A survey on distribution of Aspergillus section Flavi in corn field soilsin Iran: Population patterns based on aflatoxins, cyclopiazonic acidand sclerotia production

Mehdi Razzaghi-Abyaneh1, Masoomeh Shams-Ghahfarokhi2, Abdolamir Allameh2,Amirmohammad Kazeroon-Shiri3, Shahrokh Ranjbar-Bahadori3, Hasan Mirzahoseini4

& Mohammad-Bagher Rezaee51Department of Mycology, Pasteur Institute of Iran, 13164, Tehran, Iran; 2Faculty of Medical Sciences,Tarbiat Modaress University, 14115-111, Tehran, Iran; 3Faculty of Veterinary Sciences, Azad University,Garmsar, Iran; 4Department of Biotechnology, Pasteur Institute of Iran, 13164, Tehran, Iran; 5Forestry andRangelands Research Institute, Tehran, Iran

Received 24 September 2005; accepted in revised form 24 November 2005

Abstract

Soil isolates of Aspergillus section Flavi from Mazandaran and Semnan provinces with totally differentclimatic conditions in Iran were examined for aflatoxins (AFs; B and G types), cyclopiazonic acid (CPA)and sclerotia production. A total of 66 Aspergillus flavus group strains were identified from three species viz.Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius in both locations. A. flavus (87.9%) wasfound to be the prominent species followed by A. nomius (9.1%) and A. parasiticus (3.0%). Only 27.5% ofA. flavus isolates were aflatoxigenic (B1 or B1 and B2), out of which approximately 75% were capable toproducing CPA. All the A. parasiticus and A. nomius isolates produced AFs of both B (B1 and B2) and G(G1 and G2) types, but did not produce CPA. Sclerotia production was observed in only 4 isolates ofA. flavus among all 66 isolates from three identified species. A. flavus isolates were classified into variouschemotypes based on the ability to produce aflatoxins and CPA. In this study, a new naturally occurringtoxigenic A. flavus chemotype comprising of two strains capable of producing more AFB2 than AFB1 hasbeen identified. A relatively larger proportion of aflatoxigenic A. flavus strains were isolated from corn fieldsoils of Mazandaran province which indicate a possible relationship between high levels of relativehumidity and the incidence of aflatoxin-producing fungi. The importance of incidence of Aspergillus sectionFlavi in corn field soils regard to their mycotoxin production profiles and crop contamination with specialreference to climatic conditions is discussed.

Key words: Aflatoxin, Aspergillus section Flavi, corn field soil, cyclopiazonic acid, geographic distribution,Iran

Introduction

Aspergillus section Flavi comprises a closely rela-ted group of fungi which are widely distributed insoil, air, organic materials and plant parts all overthe world [1, 2]. Among these, three speciesAspergillus flavus, A. parasiticus and A. nomius

have received major considerations regarding totheir ability for producing potent carcinogenicaflatoxins [3, 4]. They invade susceptible cropssuch as corn, peanuts, cotton and tree nuts beforeor after harvesting which might be resulted inaflatoxin contamination of food and feed [2].A. flavus may also produces other important

Mycopathologia (2006) 161: 183–192 � Springer 2006DOI: 10.1007/s11046-005-0242-8

mycotoxins, such as cyclopiazonic acid (CPA)which is toxic to a variety of animals and has beenimplicated in human poisoning [5, 6]. A. flavuspopulations vary considerably in their capacity toproduce AFs and CPA, as many isolates produceonly one mycotoxin or neither mycotoxin [7–9]. Itis seemed that AF-producing fungi are a mosaic ofspecies belonging to divergent clades whichreproduce in diverse ecological niches throughoutwarm climates [2]. Data from different geograph-ical areas have revealed differences in proportionof A. flavus isolates that produce various amountsof aflatoxins from low to high [7]. A. flavus pro-duces only AFB (B1, B2 or both), while A. para-siticus and A. nomius produce both AFs B (B1 andB2) and G (G1 and G2) types [10]. Despite the widerange of ecological niches considered for aflatoxi-genic Aspergillus species, it is stated that agricul-tural soil serves as the main reservoir of these fungi[7, 11–13]. Conidia and special structures knownas sclerotia serve as infective propagules ofAspergillus species in soil [12]. These propagulescan contaminate various crops directly throughdirect contact (e.g. peanuts) or indirectly throughdispersal by wind or insects (e.g. corn and cot-tonseed) [14, 15]. Knowledge of regional differ-ences in population patterns and toxigenicity ofAspergillus section Flavi and their association withdominant crops in a region may be important forunderstanding population dynamics and suitablecontrol measures used for field reduction of pre-harvest aflatoxin contamination [7]. On the otherhands, new naturally occurring isolates withunique aflatoxin-producing profiles could be con-sidered for obtaining more precise data aboutaflatoxin biosynthesis specially regard to not fullyunderstood final steps of the pathway where sep-aration of AFs B and G types occurs [16–18].

Corn is considered as a major crop which iscultivated in different geographic regions of Iran.Its annual production reaches to several 1000 tonsand it is used for human and animal consumption.

Recently, A. flavus has been isolated from theharvested maize grains in four different regions ofIran [19]. However, the mycotoxin-producingability of these fungi has not been studied.

The aim of present study was to compare theAspergillus section Flavi population in soil samplesof corn fields collected from two totally differentclimatic conditions in Iran with emphasis onaflatoxin and CPA producing ability; Mazandaran

province near the Caspian sea with high mean ofannual rainfall (672 mm) and relative humidity(70%) and the Semnan province with low rainfall(150 mm) and relative humidity (30%). Selectionof different climatic regions provides this oppor-tunity to compare the vulnerability of corn seedsto soil-borne fungi habitant to either dry or moistclimates.

Different species with considerable variation intheir mycotoxin profile including a newly isolatedtoxigenic A. flavus chemotype with unique abilityfor producing more aflatoxin B2 than aflatoxin B1

were identified.

Materials and methods

Chemicals

Aflatoxins (AFs) and cyclopiazonic acid (CPA)standards were the products of Sigma ChemicalCo., St. Louis, MO, USA. TLC silica gel 60 F254

were purchased from E. Merck, Germany. Allother solvents and reagents were of analyticalgrade prepared from E. Merck, Germany.

Corn field soils

All together, 100 soil samples were collected fromtwo different climatic regions during June–July2004. Fifty samples from each province were col-lected from 10 locations (5 samples each) (Table 1).Selected regions are known as: Semnan provincewith warm-dry climate (neighboring central desertof Iran) and Mazandaran province with warm-moist climate (Caspian sea littoral) which theirposition in Iran map is shown in Figure 1. Eachfield soil comprised from five subsamples each ofapproximately 100 cm3 which were obtained usinga sterile trowel at 15 m intervals. The subsampleswere collected from the 5 cm top of the surface soiland then mixed thoroughly in a Nylon bag. Thesamples were maintained on sterile petri dishes at4 �C until complete dryness.

Fungal isolation and identification

Aspergillus parasiticus NRRL 2999 as a standardaflatoxigenic strain with capability to produceboth aflatoxins B (B1 and B2) and G (G1 and G2)types was used throughout the study.

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Isolation of Aspergillus section Flavi was per-formed using spread plating method according toKumeda et al. [20] with some modifications. Thedried soil samples were passed through testingsieves (1 mm mesh). A 20 g portion of each sievedsoil was diluted with 180 ml distilled water con-taining 0.05% agar in a 500-ml flask and thenshaked vigorously for 5 min at room temperature.Ten-fold serial dilutions of soil samples from 10)1

to 10)5 were prepared in sterile distilled water. Theamounts of 0.5 ml of each dilution were separatelyspread onto 10 cm petri dishes containing isola-tion culture media i.e., Dichloran rosebengal chl-oramphenicol agar (DRCA) and Aspergillusflavus/parasiticus agar (AFPA). The cultures werethen incubated at 30�C and were checked period-ically for colonies with brilliant orange-yellowreverse coloration on AFPA (during 42–48 h) andyellowish-green colonies on DRCA (until maxi-mum 2 weeks). The suspected colonies were sub-cultured to Potato dextrose agar (PDA) plates andthen purified using the hyphal tipping method [21].

Aspergillus species were identified based on acombination of different criteria including mor-phological observations, sclerotia production andaflatoxin and cyclopiazonic acid profiles according

to Raper and Fennel [1] and Pitt and Hocking [4].Morphological characteristics and sclerotiaproduction was notified after culturing the isolateson Czapek dox agar (CZA) plates for 10 days at30 �C. The cultures were then checked periodicallyuntil 3 weeks for sclerotia production by the iso-lates. All Aspergillus isolates were maintained onPDA slants at 4 �C.

Extraction and analysis of mycotoxins

Aflatoxin production was first screened based onblue fluorescence observation on aflatoxin-pro-ducing ability (APA) medium supplemented with0.3% b-cyclodextrin under U.V. light (365 nm)according to Fente et al. [22].

For assessment of aflatoxins and CPA, all theisolates were cultured on Yeast extract-sucrose(YES) broth medium which composed of 2% yeastextract and 18% sucrose purchased from E.Merck, Germany. The medium was divided in5 ml aliquots in 25 ml capacity erlenmayer flasksand sterilized by autoclaving at 121 �C for 15 min.The cultures were incubated at 30 �C for a periodof 4 days in a shaker incubator with 120 rev/min.after inoculation with 106 fungal conidia/ml. Both

Table 1. Incidence of Aspergillus section Flavi in corn field soils from Semnan and Mazandaran provinces of Iran

Provinces Locations* Number of sampled fields Aspergillus section Flavi (No. of isolates)

A. flavus A. parasiticus A. nomius

Mazandaran M1 5 5 0 1

M2 5 4 0 0

M3 5 3 0 0

M4 5 1 0 1

M5 5 6 0 0

M6 5 3 0 0

M7 5 7 0 0

M8 5 5 0 0

M9 5 3 1 1

M10 5 2 0 1

Semnan S1 5 1 0 0

S2 5 0 0 0

S3 5 4 0 0

S4 5 1 0 0

S5 5 0 1 1

S6 5 3 0 0

S7 5 5 0 0

S8 5 3 0 0

S9 5 2 0 0

S10 5 0 0 1

Total 20 100 58 2 6

* The position of all locations is shown in Figure 1.

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aflatoxins and CPA were extracted from the cul-tures using chloroform as a solvent [23, 24]. Thechloroformic extracts were then concentrated by arotary evaporator (EYELA N-1000, Japan) nearto dryness and analyzed using thin layer chroma-tography (TLC) on 20�20 cm silica gel 60 F254

plates (E. Merck, Germany) in comparison withrelated standards. For CPA, the plates were pre-treated by dipping in 2% aqueous oxalic acid anddrying for 1 h at 100 �C according to Dorner [24].TLC plates were developed using chloroform–methanol (98:2, vol/vol) for aflatoxins [23] andtoluene–ethyl acetate–formic acid (5:4:1, vol/vol/vol) for CPA [24]. Aflatoxins B and G types wereobserved under U.V. light (365 nm) as blue andgreen spots, respectively. Aflatoxin concentrationwas measured using CAMAG TLC Scanner 3 at365 nm by comparison of under-curved areas of

standards and the samples. Estimation of theAFB1/AFB2 ratio in selected isolates were carriedout spectrophotometrically after eluting the afla-toxin spots from silica gel plates according to theprocedure of Nabney and Nesbitt [25]. Also, afterinitial qualitative and quantitative assays, all TLCplates were subjected to spray with 25% sulfuricacid which allows to color change of aflatoxinspots from blue-green to yellow. CPA was visual-ized in daylight after spraying with Erlich’s reagent(1 g p-dimethylaminobenzaldehyde in 75 ml etha-nol and 25 ml concentrated HCl) as blue spot withRf equal to 0.68.

Statistics

The results were analyzed by SPSS version 10programme for Windows (SPSS Inc., Chicago,

Figure 1. Relative position of the two provinces and sampling locations (black circles) is shown in Iran map.

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Illinois). Analyses of variance (ANOVA) andTukey’s multiple comparison test at 5% signifi-cance level were used to compare the means offungal incidence and toxin production data.P<0.05 was considered significant.

Results

Incidence of Aspergillus section Flavi in corn fields

In this study, out of 100 soil samples collected, 55were found to be contaminated with Aspergillussection Flavi species. A total of 66 Aspergillussection Flavi strains were isolated from bothSemnan (22 isolates) and Mazandaran (44 isolates)provinces (Table 1). As shown in Table 1, major-ity of the isolates were identified as A. flavus.Among the Aspergillus section Flavi isolates, 58(87.9%) were identified as A. flavus, 2 (3.0%)A. parasiticus and 6 (9.1%) as A. nomius based onmorphological characteristics and mycotoxin pro-files. These strains are now available in the CultureCollection of Mycology Department of PasteurInstitute of Iran as A. flavus PICC-AF1 to PICC-AF58, A. parasiticus PICC-AP1 and PICC-AP2and A. nomius PICC-AN1 to PICC-AN6. TheA. parasiticus isolates were rare in these fields.Only one A. parasiticus was isolated from Semnanprovince and the other one was obtained fromMazandaran region. A. nomius isolates fromMazandaran and Semnan were 4 and 2 isolates,respectively.

Comparison of the population of these isolatesin dry and moist regions indicates that a largerproportion of A. flavus isolates belong to Maz-andaran (39 out of 58) as compared to Semnanregion (19 out of 58) (Table 1). A significant dif-ference was noticed in distribution pattern ofAspergillus isolates in the corn field soil samples(P<0.001).

Morphological studies

Study of fungal morphology carried out on theseisolates indicated that conidia in A. flavus and A.nomius are variable in shape. These conidia werefound to be relatively thin with usually finelyroughened walls. However, in A. parasiticusspherical uniform conidia with heavy rough wallsusually borne in radiated heads were observed. As

a consequence of producing shorter conidio-phores, A. parasiticus produced dark yellowish-green colonies which were more compact thanboth A. flavus and A. nomius (Figure 2). BothA. flavus and A. nomius isolates showed brightyellowish-green colonies. These colonies werefound to be morphologically indistinguishablefrom each other. Sclerotia which is considered as areliable macroscopic criteria for Aspergillus speciesdifferentiation was absent in A. nomius. These twospecies were identified as separate species based onother characteristics particularly mycotoxin pro-files.

Regarding to sclerotia-producing ability, only 4A. flavus isolates were capable to produce sclerotiaat day 10 of growth on CZA medium at 30 �C(Table 2). The sclerotia were spherical, relativelylarge, white in color at first becoming brownish-black in age. Figure 2 shows white and blackspherical sclerotia in A. flavus colony. Only one ofthese isolates was considered as aflatoxigenic afterculturing on YES medium. In contrast neitherA. parasiticus nor A. nomius could produce scle-rotia under similar conditions.

Mycotoxin production pattern

The ability of Aspergillus species to produce bothB and G types of aflatoxins as well as CPA aresummarized in Table 2. Preliminary screening ofaflatoxigenicity of the isolates was carried out on aspecific medium named aflatoxin-producing ability(APA) medium. Only 12.5% of aflatoxigenic A.flavus isolates showed blue fluorescence on APAmedium, whereas, all the A. parasiticus isolatesand half of the A. nomius isolates had AF potentialunder U.V. light (365 nm).

As shown in Table 2, all the A. parasiticus andA. nomius isolates produced four major aflatoxins(B and G groups) on YES medium. The A. para-siticus isolates produced relatively higher levels ofAFB1 compared to A. nomius isolates. AFB1 levelin these two species was in the range of 1.85–2.77 lg/ml and 0.15–1.18 lg/ml, respectively(Table 2). AF production in A. flavus isolatesshowed more variation as compared to otherspecies (Table 2). The concentration of AFB1

produced by A. flavus isolates was varied from 0.08to 2.29 lg/ml in YES medium. Approximately27.5% (16 out of 58) of A. flavus isolates wereconsidered as AF producers out of which 2 isolates

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produced only AFB1 and the other 14 isolatesproduced both B1 and B2 aflatoxins. It is inter-esting to note that two A. flavus isolates werecapable of producing more AFB2 than AFB1

(approximately 3-folds). None of A. flavus isolates

were able to produce AFG group. There was asignificant difference regarding to AFB contentsamong these isolates (P<0.01).

As shown in Table 2, CPA production waslimited to A. flavus isolates collected from bothSemnan and Mazandaran provinces. Table 3shows that A. flavus isolates from corn field soilscould be classified among five chemotypes basedon AF and CPA production patterns. All chemo-types except chemotype II were reported fromboth climatic regions studied. The isolates of thislatter new classified chemotype were only identi-fied in Mazandaran province. A major proportionof A. flavus isolates (75%) produced both myco-toxins. There was a positive correlation betweenaflatoxin B1 and CPA production by A. flavusisolates (P<0.001). A remarkable percentage ofA. flavus isolates were non-toxigenic related toboth AFs and CPA (50%). A few A. flavus isolatescould produce AFB group which were failed toCPA production (6.9%), while a considerablenumber of the isolates were also found to produceonly CPA (22.4%). Despite the production ofaflatoxins B and G types, none of the A. parasiti-cus or A. nomius isolates were able to produceCPA on YES medium as indicated in Table 2.

Discussion

Production of aflatoxins is mainly reported tooccur by some strains belonging to three majorspecies A. flavus, A. parasiticus and A. nomius [4].However, in recent years, some other Aspergillusspecies including A. bombycis, A. pseudotamariiand A. ochraceoroseus as well as two Emericellaspecies were found to be capable of producingaflatoxins, but, they have less frequent distributionin nature [26]. Although aflatoxigenic Aspergillusstrains have been reported from various crops andagricultural commodities, agricultural soil servesas the main reservoir of this fungi all over theworld [7, 11–13]. Surveys of Aspergillus sectionFlavi revealed that the diversity and proportions ofisolates from different geographical regions whichare able to produce different types of aflatoxins atvarious levels may be quite different [7, 20, 27, 28].

Preliminary study carried out on pistachio nutsin Iran showed the frequent contamination of nutswith aflatoxigenic Aspergillus species [29]. Thesefungi were identified as A. parasiticus and A. flavus

Figure 2. Colony appearance of (A) A. parasiticus, (B) A. flavusand (C) A. nomius after 10 days cultivation on Czapek dox agarat 30 �C. A. parasiticus is highly compact and show darkeryellowish-green appearance compared with two other species.As shown, A. flavus and A. nomius are indistinguishable mor-phologically. Sclerotia production is only seen for A. flavus.

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according to their aflatoxin profiles and morpho-logical characteristics.

In the present study, distribution of Aspergillussection Flavi was studied in corn field soils withemphasis on the climatic differences and the spe-cial reference to mycotoxigenic species. Threemajor aflatoxigenic species including A. flavus,A. parasiticus and A. nomius were isolated fromboth Semnan and Mazandaran provinces withvarious distribution patterns. These species wereidentified based on a combination of morpholog-ical characteristics, sclerotia-producing ability aswell as aflatoxins and CPA profiles. A. flavusencountered more frequently as compared to bothA. parasiticus and A. nomius in soil samplesregardless the geographic source of soils. Similarresults have been reported from other countries[27, 28]. It has been reported that A. parasiticus iscertainly widely distributed in soils from tropicaland subtropical regions and its presence in food-stuffs and other substrates is less frequent [4].Likewise, A. nomius is considered as a rare speciesisolated from alkali bees in the USA [4], maize fieldsoils and different commodities, nuts and oilseedsfrom Thailand [30–32] and also from sugarcanefield soil in Japan with a less occurrence than theother isolated species A. flavus and A. parasiticus[20].

Data from different geographic areas demon-strated a great variability in the mycotoxin-pro-ducing potential of A. flavus and closely relatedspecies [7]. Based on TLC analysis, we showed thatall A. parasiticus and A. nomius isolates were ableto produce four major aflatoxins from both B andG types. These results are in accordance with otherreports showing that these two species have theability for producing both B and G aflatoxins [4,19, 20]. However, only 28% of A. flavus isolates

were capable of producing aflatoxins B group.Vaamonde et al. [28] reported that A. flavus strainsfrom various crops may be different in their APA.They showed that 29% of the total A. flavus iso-lates were able to produce aflatoxin. Horn andDorner [7] showed that the majority of A. flavusisolates (88%) from soils of USA were aflatoxi-genic. It has been shown that modern agriculturalmanagements are responsible for creating uniqueecological niches which select toxigenic fungi withan unknown mechanism. Hence, the aflatoxigenicand non-aflatoxigenic fungi can occur concur-rently in a natural habitat [33]. The presence ofthese fungi in a ecological nich can cause seriousproblems regard to their consistent interactionstoward toxigenic–non-toxigenic interconvertion.The incidence of conidia from non-aflatoxigenicfungi in air is always higher than the toxigenicones, so it is seemed that aflatoxigenic isolates arethe mutants of non-toxigenic isolates in an envi-ronment. These mutations probably occur as aconsequence of habitat diversity in air, soil,organic matters, rhizosphere, etc. [34]. It has beenshown that natural mutations often occur in con-version of saprophytic Alternaria alternata to itspathogenic form in the nature [35]. Thus, thehigher frequency of non-aflatoxigenic A. flavusstrains isolated in our study may be considered asa consequence of occurring such above-mentionedevents. Interestingly, we isolated two A flavusstrains with unique ability to produce more afla-toxin B2 than aflatoxin B1, a phenomenon whichhas not been reported earlier. Such isolates arepotential candidates for obtaining more dataabout phylogeny of aflatoxin-producing fungi andalso for getting more documented data regard tonot-fully understood final steps of aflatoxin bio-synthetic pathway where toxin separation between

Table 2. Production pattern of aflatoxins B and G types, CPA and sclerotia by Aspergillus section Flavi from Mazandaran andSemnan provinces, Iran

Aspergillus

section Flavi

Aflatoxin

production

(on YES)

AFB1 concentration range Fluorecence

on APA

CPA

production

(on YES)

Sclerotia

production

(on CZA)

B1 B1, B2 B1, B2, G1, G2 On YES (lg/ml) On APA (lg/g)

A. flavus 2/58 14/58 0/58 0.08–2.29 0.00–1.69 2/58 25/58 4/58

A. parasiticus 0/2 0/2 2/2 1.85–2.77 0.41–1.05 2/2 0/2 0/2

A. nomius 0/6 0/6 6/6 0.15–1.18 0.00–0.51 3/6 0/6 0/6

A. parasiticus NRRL 2999 produced AFB1 at concentrations of 5.86 lg/ml and 0.58 lg/g on YES and APA media, respectively.

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B and G types takes place. Meanwhile, furtherstudies are currently performed to verify thisfinding by more sensitive methods such as HPLC.

CPA production was limited to A. flavus iso-lates and none of the A. parasiticus and A. nomiusisolates could produce CPA. Based on reports byHorn and Dorner [7] and Vaamonde et al. [28],various proportions of A. flavus isolates werecapable of producing CPA. Furthermore, a posi-tive correlation has been established betweenaflatoxin and CPA production by A. flavus iso-lates. We classified A. flavus isolates into fivechemotypes (I–V) based on aflatoxins and CPAproduction patterns including a new chemotypewhich was able to produce higher amounts ofaflatoxin B2 than aflatoxin B1 (Table 3). The typeV (aflatoxin and CPA negative) was found to bethe most prominent chemotype which comprisedmore than half of A. flavus isolates.

The occurrence of aflatoxigenic A. flavus iso-lates from Mazandaran and Semnan provinceswere recorded as approximately 30% and 20%,respectively. Overall, about 75% of aflatoxigenicA. flavus isolates were obtained from Mazandaranprovince with high relative humidity by average of70%. All sclerotia-producing A. flavus strains (4out of 58 isolates) were isolated from Mazandaranprovince. In the present study, a few number ofA. flavus isolates could produce sclerotia out ofwhich only one isolate was considered as aflatoxi-genic. These results may further suggest that scle-rotia formation is poorly related to aflatoxinproduction.

Although factors responsible for the toxigenic-ity profile of Aspergillus section Flavi populationsin a geographic region have not been defined, butvarious factors such as temperature, soil condition,day length, insect levels, crop sequence history,rainfall frequency and management practices may

influence the relative proportions of AspergillusFlavi communities [36]. The occurrence of a rela-tively large number of toxigenic A. flavus andA. parasiticus in Mazandaran province comparedto Semnan province in the present study may beindicate a possible role of high relative humidity insupporting the growth of aflatoxigenic fungi.

Overall, aflatoxigenic fungal strains from threespecies viz. A. flavus, A. parasiticus and A. nomiusmay present in agricultural soils where their air-borne conidia can contaminate different cropseither directly or indirectly. This study is the firstreport on the distribution of A. nomius in Iran.Existence of a population of non-toxigenic A. fla-vus may be important in biological control of pre-harvest aflatoxin contamination of crops throughtheir application to the soil. A better understand-ing of genetic variability, population diversity, andfungal–fungal or host–fungal interactions withinand between field Aspergillus section Flavi popu-lations is promising for planning suitable strategiesin effective management of these important groupof fungi in the field.

Acknowledgements

This work was financially supported by PasteurInstitute of Iran (Grant No.178). The authors arevery grateful to Prof H.G. Raj, Patel Chest Insti-tute, Delhi, India for providing aflatoxigenicA. parasiticus NRRL 2999 strain.

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Address for correspondence: Mehdi Razzaghi-Abyaneh, Assis-tant Professor of Mycology, Pasteur Institute of Iran, 13164Tehran, IranPhone: +98 21 6953311-20; Fax: +98 21 6465132E-mail: mrab442@yahoo.com

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