Mycopathologia 155: 207211, 2001. 2002 Kluwer Academic Publishers. Printed in the Netherlands. 207

Mycoflora of freshly harvested flint corn from Northwestern Provinces inArgentina

H.H.L. Gonzalez1,2, S.L. Resnik3,4 & A.M. Pacin4,51Consejo Nacional de Investigaciones Cientficas y Tecnicas, Argentina; 2 Facultad de Ingeniera, Universidad deBuenos Aires, Argentina; 3 Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina; 4Comision de Investigaciones Cientficas de la Provincia de Buenos Aires, Argentina; 5 Universidad Nacional deLujan Centro de Investigacion en Micotoxinas, Argentina

Received 20 February 2001; accepted in revised form 22 June 2002

Abstract

A mycological survey was carried out for the first time, on red flint corn samples from the northwestern Andinianregion of Argentina in the 1999 and 2000 harvest seasons. Species of the genus Fusarium were the most prevalentcomponent of the flint corn mycoflora present in all provinces. F. verticillioides was the predominant Fusariumisolated in the 1999 harvest season in the the region, and was found at higher incidence level than those observedon commercial semident corn hybrids harvested in the main corn production area in Argentina (Pampean region).During the 2000 harvest season, Fusarium graminearum was most commonly isolated species in Salta province.

Key words: flint corn, Fusarium verticillioides.

Introduction

Information about fungi associated with cereal grainsis important in assessing the risk of mycotoxin con-tamination. A number of these fungal species, belong-ing mainly to the genera Fusarium, Aspergillus andPenicillium, have been reported to produce mycotox-ins that cause mycotoxicoses of domestic animals andman [1].

Flint corn (Zea mays L. var indurata (Sturtevant)Bailey) is a cereal grain produced in, and exportedfrom, Argentina. Flint type is similar to pop corn butwith larger grain, and it was probably developed frompop types by selection for grain size and greater yield.Flint corn is produced in areas where cold toleranceis required or where storage and germination condi-tions are poor, most of this corn type accounts for14% of commercial world production [2], and cur-rently in Argentina, is used in corn-based foodstuffssuch as corn flakes or corn meal (polenta) as wellas in corn-based feeds for poultry [3], because thestrong redish yellow colour is important to poultryskin pigmentation.

* Published in 2002.

The main concentration of the cultivated corn areasare between latitudes 32 and 35 of South, and the ma-jor corn-producer provinces are Buenos Aires, SantaFe and Crdoba. Currently information is only avail-able on the fungal species associated with semidentcommercial hybrids in the main corn production area[4, 5]. The production area of flint corn in Argentinahas an important regional production at the Northwest-ern provinces, which is a marginal region for corngrowing, with c.a. of 470,000 tons of production,mainly exported for the corn flakes industry [6].

Samples of semident corn in Argentina have beenshown to be contaminated with aflatoxins and zear-alenone [7] and some field-trial semident corn samplesfrom the main production area have been infestedby Fusarium verticillioides and contaminated withthe toxic metabolites of this fungus, the fumonisins[8], and zearalenone [9]. A preliminary study of my-cotoxin (fumonisins, deoxynivalenol, zearalenone andaflatoxins) presence in corn-based products (corn mealand corn flakes) has also shown that fumonisin con-tamination is the main mycotoxin problem concerningfor human consumption in Argentina [10].

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The aims of this study were to identify the fungiassociated with red flint corn kernels harvested in anextensive region of corn production in NorthwesternArgentina in 1999 and 2000, to establish the speciesdistribution of mycotoxicological interest, to comparethe mycoflora at different provinces, and to improvethe existing information on the mycological behaviourduring the harvest seasons in order to undertake a basicrisk evaluation of the occurrence of mycotoxins.

Materials and methods

Corn samples. Red flint corn samples belonging tothe most prevalent commercial hybrid sown: Cargill350, were collected as follows: at harvest, subsamplesnot greater than 50 g were periodically taken fromeach lot, homogenized, quartered and divided to give5 Kg samples. These samples were used in the my-cological analysis. The red flint corn kernels sampleswere collected at the northwestern production area inArgentina corresponding ecologically to the North-western Andinian region (NOA). In 1999, 47 sampleswere taken from Salta and 37 from Tucumn provincesand at 2000 harvest season 43 and 23 respectively.

Isolation of fungi. Subsamples of corn kernels fromeach sample were surface-sterilised in a commercial5% aqueous solution of sodium hypochlorite for 1minute, rinsed twice with sterile distilled water, anddried in a laminar flow cabinet. One hundred cornkernels per sample were placed, 20 kernels per plate,on YGCA (Yeast extract-Glucose-ChloramphenicolAgar, Merck No. 16000). The plates were incubatedin the dark at 28 C for 47 days and the result-ing fungal colonies subcultured onto PDA (Potato-Dextrose Agar, Merck No. 10130) and identified.Where several fungi were isolated from a single ker-nel, all were recorded.

Identification of fungi. Isolates of fungi were identi-fied according to the following authorities: Fusariumspp., according to Nelson et al. [11]; Penicillium spp.,Aspergillus spp., and other fungi according to Pitt andHocking [12]. The isolation frequency (Fr) and relat-ive density (RD) of species were calculated accordingto Gonzlez et al. [4] as follows:Fr (%) =[

No. of samples of occurrence of a speciesTotal No. of samples

] 100

RD (%) =[

No. of isolates of a speciesTotal No. of fungi isolated

] 100

Statistical analysis. Asymptotic tests for equality ofproportions were used to compare the relative densit-ies of fungal species [13], and the Fischer exact testwas used to analyze possible differences in the isola-tion frequencies of fungal species. The analysis wasperformed by using the Statistix 4.1 package [14].

Results and discussion

Fungi associated with corn kernels. The potentiallytoxigenic internal mycoflora associated with flint cornkernels collected in the Northwestern provinces ofSalta and Tucumn, Argentina during 1999 and 2000harvest seasons are shown in Table 1. Based on Fr aswell as RD, Fusarium showed the highest mean incid-ence. Other genera isolated as significant componentsof the internal mycoflora included Nigrospora, Peni-cillium and Trichoderma. Rhizopus was also isolatedfrequently as internal mycoflora. Species of the genusAspergillus were isolated at both low Fr and RD inSalta and Tucumn during the 1999 harvest in com-parison to the levels observed during the 2000 harvestseason.

The incidence of Fusarium species in red flintcorn kernels collected in Salta and Tucumn during1999 and 2000 harvest seasons is given in Table 2.It can be seen that F. verticillioides was by far themost prevalent Fusarium spp in the internal mycoflora,at the 1999 harvest season with respect both to theRD and Fr. The Fr of F. verticillioides in this regionwas higher than that observed in freshly harvestedsemident corn hybrids from Buenos Aires, Crdobaand Santa Fe provinces by Gonzlez et al. [4], andhigher than in commercial hybrid corn harvested inEntre Ros [5]. The prevalence of F. verticillioides asan internal seedborne fungus in commercial Argen-tinian flint corn kernels is similar to the situation inother corn-producing countries for other corn types[1518]. The well known ear-rot pathogen of corn, F.graminearum, was isolated as the second most preval-ent Fusarium species in the flint corn collected in Saltaand Tucumn during 1999; but was the most preval-ent Fusarium species recovered from red flint corn inSalta during the 2000 harvest. Fusarium semitectumwas only isolated in Salta at low RD and Fr in both

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Table 1. Mycotoxigenic fungal genera recovered from flint corn kernels collected in Salta and Tucuman provinces,Argentina, during 1999 and 2000 harvest seasons.

Salta Tucuman1999 2000 1999 2000

Genera RD Fr RD Fr RD Fr RD Fr

Alternaria 1.1 18.9 nd nd 0.4 9.3 2.5 10.2Aspergillus 0.06 5.7 1.5 24.1 0.03 2.3 0.3 10.2Fusarium 88.7 100.0 60.0 96.6 85.5 100.0 36.0 79.6Penicillium 1.0 28.3 1.2 27.6 3.6 23.3 2.3 22.5

RD: Relative density (%); Fr: Frequency of isolation (%) nd: not detected.

Table 2. Fungal species recovered from flint corn kernels collected in Salta and Tucuman provinces, Argentina, during 1999 and 2000harvest seasons.

Salta Tucuman1999 2000 1999 2000

Species RD Fr RD Fr RD Fr RD Fr

Alternaria alternata 1.2 21.3 nd nd 0.4 10.8 0.4 13.0Arthrinium phaeospermum 0.4 4.3 nd nd nd nd nd ndAspergillus flavus 0.02 2.1 0.4 9.3 0.03 2.7 0.4 13.0Aspergillus niger 0.05 2.1 nd nd nd nd nd ndBipolaris sorokiniana 0.02 2.1 nd nd nd nd nd ndCladosporium cladosporioides 0.02 2.1 0.3 14.0 0.2 16.2 0.05 4.4Curvularia lunata 0.5 8.5 2.8 16.3 0.7 10.8 16.1 39.1Chrysonilia sitophila nd nd 1.5 2.3 nd nd 1.8 8.7Diplodia zeae 0.4 10.6 3.9 9.3 0.06 2.7 8.1 26.1Epicoccum nigrum 0.02 2.1 nd nd 0.06 2.7 nd ndFusarium graminearum 10.5 74.5 62.6 76.7 13.4 62.2 9.1 69.6Fusarium semitectum 0.07 2.1 0.1 4.7 nd nd nd ndFusarium verticillioides 74.3 100.0 6.6 44.2 69.9 91.9 18.8 60.9Mucor racemosus 0.1 4.3 0.2 2.3 0.09 2.7 1.4 4.4Nigrospora oryzae 4.0 14.9 20.8 58.1 7.4 35.1 38.0 69.6Penicillium funiculosum 1.2 31.9 0.7 20.9 4.3 27.0 1.4 30.4Phoma glomerata nd nd 0.1 2.3 nd nd 0.3 4.4Rhizopus stolonifer 1.0 29.8 nd nd 1.4 54.1 0.2 17.4Trichoderma harzianum 2.6 6.4 nd nd 1.2 5.4 4.1 4.4

RD: Relative density (%); Fr: Frequency of isolation (%); nd: not detected.

Table 3. Meteorological data for the 1998/99 and 1999/00 flint corn crop seasons in Salta and Tucuman provinces,Argentina .

Provinces Total rainfall (mm) Maximum temperature (C) Minimum temperature (C)1998/99 1999/00 1998/99 1999/00 1998/99 1999/00

Salta 120.3 96.8 25.1 25.1 12.6 12.6Tucuman 124.3 125.6 27.3 27.6 15.6 15.9

Average data for period AugustMarch. Data supplied by the Servicio Meteorologico Nacional, Argentina.

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Table 4. p-values for the RD and Fr comparison between 1999 and 2000 harvest seasonsat Salta and Tucuman provinces, Argentina.

Salta TucumanRD Fr RD Fr

Alternaria alternata np np 0.8646 1.0000Aspergillus flavus 0.0003a 0.1888 0.0168b 0.2880Fusarium graminearum 0.0000a 0.8120 0.0000a 0.5916Fusarium semitectum 0.7567 0.6043 np npFusarium verticillioides 0.0000a 0.0000a 0.0000a 0.0064aPenicillium funiculosum 0.0244b 0.3401 0.0000a 1.0000

a p< 0.01 (highly significant differences).b p< 0.05 (significant differences).np: test not performed.

Table 5. p-values for the RD and Fr comparison between Salta and Tucuman provincesin 1999 and 2000 harvest season.

1999 2000RD Fr RD Fr

Alternaria alternata 0.0000a 0.2479 np npAspergillus flavus 0.7844 1.0000 0.7353 0.6873Fusarium graminearum 0.0001a 0.2445 0.0000a 0.5641Fusarium semitectum np np np npFusarium verticillioides 0.0000a 0.0815 0.0000a 0.3015Penicillium funiculosum 0.0000a 0.6413 0.0306b 0.6413

a p < 0.01 (highly significant differences).b p < 0.05 (significant differences).np: test not performed.

harvest seasons. It could be possible that the slightlylower rainfall measured at Salta in the 1999/2000 cropseason, could be the reason for the differences betweenF. graminearum and F. verticillioides incidence levelsin this province (Table 3).

The only species identified among the Penicilliumisolates, at both harvest seasons, was P. funiculosumThom (Table 2). Similarly, Hesseltine et al. [19] foundthat 28.6% of semident corn kernels from samplesharvested in North Carolina, USA, were infected byPenicillium spp., and 1.6% with P. funiculosum. Inthe Southeastern United States, P. funiculosum, P.purpurogenum and P. citrinum are characteristic ofpreharvest semident corn colonization. Nevertheless,the flint corn samples harvested in NOA during 1999and 2000, revealed higher Fr for P. funiculosum thanthose observed by Hesseltine et al. [19].

The incidence of Alternaria alternata is shownin Table 2. This fungus is one of the most commonairborne fungi which colonizes cereal crops such ascorn and subepidermal penetration may occur and isassociated with ear rot [20].

The predominant Aspergillus spp isolated intern-ally was A. flavus in Salta and Tucumn at both harvestseasons and the other Aspergillus spp identified wasA. niger, which was isolated only in Salta provinceduring the 1999 harvest season (Table 2).

Of the fungi involved in dry ear-rots (cob, kernelsand stalk rot) Nigrospora oryzae [20] was isolated atrelatively high Fr in the 2000 harvest season in bothprovinces; meanwhile Diplodia zeae (= Stenocarpellamaydis), associated with white ear, root and stalk rot[20], was isolated at low Fr and RD in both harvestseasons. It has been noted that in Argentina Diplodiaspp. are relatively important as ear-rot pathogens in thepampean region only in dry seasons [4]. Chrysoniliasitophila and Phoma glomerata were only recorded inSalta and Tucumn in the 2000 harvest season.

In Tables 4 and 5 the p values are listed for com-paarisons of RD and Fr for selected fungal speciesbetween Salta and Tucumn and between 1999 and2000. Due to the relatively small number of samplescollected at both provinces, the Fisher exact test wasperformed for Fr.

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The statistically significant differences in isolationfrequencies can not be explained simply by the tem-peratures and total rainfall observed (Table 3). Forexample highly significant differences were observedin the RD and Fr of F. verticillioides at Tucumn in thecomparison between both harvest seasons, howeverthe total rainfall and temperature were quite similar inboth seasons (Tale 3) and so, other factors could influ-ence the mycoflora, one possibility being the rainfallfrequency [21].

In conclusion, this is the first report of the contam-inating mycoflora in a marginal corn producing area inArgentina; and the study has also provided for the firsttime, information on the internal mycoflora of freshlyharvested flint corn kernels for this area. Fungi asso-ciated with flint corn that should be of concern due totheir toxigenic potential include F. verticillioides, F.graminearum, P. funiculosum, Aspergillus flavus andAlternaria alternata. The recovery of F. verticillioidesin the flint corn harvested in the northwestern Argen-tinian region is higher than that observed in the maincorn production area, and mycotoxins produced bythis fungus, particularly the fumonisins may also bepresent.

Acknowledgements

The authors acknowledge the financial support fromCargill Foundation, Consejo Nacional de Investiga-ciones Cientficas y Tcnicas, Comision de Investiga-ciones Cientficas de la Provincia de Buenos Aires,Universidad de Buenos Aires, Universidad Nacionalde Lujn and Agencia Nacional para la PromocinCientfica y Tecnolgica. The authors also acknow-ledge with great thanks the useful co-operation ofMrs. M.E. Mdena, Lic. L.E. Broggi and Bqco. M.D.Castillo.

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Address for correspondence: H.H.L. Gonzlez, Departamento deIndustrias, F.C.E. y N., Universidad de Buenos Aires, Ciudad Uni-versitaria, (1428), Nez, Buenos Aires, ArgentinaPhone and Fax: (54-11) 4631-1148 e-mail: lucas@di.fcen.uba.ar