5 � 3 mm) of actinomycete or fungus

agar culture. Inoculated flasks were incubated on a shaker for two

weeks. After incubation, cultures were separately ground in an

Omni mixer at 4000 rpm for 1 min before being used to test their

ability to reduce disease incidence.

Inoculum preparationA. brassicicola (Schw.) Wiltshire (isolate Aba-31) was grown on 10%

V-8 agar at 248C under light for four to six days for the production

of conidia. A conidial suspension was prepared by placing two

pieces of culture blocks (ca. 5 � 5 � 3 mm) in 5 ml sterile distilled

water in a test tube and by agitating the test tube for 30 s with a

Vortex mixer. The concentration of conidia was adjusted to 3

conidia ml�1 with a Pipetman microliter pipette (West Coast Scien-

tific, Oakland, CA) [16].

Disease control assay of liquid culturesSeeds of mustard cabbage [Brassica juncea (L.) Coss.] were grown in

8-cm pots containing a mixture of peat moss and vermiculite (9:1,

v/v). Two leaves of a four-week-old plant were sprayed to run off

with liquid culture of a test microorganism daily three times before

inoculation on the fourth day.

Each mustard cabbage leaf was inoculated with five 2-ml drops of

conidial suspension of A. brassicicola along the edge of the leaf, and

a 10-ml drop of molten agar consisting of 1% agar and 1% V-8 juice

at 608C was added to each inoculum drop to fix the inoculum on

the target site [17]. Leaves of mustard cabbage sprayed with liquid

medium were inoculated with conidia of A. brassicicola and used as

controls. Inoculated plants were placed in moist chambers and

680 www.elsevier.com/locate/nbt

kept in the greenhouse. The number and the size of lesions that

developed at the inoculated sites were recorded three days after

inoculation. Two leaves were used for each treatment, and all the

experiments were repeated at least twice.

DNA extractionThe DNA of A. flavus isolate V5F-13 was extracted from 0.1 g three-

day-old mycelia grown on cellophane placed on PDA [18] by the

plant genomic DNA extraction kit (GeneMark Technology Co.,

Taichung, Taiwan). The ITS region was amplified with primers ITS1

and ITS4 [19]. PCR was performed in a 50 ml volume reaction

containing 2 ml DNA, 1 pmol of upstream and downstream pri-

mers and 2.5 units of SuperTaq polymerase (Protech Technology

Enterprise Co., Ltd, Taipei, Taiwan) with buffer system recom-

mended by the manufacturer. Cycling conditions of PCR were:

initial denaturation at 948C for 2 min, 30 cycles at 948C for 30 s,

558C for 30 s, 728C for 1 min, and a final elongation at 728C for

6 min. The PCR product was analyzed by electrophoresis in a 1.2%

agarose gel. In the same manner the large ribosomal subunit 28S

was analyzed with primer pairs LROR and LR7 [20]. The annealing

temperature was changed to 508C.

BioassayA 10-ml aliquot of conidial suspension (2 � 104 spores ml�1) of A.

brassicicola was mixed with 10 ml of culture or extract in a cavity of

a sterile eight-cavity slide. Slides with spores were kept moist by

placing each on an L-shaped glass rod in a 9-cm Petri plate

containing 10 ml sterile distilled water. Germination was recorded

after incubation at 288C for four hours, and 100 spores were

counted in each of three replicates.

Extraction of inhibitory substanceEach flask of liquid culture of A. flavus was tested for the ability to

inhibit spore germination of A. brassicicola to assure the presence

of the inhibitory substance before freeze drying. One gram of dry

powder, obtained from approximately 50 ml of culture, was

extracted with 50 ml of water, ethanol, methanol, acetone, ethyl

acetate or ether in a 250-ml flask by shaking on a shaker for 24

hours. The mixture was centrifuged at 1500 � g for 5 min to obtain

clear extract. For bioassay and characterization of the inhibitory

substance, 10 ml extract was evaporated to 2 ml followed by the

addition of 2 ml water and evaporation to 2 ml again. For control,

water was similarly extracted with each solvent and tested for the

ability to inhibit spore germination.

Characterization of the inhibitorTo study the ability of different adsorptive materials to remove the

inhibitor from the culture extract of A. flavus, 5 g of Diaion SK1B

cation exchange resins (equivalent to Amberlite 1R-120), Diaion

SA 12A anion exchange resins (equivalent to Amberlite 1RA-420;

Tai-Young Chemical Co., Kaohsiung, Taiwan) or activated char-

coal (Sigma–Aldrich) was washed with 50 ml of distilled water

three times by shaking over a six-hour period to remove possible

inhibitory substances [21]. Ten milliliter extract was shaken with

1 g cation exchange resins, anion exchange resins or activated

charcoal in a 150-ml flask at 100 strokes min�1 for 24 hours and

filtered through a Whatman no. 1 filter paper. The filtrates were

then used for germination tests.

New Biotechnology �Volume 28, Number 6 �October 2011 RESEARCH PAPER

TABLE 2

Effectiveness of different solvents in extracting inhibitory sub-stances from freeze-dried powder of liquid culture of Aspergillusflavus against germination of Alternaria brassicicola conidia

Solvent Germination (%)

Extract Control

Water 57 100

Ethanol 0 98

Methanol 0 100

Acetone 89 98

Ethyl acetate 86 99

Ether 85 91

Water (control) 95

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To determine whether the effect of the inhibitor is fungistatic or

fungicidal, conidia of A. brassicicola in 1 ml of the extract (105

spores ml�1) in a 1.5 ml centrifuge tube were incubated at 288C.

After 24 or 48 hours, the inhibitor was removed by centrifugation

of the spore suspension at 1500 � g for 5 min and replacement of

the supernatant with the same amount of sterile distilled water.

Conidia in 0.1 ml of the suspension were then spread on a 1.5%

water agar plate. Germination was recorded after incubation at

288C for 24 hours.

ResultsEffect of liquid cultures of isolated microorganisms on diseaseincidenceFrom the six soil samples, 71 fungi, 69 actinomycetes and 101

bacteria which were able to use vegetable tissues for multiplication

in soils were isolated. Most liquid cultures of isolated microorgan-

isms were ineffective in reducing the disease incidence, when

sprayed on mustard cabbage leaves. However, the cultures of

one bacterium and five fungi were able to reduce disease incidence

of black leaf spot caused by A. brassicicola from 90% in the control

to 30% or less, and lesion diameter from 18 mm to 10 mm or less

(Table 1). Although all of them were able to reduce the germina-

tion rate of A. brassicicola conidia to certain extent, only the

culture of the fungus V5F-13 was capable of reducing the germina-

tion rate from more than 90% in the control to 0%. It was,

therefore, selected for further studies.

Identification of the fungusOn Czapek’s agar, the fungus V5F-13 grows 3.8–4.0 cm in diameter

in 14 days at 248C. The mycelium was white, but the colony

appeared pyrite yellow. Stipes measured 220–370 mm, pale brown

and rough. Vesicles were subglobose or pyriform, 12.0–28.0 mm

wide. Aspergilla were usually biseriate, occasionally uniseriate or

with both forms in the same vesicle. Phialides (7.8–10.4 � 2.1–

5.0 mm) covered about 3/4 of the whole vesicles surface. Conidia

were globose, rough and 3.5–5.6 mm in diameter. The morpholo-

gical characteristics of V5F-13 fitted the description of A. flavus

Link [22]. Its ITS sequence (HQ 395774) matched the ITS sequence

of FJ216392 of A. flavus in GenBank with a 99.6% similarity. Its 28S

sequence (HQ 395773) also matched the 28S sequence EU071389

TABLE 1

Effect of six liquid cultures of soil microorganisms from the firstselection on disease incidence of black leaf spot of mustardcabbage caused by Alternaria brassicicolaa

Isolate Diseaseincidence (%)

Lesion size(mm, diam.)

V3B-34 20 10

V3F-3 0 0

V4F-13 30 10

V5F-13 0 0

V5F-14 0 0

V5F-15 0 0

Liquid medium (control) 90 18

a Two leaves were sprayed once with a liquid culture daily for three days before inoculation

on the fourth day. Each leaf was inoculated at five locations, and the disease incidence and

lesion size were recorded after three days.

of A. flavus in GenBank with a 99.6% similarity. Therefore, the

fungus V5F-13 was identified as A. flavus. A culture of the isolate

was deposited at Culture Collection and Research Center, Food

Industry Research and Development Institute, Hsinchu, Taiwan,

as BCRC34704.

Isolation of the inhibitory secondary metaboliteFor studying the nature of the inhibitory secondary metabolite,

the inhibitory substance was isolated by freeze drying the liquid

culture of A. flavus and extracting the dry powder with different

organic solvents. After the evaporation of solvents, the extracts

were tested for their ability to support spore germination of A.

brassicicola. Extracts of ethanol or methanol showed complete

inhibition of germination of A. brassicicola conidia. Water extract

was moderately inhibitory, while extracts of acetone, ethyl acetate

or ether showed much less inhibitory or close to not inhibitory.

Controls with each solvent were not inhibitory (Table 2).

Characterization of the inhibitory secondary metaboliteExposure of A. brassicicola conidia to the ethanol culture extract of

A. flavus for 24 or 48 hours inhibited germination completely in

the extract. However, these condia germinated nearly completely

after being transferred to water agar (Table 3), indicating that the

effect of the inhibitory metabolite was fungistatic. When the pH of

the extract was adjusted from the original 5 to 4 or 3 with 1 N HCl,

or 6 with 1 N NaOH, germination of A. brassicicola was still

inhibited completely. However, when the pH was adjusted to 7,

8 or 9, germination increased. The extract was no longer inhibitory

when its pH was adjusted to 10 (Table 4). When the pH of the

extract was adjusted to 2 or 12 for 24 hours and then re-adjusted

back to 5, the extract still completely inhibited conidial germina-

TABLE 3

Ability of Alternaria brassicicola conidia to germinate afterexposure to culture extract of Aspergillus flavus for 24 or 48 hours

Exposure time (h) Germination (%)

Extract Water agar

24 0 81

48 0 82

Water (control) 98 97

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RESEARCH PAPER New Biotechnology �Volume 28, Number 6 �October 2011

TABLE 6

Effects of exchange resins and activated charcoal treatments ofculture extract of Aspergillus flavus on its inhibitory activityagainst germination of Alternaria brassicicola conidia

Treatment pH value after

Treatmenta Germination (%)

Extract without treatment 5.0 0

Cation exchange resins 4.6 0

Anion exchange resins 4.9 0

Activated charcoal 6.1 82

Water (control) 5.0 96a The pH value was adjusted to the original 5.0 before spore germination test.

TABLE 5

Effects of extreme pH and high temperature treatments of cultureextract of Aspergillus flavus on its inhibitory activity againstgermination of Alternaria brassicicola conidia

Treatment Treatment

Duration Germination (%)

pH 2a 24 hours 0

pH 12a 24 hours 0

1008C 30 min 0

Autoclave 15 min 0

Non-treated extract 0

Water (control) 97a The pH value was adjusted back to the original 5 after treatment.

TABLE 4

Conidial germination of Alternaria brassicicola in culture extractof Aspergillus flavus adjusted to various pH values

pH value after adjustment Germination (%)

Extract Water (control)

3.0 0 91

4.0 0 89

5.0 (original) 0 91

6.0 0 94

7.0 20 93

8.0 20 93

9.0 56 87

10.0 100 93

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tion of A. brassicicola (Table 5). Conidia of A. brassicicola still failed

to germinate after the extract was heated at 1008C for 30 min or

autoclaved for 15 min (Table 5).

The inhibitory effect of the extract on germination of A. brassi-

cicola was not affected after treatment with cation exchange resins

or anion exchange resins. However, the extract was essentially no

longer inhibitory after treatment with activated charcoal (Table 6).

No positive bands were observed following SDS–PAGE of the

inhibitory extract indicating that the inhibitory metabolite is

not a protein.

DiscussionA. flavus V5F-13 was among the microorganisms capable of colo-

nizing vegetable tissues mixed in soil. Because soil contains abun-

dant and diverse microorganisms [23], the ability to colonize plant

tissues under such environment is a good indication that A. flavus

possesses a strong competitive saprophytic ability [14]. This may

explain its widespread, occurrence in soil [6–10]. The A. flavus

isolated in this study produced stable fungistatic substance when

cultured in liquid medium prepared from the same kinds of

vegetable tissues used in the soil amendment. This indicates the

possibility of production of such fungistatic substance by A. flavus

during its colonization of plant tissues in soil. It is considered

possible that suppression of substrate competitors by this fungi-

static substance may account at least in part for the strong com-

682 www.elsevier.com/locate/nbt

petitive saprophytic ability of A. flavus in soil. Such possibility

deserves further investigation.

The inhibitory substance produced by A. flavus was very stable

under high or low pH value or high temperature, indicating that

the compound may last very long in nature after its application to

the plant leaves. This may explain why the inhibitory substance

was very effective in controlling black leaf spot of mustard cabbage

even though the inhibitor was only fungistatic against A. brassi-

cicola. Although the compound was effective in controlling black

leaf spot of mustard cabbage in the greenhouse, its possible

development into a commercial product is far from certain. Its

disease control efficacy under field conditions and its toxicity to

the non-target organisms in nature remain to be investigated.

The substance produced by A. flavus was effective against A.

brassicicola at pH 3–6, but was less effective at pH 7–9 and com-

pletely ineffective at pH 10. However, when the pH of the extract

was changed back to pH 5 after 24 hours at pH 12, it was still

strongly inhibitory to A. brassicicola. This suggests that the effect of

high pH was probably on the susceptibility of the test organism,

rather than on the inhibitor per se. Several antibiotics are more

active against microorganisms in acidic medium than in the more

alkaline medium [24].

Results from the extraction of freeze dry powder of liquid

culture of A. flavus show that the inhibitory substance produced

by this fungus is soluble in ethanol or methanol, moderately

soluble in water and insoluble in acetone, ethyl acetate or ether.

The inhibitory effect of the extract was not affected by treatment

with cation or anion exchange resins, indicating that the inhibitor

has no positive or negative charge on its molecule. SDS–PAGE of

the inhibitory substance suggests that the inhibitor is not a pro-

tein. On the basis of its solubility in different solvents and non-

exchangeability with cation and anion exchange resins, the inhi-

bitory substance appears to be a hydrophilic compound with

hydrophobic functional groups. This is the first report of the

production of a fungistatic substance by this important deleterious

fungus.

AcknowledgmentsThe authors thank Dr. S. S. Tzean, Department of Plant Pathology

and Microbiology, National Taiwan University for assistance in

fungus identification. This study was supported in part by a grant

from the National Science Council of Taiwan (NSC 97-2321-B-005-

007-MY3).

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