Aflatoxin-Producing us Species From Thailand

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Aflatoxin-producing Aspergillus species from Thailand

Kenneth C. Ehrlich , Kerri Kobbeman, Beverly G. Montalbano, Peter J. Cotty

Southern Regional Research Center\ARS\USDA, 1100 RE Lee Blvd., PO Box 19687, New Orleans LA 70124, USA

Received 7 April 2006; received in revised form 24 August 2006; accepted 25 August 2006

Abstract

Aflatoxin-producing Aspergillus species were isolated from soil samples from ten different regions within Thailand. Aspergillus flavus waspresent in all of the soil samples. Unlike previous studies, we found no A. parasiticus or A. flavus capable of both B- and G-type aflatoxin

production in any of the samples. A. pseudotamarii, which had not been previously reported from Thailand, was found in four soil samples. In two

of the samples A. nomius was determined to be the most abundant aflatoxin-producing species. Based on sequence alignments for three DNA

regions (Taka-amylase A (taa), the rRNA internal transcribed spacer (ITS), and the intergenic region for the aflatoxin biosynthesis genes aflJand

aflR) the A. nomius isolates separated into three well-supported clades. Isolates from one of the A. nomius clades had morphological properties

similar to those found for S-type isolates capable of B and G aflatoxin production and could easily be mistaken for these isolates. Our results

suggest that such unusual A. nomius isolates could be a previously unrecognized agent for aflatoxin contamination in Thailand.

Published by Elsevier B.V.

Keywords: Aflatoxin; Phylogenetics; Aspergillus nomius; Flavus; Pseudotamarii; Sclerotia

1. Introduction

Thailand suffers from endemic aflatoxin contamination of

maize and groundnuts (Pitt et al., 1993). Previously, several

studies showed that Aspergillus flavus was the main aflatoxin-

producing species isolated from maize obtained from markets

(Saito and Tsuruta, 1993). In addition to A. flavus, other

aflatoxigenic Aspergillus species, namely A. parasiticus and A.

nomius were also reported to be present, but in much lower

amounts. Atypical A. flavus isolates that produce both B- and G-

type aflatoxins were also reported to be present on maize and

peanuts (Pitt et al., 1993; Saito and Tsuruta, 1993). Atypical A.

flavus-like isolates (also called strain SBG orA. flavus Group II)have been found in West Africa, Argentina, and Australia

(Fernandez Pinto et al., 2001; Geiser et al., 1998; Saito and

Tsuruta, 1993). A. flavus normally produces only B-type

aflatoxins while A. parasiticus and A. nomius produce B- and

G-type aflatoxins.

The ability to use sequence data to determine phylogenetic

relationships has allowed the reassessment of relationships among

organisms that had been solely based on morphological criteria

(Taylor et al., 2000). ForAspergillus species capable of aflatoxinproduction, DNA sequence analysis has also allowed identifica-

tion of new species and subspecies within established species

designations (Ito et al., 2001; Peterson, 1997; Peterson et al.,

2001). It has also provided the means to measure the degree to

which these otherwise asexual fungi exchange genetic material

(Geiser et al., 2000, 1998).

In the present study, we examined the Aspergillus popula-

tions in soil samples from different regions in Thailand and

were unable to find A. parasiticus isolates and the previously

reported atypical A. flavus isolates capable of B and G aflatoxin

production. However, isolates resembling such atypical A.

flavus were found, which, by phylogenetic analysis, weredetermined to be an unusual type of A. nomius. We also show

for the first time that A. pseudotamarii is among the aflatoxin-

producing species from Thailand.

2. Materials and methods

2.1. Sampling locations and Aspergillus isolation

Soil samples (approx. 250 g each) from the top 5 cm were

collected during late December and January 2000 (the dry

season) at 10 different locations within Thailand (Fig. 1). The

International Journal of Food Microbiology 114 (2007) 153 159

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Corresponding author. Tel.: +1 504 286 4369; fax: +1 504 286 4419.

E-mail address: [email protected] (K.C. Ehrlich).

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sampling sites chosen were intended to be representative of a

variety of geographical locations ranging from the north, south,

and east of Thailand. Sampling sites included: (1) a rice field

near Samoeng (SA), a small city west of Chiang Mai; (2) a

forested area near Sanpatong (SP), a city slightly south of

Chiang Mai; (3) a teak forest (TK) north of Lampang; (4) a

maize-growing area north of Nan (NN); (5) a maize field near

old Sukhothai (SU); (6) a non-agricultural region near a historic

kiln site in old Sukhothai (SK); (7) a field in a silk-producing

region near Ubon Ratchatani (UR); (8) a rice-growing region

46 km north of Bangkok near Pathum Thani (PT); (9) Khao Yai

National Park (KY), a rainforest east of Bangkok; and (10) a

Table 1

Summary of Aspergillus section Flavi isolates obtained from soil samples from different regions in Thailand

Region Code Soila CFU

per g

No. of

isolates

examined

Sclerotial

typebAflatoxin production

%S %L %B %B+G % A. pseudotamarii isolates c

Sukhothai Kiln SK NA 6766 53 24 15 53 0 15

Khao Yai KY NA 3 54 2 44 11 4 2

Sukhothai SU AG 2 33 6 79 55 0 0

Teak forest TK NA 3 50 8 56 40 6 2

Nan NN AG 46 49 26 41 6 45 0

Koh Samui Ubon KS NA 168 41 0 15 10 39 7

Ratchathani UR AG 8 24 4 54 46 4 0

Samoeng SA AG 97 37 0 84 76 0 0

Sanpatong SP AG 96 36 47 6 53 0 0

Pathum Thani PT AG 83 41 2 80 22 10 0

a AG = agricultural area, NA = non-agricultural area.b S = small sclerotia (average sizeb400 mM); L = large sclerotia (average sizeN400 mM).c

A. tamarii were identified by the characteristic olive-brown color of mature colonies on Czapek-Dox agar plates as well as colony and conidial morphology(Ito et al., 2001).

Fig. 1. Map of Thailand showing locations (indicated by arrows) where soil samples were collected. The letters in brackets following the name are abbreviations used

for the sites.

154 K.C. Ehrlich et al. / International Journal of Food Microbiology 114 (2007) 153159


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non-agricultural area on the island of Koh Samui (KS) in

southern Thailand. Samples were imported into the United

States under a permit from the Animal and Plant Health

Inspection Service to PJC.

Soils were dilution-plated onto a Modified Rose-Bengal Agar

(MRBA), and approximately 50 aflatoxin-producing colonies

from each soil sample were collected. Initially isolates were

assigned as previously described (Cotty, 1994) to the different

known aflatoxin-producing species based on whether or not they

produced only B-aflatoxins or both B and G-aflatoxins, their

characteristic growth patterns on various media (A. flavus and

parasiticus Agar (AFPA, Oxoid, Inc, Ogdensburg NY), Czapek-

Dox Agar, (BD Diagnostics, Sparks, MD) and 5% V8 juice

(Campbell Soup Company, Camden, NJ)/2% Agar), their spore

ornamentation, and colony and sclerotial morphology. Based on

this initial screening, colonies were separated into five

categories. Isolates which produced only B aflatoxins and had

abundant small sclerotia were classified as S-type A. flavus.

Isolates which produced only B aflatoxins and had sclerotia

averaging over 400 m in diameter were classified as L-type A.

flavus. Isolates that produced both B and G aflatoxins and had

sclerotia similar to S-type A. flavus were initially classified as

Group II-type A. flavus (Geiser et al., 2000). Isolates that were

morphologically similar to A. tamarii and produced only B

aflatoxins were classified as A. pseudotamarii. Isolates that

produced B- and G-aflatoxins and reacted on AFPA agar plates

Fig. 2. Phylogenetic analysis showing one of the most-parsimonious trees for each of the three DNA alignment datasets and the combined gene dataset. A, ITS, rRNA

internal transcribed spacer DNA; B, AFLR, aflRaflJ intergenic region; C, TA, partial sequence of the gene encoding Taka-amylase A, and D, the combined dataset.

Numbers below branches represent bootstrap percentages based on 1000 replicates and numbers above the branches are the number of steps. CI, consistency index; HI,

homoplasy index; RI, retention index; RC, rescaled consistency index. APT=A. pseudotam arii; AP=A. parasit icus; AN=A. nomius; AN1, AN2, AN3=three clades

ofA. nomius based on bootstrap supportN95% for the separate branches. Two-letter codes for the different isolates are defined in Table 1 and refer to the different

sampling sites.

155K.C. Ehrlich et al. / International Journal of Food Microbiology 114 (20 07) 153159


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like typicalA. nomius were assumed to beA. nomius. Qualitative

determination of aflatoxin production was made on silica gel

thin layer plates as previously described (Cotty, 1994).

2.2. Measurement of aflatoxin production on defined media

Representatives of each category were selected for quanti-

tative determination of aflatoxin production after growth on

chemically defined media pH 4.75 containing either 22.5 mM

ammonium sulfate (AM), 22.5 mM sodium nitrate (NO) or

45 mM urea (UR) as sole nitrogen source (Cotty and Cardwell,

1999; Ehrlich and Cotty, 2002). Aflatoxin yields were deter-

mined on day 4, at which time production plateaued under the

growth conditions used. Aflatoxin production results are from

two separate experiments. In each experiment aflatoxin deter-

minations were done in triplicate. Statistical analyses were

performed on the log transform of the aflatoxin yield data by the

ANOVA Mixed Data procedure for calculating Least Squared

Means (LSM) and probabilities using SAS System for WindowsVersion 8 software (SAS Institute, Cary NC). Mean separations

were performed on the log of the aflatoxin B1 yield data, the

ratio of aflatoxin B1 to aflatoxin G1, and the ratio of aflatoxin

yield in AM to aflatoxin yield in NO medium. Common

variance and a normal distribution of data were found for the

isolates. Cultures for DNA isolation were grown for 24 h on

yeast extract sucrose (YES) medium and DNA was isolated as

previously described (Ehrlich and Cotty, 2002).

2.3. Sequencing and phylogenetic analyses

Portions of the rRNA internal transcribed spacer (ITS) and

its flanking regions (White et al., 1990), the Taka-amylase gene

Table 2

Aflatoxin production by and growth of the different Aspergillus isolates in AM, NO and UR media

Phylogenetic

groupaAflatoxin

typesbIsolatec Mediad AM/NO

ratio of

total

aflatoxin

Growth

at

42 CeAM NO UR

Total aflatoxin

(g/g)

Ratio

B/G

Total aflatoxin

(g/g)

Ratio

B/G

Total aflatoxin

(g/g)

Ratio

B/G

AN1 BG NRRL13137 533 5.5 1403 0.2 1582 1.0 0.4 No

AN1 BG NN03 3501 5.2 1137 0.6 1246 5.1 3.1 No

AN1 BG KS13 350 5.6 1381 0.1 1705 1.1 0.3 No

AN1 BG PT04 2075 9.5 1606 0.2 3280 1.5 1.3 No

AN1 BG KY22 736 5.4 685 0.1 1937 1.0 1.1 No

AN1 BG UR05 1280 8.2 1398 0.2 2058 1.5 0.9 No

AN2 BG KS02 1249 7.6 2303 0.4 2993 1.6 0.5 No

AN2 BG KS10 1174 6.8 1595 0.3 3023 1.6 0.7 No

AN2 BG PT02 722 7.7 862 0.4 3004 2.4 0.8 No

AN3 BG NN20 149 3.5 614 0.1 3179 0.8 0.2 No

AN3 BG TK05 0 3.0 148 0.1 261 0.4 0.0 No

AN3 BG TK32 92 2.4 877 0.3 3092 0.7 0.1 No

APT B NRRL443 5 NA 21 NA 11 NA 0.3 No

APT B KY38 20 NA 1 NA 510 NA 13.5 No

APT B TK31 32 NA 90 NA 237 NA 0.4 NoAPT B SU16 59 NA 2 NA 98 NA 31.5 No

APT B SK53 30 NA 0 NA 167 NA 111.9 No

L B AF13 711 NA 562 NA 273 NA 1.3 Yes

L B PT18 2 NA 188 NA 17 NA 0.0 Yes

L B SA35 500 NA 56 NA 66 NA 9.0 Yes

L B SK16 1 NA 187 NA 172 NA 0.0 Yes

L B TK04 180 NA 143 NA 82 NA 1.3 Yes

L B UR24 54 NA 2 NA 2 NA 27.7 Yes

S B AF70 639 NA 36 NA 556 NA 17.6 Yes

S B SP09 2538 NA 523 NA 1564 NA 4.8 Yes

S B SU09 2143 NA 347 NA 791 NA 6.2 Yes

S B SU19 3642 NA 2055 NA 2107 NA 1.8 Yes

S B UR03 2903 NA 845 NA 1665 NA 3.4 Yes

S B SK20 1512 NA 169 NA 599 NA 8.9 Yes

S B SK30 2222 NA 725 NA 607 NA 3.1 YesSBG BG BN8 59 15 954 0.6 1466 3.2 0.1 Yes

AP BG NRRL2999 3257 18 331 1.4 5201 5.1 9.8 Yes

a TypesAN1, 2, 3, A. nomius clades 1, 2 and seen in Fig. 2 for the aflJaflR phylogenetic tree; S, L = A. flavus strain S and L; APT = A. pseudotamarii; SBG =

strain SBG (Cotty and Cardwell, 1999); AP = A. parasiticus.b B and G indicate aflatoxins B1+B2 and G1 respectively.c An asterisk indicates the strain used for the species definition.d AM, NO, and UR indicate minimal media used for Aspergillus growth containing ammonium sulfate, sodium nitrate, and urea as the nitrogen source, respectively

(see Materials and methods). Aflatoxin yields are the averages of three replicate cultures grown with shaking at 31 C for four days.e Growth was on Czapek-Dox agar plates at 42 C for 5 days.



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(Egel et al., 1994), and the aflJaflR intergenic region (Ehrlich

et al., 2003) were amplified by Taq polymerase (Amplitaq,

Applied Biosystems, Foster City CA) with primers identical to

those used previously (Egel et al., 1994; Ehrlich et al., 2003;

White et al., 1990). Sequencing was performed in both direc-

tions on 10 to 20 ng of purified PCR-produced DNA (Qiagen,

Valencia, CA) using the PCR primers for each gene. Se-

quencing was done by the Auburn Genomics and Sequencing

Facility, Auburn University AL 36849. DNA sequence mani-pulations and alignments were made using DNAMAN (Lynnon

Biosoft, Vandreuil, Quebec, Canada). Phylogenetic analyses

were performed using PAUPver4b10 (Sinauer Associates,

Sunderland, MA). GenBank Accession Numbers for the se-

quences are: Taka-amylase (taa), DQ467904DQ467935;

aflRaflJ intergenic region, DQ467936DQ467967; ITS,

DQ467968DQ467999.

3. Results

3.1. Preliminary determination of Aspergillus populations in

soil samples

Aflatoxin-producing aspergilli were isolated from all of the

soil samples (Table 1). The sites that were sampled included

both non-agricultural and agricultural areas. Of the 400 isolates

screened on MRBA plates, approximately half produced

detectable quantities of aflatoxin. In most of the soils there

were more isolates that produced only B aflatoxins than B and G

aflatoxins. However, in two of the soil samples (NN and KS),

there were more B and G-producing isolates than isolates that

produced only B aflatoxins. For most of the samples, isolates

that produced small sclerotia were less abundant than isolates

that produced large sclerotia. The exception was the area near

Sanpatong (SP) in which most isolates were classified as the S-

type. Most of the isolates with S-type sclerotial morphology

produced only B toxin, but all of the S-type isolates from the

maize field north of Nan (NN) in northern Thailand produced

both B and G toxins. Such isolates were also present in low

abundance from Khao Yai National Park (KY) and a teak (TK)

forest near Lampang. Other B and G-producing isolates had

growth characteristics, conidial and sclerotial morphology

characteristic of A. nomius. Several isolates produced aberrant

sized sclerotia. In a few cases the sclerotia had a tan color ratherthan the normal black and in others, were unusually long (1.0 to

4.0 mm). No isolates with the characteristic spore ornamenta-

tion ofA. parasiticus were found from any of the soils in which

over 400 aflatoxin-producing and atoxigenic fungal colonies

were screened.

3.2. Phylogenetic analysis of selected aflatoxin-producing

colonies

Portions of three genes were sequenced from a subset of

isolates chosen from each of the five initial species categories

listed in Materials and methods. DNA alignments weresubjected to phylogenetic analysis (Fig. 2). This analysis

showed that the isolates that produced both B and G aflatoxins

and S-type sclerotia, and initially classified as Group II A.

flavus, were more closely related to A. nomius, as were the

isolates identified in the preliminary screening as A. nomius by

their reactions on AFPA plates and their morphological charac-

teristics. Most of these latter isolates had either no sclerotia or

large or elongated sclerotia. The isolates with L-type and S-type

sclerotia that only produced B-aflatoxins were related to typical

A. flavus found in North America and other regions of the world.

The aflatoxin-producing isolates that resembled A. tamari were

identified as A. pseudotamarii by their alignment with the se-

quence of the type culture for this species.

Table 3

Summary of aflatoxin production data by Aspergillus typea

Type No.b Media Ratio

aflatoxin

in AM to

aflatoxin

in NO

mediumc

AM NO UR

Aflatoxin

yieldscRatio

B/GcAflatoxin

yieldscRatio

B/G>cAflatoxin

yieldscRatio

B/Gc

AN1 6 1301 a 6.6 a 1211 a 0.2 a 2055 a 1.8 a 1.1 a

AN2 3 1048 a 7.3 a 1586 a 0.3 ab 2993 a 1.86 a 0.7 a

AN3 3 80 b 2.9 b 546 a 0.2 a 2177 a 0.62 b 0.1 b

APT 5 114 b NAd 69 d NA 274 bc NA 1.7 c

AFL 6 241 b NA 190 c NA 102 b NA 1.3 a

AFS 6 2228 a NA 672 a NA 1127 a NA 3.3 c

AP 1 3257 a 18.1 a 331 a 1.4 c 5201 a 5.12 c 9.8 c

SBG 1 59 b 14.5 a 954 a 0.6 bc 1466 ac 3.21 c 0.1 b

a The Aspergillus types are listed for each isolate in Table 2. Each represents a N95% bootstrap-supported clade in the phylogenetic tree shown for the aflJaflR

intergenic region alignment dataset.b No.number of isolates compiled for the statistical calculation. Aflatoxin yields (g/g dry weight mycelia) were determined on triplicate cultures on isolates given

in Table 2. Values given are the means of the data.c Values followed by the same letter are not statistically different based on the log transform of total aflatoxin yields for the isolate in each medium based on the

Mixed Method for ANOVA using SAS software. Statistical analysis of the ratio of B aflatoxin to G aflatoxin in each medium and the ratio of total aflatoxin production

in AM medium to aflatoxin production in NO medium was performed with log transformed data.d NAnot applicable.



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Three distinct clades of A. nomius could be distinguished

based on the aflJaflR gene dataset and the combined gene

dataset, but only two distinct clades were apparent with the TA

and ITS datasets. Furthermore, the isolate affiliations of A.

nomius for the ITS, TA, and aflJaflR datasets were different.

Based on partition homogeneity tests (PHT) using parsimony

informative sites only, partitions consisting of alignmentdatasets for the three genes were significantly incongruent

(P=0.001 based on 1000 bootstrap replicates) when the 12 A.

nomius isolates (types AN13) were included or when the

datasets included alignments forA. nomius types AN2 and AN3

only. Congruency of the gene partitions was found (P=1.0)

when the true A. nomius isolates (AN1) were examined

independently. The three isolates in A. nomius clade 3 (AN3)

and some of the isolates in clade 1 (AN1) produced small

sclerotia. However, production of small sclerotia was not a

consistent characteristic of isolates in AN1. None of the isolates

that produced B and G aflatoxins resembled A. parasiticus, West

African SBG (Cotty and Cardwell, 1999) or A. flavus Group II(Geiser et al., 2000) isolates and sequence analysis of the 26

isolates also failed to identify any of this type.

3.3. Aflatoxin production by different isolates is affected by

nitrogen source in the media

Aflatoxin production results are shown in Table 2 and

summarized by type of Aspergillus in Table 3. On urea-

containing minimal medium (UR) most A. nomius isolates

produced as much as 710 fold more aflatoxin than did A.

pseudotamarii and A. flavus L isolates and about two-fold more

than did A. flavus S isolates (Table 3). For most of the A. nomius

isolates, aflatoxin production in ammonium-containing medium(AM) was lower than in nitrate-containing medium (NO). This

result is similar to the results found previously for the atypical B

and G-producing S-type isolates from West Africa (Ehrlich and

Cotty, 2002). For A. nomius isolates in clades AN2 and AN3

(Fig. 1) and SBG isolates (Ehrlich and Cotty, 2002) less aflatoxin

was produced in AM medium than in NO medium, but for all

other isolates, more aflatoxin was produced by growth in AM

medium (Table 2).

4. Discussion

Some aflatoxigenic aspergilli produce numerous S-typesclerotia on certain agar media. These include S-type A. flavus,

as well as A. flavus Group II and an unnamed taxon related to A.

flavus which produces B and G-aflatoxins. A. flavus is found

world-wide in temperate regions, while, so far, isolates of the

atypical A. flavus have been found mainly in Africa, Australia,

and Argentina (Cotty and Cardwell, 1999; Fernandez Pinto

et al., 2001; Geiser et al., 1998). Previous reports suggested that

such isolates are in Thailand, but gave no phylogenetic infor-

mation to support this claim, since molecular methods enabling

distinctions among species in Aspergillus section Flavi had not

been widely used when these papers were written (De Leon

et al., 1995; Pitt et al., 1993; Saito and Tsuruta, 1993; Saito

et al., 1986; Sripathomswat and Thasnakorn, 1981).

In this paper we identified B and G aflatoxin-producing

aspergilli in soil samples from different regions of Thailand that

had S-type sclerotia that were not related to the unnamed taxon

from West Africa or the Group II A. flavus from Australia or

Argentina. Phylogenetic analyses clearly place these isolates

within the broad phylogenetic classification assigned to A.

nomius (Kurtzman et al., 1987). Based on these results, weconsider it possible that the S-type, aflatoxin B and G-producing

isolates previously found in Thailand were actually atypical A.

nomius. In that study, typical A. nomius were reported as being

present in low frequency in the population of mycoflora from

commodities in Thailand (Pitt et al., 1993). Among the isolates

examined in the current study, A. nomius was abundant in the

soils of some regions of Thailand. It is clear that A. nomius can

produce morphologically diverse sclerotia including the

abundant S-type sclerotia associated with A. nomius in the

current study, and the originally described indeterminate

sclerotia considered characteristic of this species (Kurtzman et

al., 1987). The small sclerotial morphotype of A. nomius couldeasily be confused with the unnamed taxon isolates reported

from Africa and the A. flavus Group II isolates from Australia

and Argentina. From two geographically separated collection

sites, namely the Northeastern upland region north of the city of

Nan and a non-agricultural region on the island of Koh Samui,

A. nomius was the predominant aflatoxin-producing Aspergillus

in the soil. The former region is a maize-growing area farmed by

the Yao (Mien) people, an indigenous hill tribe population in

Northern Thailand, while the latter region is a coastal mixed

palm and hardwood forest with few agricultural areas. While

our study is not meant to be an intensive survey of the

mycoflora of Thailand it points to two conclusions: 1. previous

studies may have overlooked a potentially important reservoirof highly aflatoxigenic fungi, namely, A. nomius, a species

which shares morphological traits with the S strain of A. flavus,

and is widespread in soils from both low mountainous, and

rainforest regions, and 2. the previously described B and G-

producing A. flavus from Thailand is probably a variant form of

A. nomius rather than A. flavus. Furthermore, the present results

confirm our previous results and the results of others, that A.

nomius is a morphologically diverse species difficult to classify

by conventional criteria (Ehrlich et al., 2005; Kumeda and

Asao, 2001; Peterson et al., 2001).

The current paper is the first report of the widespread

distribution of A. pseudotamarii and A. nomius isolates inThailand. A. pseudotamarii isolates were found at four of the

ten sites sampled. Previously, A. pseudotamarii was only

reported from Japan (Ito et al., 2001). A. nomius was found at

six of the ten sites. The diversity of Aspergillus section Flavi

populations found in the different soil samples (Table 1) did not

seem to correlate with agricultural use of the land. A previous

study found that aflatoxin-producing isolates were common in

non-cultivated regions of the Sonoran desert of the United

States and contaminated seeds of native plants there (Boyd and

Cotty, 1997). The abundance ofA. nomius isolates in the samples

demonstrates that A. nomius is more widespread than may be

commonly thought, and as such, must be considered a potential

etiological agent of contamination events in those regions. A.



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nomius may be particularly important because the A. nomius

isolates are capable of producing greater amounts of aflatoxins

than typicalA. flavus isolates. Previous reports found associations

ofA. nomius with pistachio nuts (Feibelman et al., 1998), wheat

(Kurtzman et al., 1987), and agricultural soils in the southern

United States (Egel et al., 1994; Kurtzman et al., 1987).

The present study demonstrates that genetically distinct A.nomius are present even in a relatively small geographical area.

Such diversity has been found forA. flavus (Bayman and Cotty,

1993). Like A. nomius, A. flavus showed a lack of homogeneity

among gene datasets, by the Partition Homogeneity Test (PHT).

Therefore, diversity among A. nomius may have arisen by

similar processes to those governing A. flavus diversity (Geiser

et al., 2000, 1998). Soil is a reservoir of diverse strains of As-

pergillus that only infect crops when conditions are favorable.

Although compared to infection by A. flavus, infection of crops

by A. nomius may be rare, even rare infection events could be

important if the infecting isolates produce large quantities of

aflatoxins as do the A. nomius isolates endemic to soils ofThailand. Knowledge of the presence and distribution of such

fungi is necessary to be able to develop viable strategies for

aflatoxin control.

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