International Journal of Food Microbiology 114 (2007) 153 159 www.elsevier.

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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 was present 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 aflJ and 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 aflatoxinproducing 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 Gtype 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 or A. 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
Corresponding author. Tel.: +1 504 286 4369; fax: +1 504 286 4419. E-mail address: ehrlich@srrc.ars.usda.gov (K.C. Ehrlich). 0168-1605/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.ijfoodmicro.2006.08.007

(Taylor et al., 2000). For Aspergillus species capable of aflatoxin production, DNA sequence analysis has also allowed identification 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 populations 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, were determined to be an unusual type of A. nomius. We also show for the first time that A. pseudotamarii is among the aflatoxinproducing 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

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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.

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 53 54 33 50 49 41 24 37 36 41 Sclerotial typeb %S 24 2 6 8 26 0 4 0 47 2 %L 15 44 79 56 41 15 54 84 6 80 Aflatoxin production %B 53 11 55 40 6 10 46 76 53 22 %B+G 0 4 0 6 45 39 4 0 0 10 % A. pseudotamarii isolates c 15 2 0 2 0 7 0 0 0 0

Sukhothai Kiln Khao Yai Sukhothai Teak forest Nan Koh Samui Ubon Ratchathani Samoeng Sanpatong Pathum Thani
a b

SK KY SU TK NN KS UR SA SP PT

NA NA AG NA AG NA AG AG AG AG

6766 3 2 3 46 168 8 97 96 83

AG = agricultural area, NA = non-agricultural area. S = small sclerotia (average size b 400 mM); L = large sclerotia (average size N 400 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).

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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. pseudotamarii; AP = A. parasiticus; AN = A. nomius; AN1, AN2, AN3 = three clades of A. nomius based on bootstrap support N95% for the separate branches. Two-letter codes for the different isolates are defined in Table 1 and refer to the different sampling sites.

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), CzapekDox 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

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Table 2 Aflatoxin production by and growth of the different Aspergillus isolates in AM, NO and UR media Phylogenetic group a Aflatoxin types b Isolate c Media d AM Total aflatoxin (g/g) AN1 AN1 AN1 AN1 AN1 AN1 AN2 AN2 AN2 AN3 AN3 AN3 APT APT APT APT APT L L L L L L S S S S S S S SBG AP BG BG BG BG BG BG BG BG BG BG BG BG B B B B B B B B B B B B B B B B B B BG BG NRRL13137 NN03 KS13 PT04 KY22 UR05 KS02 KS10 PT02 NN20 TK05 TK32 NRRL443 KY38 TK31 SU16 SK53 AF13 PT18 SA35 SK16 TK04 UR24 AF70 SP09 SU09 SU19 UR03 SK20 SK30 BN8 NRRL2999 533 3501 350 2075 736 1280 1249 1174 722 149 0 92 5 20 32 59 30 711 2 500 1 180 54 639 2538 2143 3642 2903 1512 2222 59 3257 Ratio B/G 5.5 5.2 5.6 9.5 5.4 8.2 7.6 6.8 7.7 3.5 3.0 2.4 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 15 18 NO Total aflatoxin (g/g) 1403 1137 1381 1606 685 1398 2303 1595 862 614 148 877 21 1 90 2 0 562 188 56 187 143 2 36 523 347 2055 845 169 725 954 331 Ratio B/G 0.2 0.6 0.1 0.2 0.1 0.2 0.4 0.3 0.4 0.1 0.1 0.3 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.6 1.4 UR Total aflatoxin (g/g) 1582 1246 1705 3280 1937 2058 2993 3023 3004 3179 261 3092 11 510 237 98 167 273 17 66 172 82 2 556 1564 791 2107 1665 599 607 1466 5201 Ratio B/G 1.0 5.1 1.1 1.5 1.0 1.5 1.6 1.6 2.4 0.8 0.4 0.7 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 3.2 5.1 AM/NO ratio of total aflatoxin 0.4 3.1 0.3 1.3 1.1 0.9 0.5 0.7 0.8 0.2 0.0 0.1 0.3 13.5 0.4 31.5 111.9 1.3 0.0 9.0 0.0 1.3 27.7 17.6 4.8 6.2 1.8 3.4 8.9 3.1 0.1 9.8 Growth at 42 C e

No No No No No No No No No No No No No No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 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.

like typical A. nomius were assumed to be A. 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 quantitative 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 determined 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 determinations 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 Windows Version 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

K.C. Ehrlich et al. / International Journal of Food Microbiology 114 (2007) 153159 Table 3 Summary of aflatoxin production data by Aspergillus type a Type No.b Media AM Aflatoxin yields c AN1 AN2 AN3 APT AFL AFS AP SBG 6 3 3 5 6 6 1 1 1301 a 1048 a 80 b 114 b 241 b 2228 a 3257 a 59 b Ratio B/G c 6.6 a 7.3 a 2.9 b NAd NA NA 18.1 a 14.5 a NO Aflatoxin yields c 1211 a 1586 a 546 a 69 d 190 c 672 a 331 a 954 a Ratio B/G> c 0.2 a 0.3 ab 0.2 a NA NA NA 1.4 c 0.6 bc UR Aflatoxin yields c 2055 a 2993 a 2177 a 274 bc 102 b 1127 a 5201 a 1466 ac Ratio B/G c 1.8 a 1.86 a 0.62 b NA NA NA 5.12 c 3.21 c

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Ratio aflatoxin in AM to aflatoxin in NO medium c 1.1 0.7 0.1 1.7 1.3 3.3 9.8 0.1 a a b c a c c 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.

(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 directions on 10 to 20 ng of purified PCR-produced DNA (Qiagen, Valencia, CA) using the PCR primers for each gene. Sequencing was done by the Auburn Genomics and Sequencing Facility, Auburn University AL 36849. DNA sequence manipulations 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 sequences 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 rather than the normal black and in others, were unusually long (1.0 to 4.0 mm). No isolates with the characteristic spore ornamentation of A. 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 were subjected 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 characteristics. 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 sequence of the type culture for this species.

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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 alignment datasets 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 for A. 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 ureacontaining 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-type sclerotia 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 information 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, we consider 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 could easily 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 reservoir of 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 Gproducing 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 in Thailand. 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 of A. 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 typical A. flavus isolates. Previous reports found associations of A. 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 for A. 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 Aspergillus 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 of Thailand. Knowledge of the presence and distribution of such fungi is necessary to be able to develop viable strategies for aflatoxin control. References
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