Directory UMM :Journals:Journal of Stored Products Research:Vol 36.Issue1.Jan2000:

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Myco¯ora and occurrence of fumonisins in freshly

harvested and stored hybrid maize

Reinaldo B. Orsi

a

, Benedito CorreÃa

a,

*, Claudia R. Possi

b

, Eliana

A. Schammass

b

, Jose R. Nogueira

b

, Silvia M.C. Dias

c

, Marisa A.B. Malozzi

c a

Departamento de Microbiologia, Instituto de CieÃncias BiomeÂdicas, Universidade de SaÄo Paulo, Brazil b

Instituto de Zootecnia, Universidade de SaÄo Paulo, Brazil c_{Instituto Biologico, Universidade de SaÄo Paulo, Brazil}

Accepted 9 June 1999

Abstract

The study of the myco¯ora in stored grain permits an evaluation of cereal storage conditions that aect grain deterioration and the risk of mycotoxin contamination. Abiotic factors can directly aect the relative frequency of fungal populations in stored grain. The aim of the present work was to study the in¯uence of abiotic factors on variations of myco¯ora of freshly harvested and stored maize in Brazil and the occurrence of fumonisins. Samples (195) of three hybrids of maize were analyzed monthly during one year. Microbiological analysis revealed a predominance ofFusariumspp, which presented the greatest total number of colony forming units per gram in the three hybrids, namely: Br 201 (11104

to 5340104 CFU/g), C 125 (18104 to 2790104 CFU/g) and Cx 322 (25104 to 2940104

CFU/g), followed byPenicillium spp, Aspergillus spp and 10 other fungal genera. Fusarium moniliforme

Sheldon was the most prevalent species (59.2% of Fusarium isolates in Br 201, 55.4% in C 125 and 69.2% in Cx 322). Fusarium spp showed signi®cant negative correlations with mean temperature and relative humidity of the air. Higher temperatures and relative humidity at the end of the study and high moisture content at the beginning of the study were observed. The CFU/g values recorded for the three predominant genera exceeded the internationally accepted tolerance limits. The mycotoxicological evaluation indicated contamination of 176 samples (90.2%) with fumonisin B1 and of 190 samples

Keywords:Myco¯ora; Fumonisins;Aspergillus ¯avus;Fusarium moniliforme; Stored maize; Fumonisins in maize Journal of Stored Products Research 36 (2000) 75±87

www.elsevier.com/locate/jspr

* Corresponding author. Tel.: +55-11-818-7295; fax: +55-11-818-7354.

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1. Introduction

In Brazil, maize has historically had an important role in both human and animal nutrition. Even before the arrival of European colonizers, the Brazilian natives already cultivated this cereal. At present, Brazil is ranked as the third largest producer of maize in the world, coming after the USA and China. Of the Brazilian annual yield of about 32 million tonnes in recent years (Tsuneshiro and Okawa, 1996), 41% is used by the poultry and swine industries. A substantial part of this crop comes from small producers and approximately 24.7% of the harvest is stored and used on the producing farms (Pedrosa and Dezen, 1991).

In the state of SaÄo Paulo, maize plantations make up 7% of the cultivated land (Francisco et al., 1997). The mean productivity of 2747 kg/ha recorded for this region during the 1990± 94 period re¯ects a poor local technology (Tsuneshiro and Okawa, 1996). The maize-producing farms in this state have a mean area of 13.3 ha; 61.9% of the farmers receive technical assistance, 44.1% use soil analysis, and 46.2% use hybrid seed (Francisco et al., 1997).

The overall world loss of grains is considered to be 5% of the total production (FAO/WHO/ UNEP, 1977). In Brazil, losses of 10±25% (Pedrosa and Dezen, 1991) have been estimated to occur throughout the trading process, partly due to contamination with toxigenic fungi and mycotoxins. Improved storage conditions would allow a 10±20% increase in the supply of food available to people (Christensen and Kaufmann, 1969).

The fumonisins are secondary metabolites produced by Fusarium moniliformeSheldon and F. proliferatum (Matsushima) Niremberg (Bacon and Nelson, 1994). They show a worldwide distribution and can be isolated from maize and maize-based food and feedstus naturally contaminated withFusarium. Of the seven fumonisins currently identi®ed (Bezuidenhout et al., 1988; Cawood et al., 1991; Musser, 1996), fumonisins B1 (FB1), B2 (FB2) and B3 (FB3) are the

most frequently detected in fungal cultures or in naturally contaminated maize, a component of human and animal feed in many countries (Sydenham et al., 1991; Doko et al., 1995; Visconti, 1996; Thiel et al., 1996).

The fumonisins have been associated with leukoencephalomalacia (ELEM) in equines (Ross et al., 1991; Sydenham et al., 1992), porcine pulmonary edema (PPE; Colvin and Harrison 1992; Osweiler et al., 1992), diarrhea and reduced body weight in broiler chicks (Brown et al., 1992), carcinogenicity in rats (Gelderblom et al., 1991) and leukoencephalomalacia and hemorrhage in the brain of rabbits (Bucci et al., 1996). In addition, epidemiological evidence suggests a correlation between the consumption of F. moniliforme contaminated maize and a high incidence of human esophageal carcinoma (Thiel et al., 1992, Rice and Ross, 1994).

Based on the above considerations, the present investigation was undertaken in order to: (a) identify the myco¯ora of local maize hybrids freshly harvested and after storage in normal facilities in SaÄo Paulo, Brazil; (b) determine the occurrence of fumonisins B1 and B2 in the

cereal grains; and (c) correlate the results with data on abiotic factors (temperature, moisture content, water activity, and relative humidity).

R.B. Orsi et al. / Journal of Stored Products Research 36 (2000) 75±87

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2. Material and methods

2.1. Samples

A total of 195 samples of three maize hybrids: Br 201 (Embrapa Milho e Sorgo, Rod. MG 424, Km 65, Sete Lagoas/MG, Brazil, CP 151, Cep 35701-970), C125 (Cargill, R. Jacarezinho, AndiraÂ/PR, Brazil, Cep 86380-000) and Cx 322 (ICI Sementes, Rod. Anhanguera, Km 296, Cravinhos/SP, Brazil, Cep 14140-000), which are commonly planted in the State of SaÄo Paulo, were used. The samples (15 of freshly harvested grains and 180 of stored grains) were collected in RibeiraÄo Preto, State of SaÄo Paulo, Brazil, a region characterized by humid tropical weather with hot and rainy summers and dry winters. After harvest, the maize grains were put in 60 kg jute sacks (5 sacks per hybrid), stacked over wooden boards and stored for 12 months in a well-ventilated warehouse, located near the production area. Monthly samples were collected from various points of each sack (Fonseca, 1991), until a total of 1 kg grains/sack was obtained (one sample per month per sack).

2.2. Moisture content and water activity

The moisture content of the maize grains was determined in the grain-storage areas immediately after sampling, using a ``Brow Duvel'' moisture meter (Model CA 2511 manufactured by Gehaka Co., SaÄo Paulo, Brazil). Water activity was determined by automated analysis with the equipment AQUALAB CX-2 (Decagon Devices Inc.). Each sample was measured ®ve times.

2.3. Recovery, identi®cation and enumeration of the myco¯ora

A 10 g sample of maize from each of the 195 samples was ground and mixed with 90 ml of sterile distilled water to obtain a 10ÿ1 _{stock dilution, from which 10-fold serial dilutions up to} 10ÿ6_{were made using the same diluent. Duplicate 1 ml volumes of each dilution were added to} Petri dishes containing 10±15 ml of Potato Dextrose Agar (Swanson et al., 1992). The plates were then incubated at 258C for 5 days and observed daily. Plates that contained 15 to 150 CFU were used for counting and the results were expressed as CFU per gram of sample (Mislivec et al., 1992). The fungal colonies recovered were identi®ed according to the methods for each genus (Raper and Fennell, 1965; Von Arx, 1974; Nelson et al., 1983; Nelson, 1992).

2.4. Determination of fumonisins

The maize samples were analyzed for fumonisins B1 (FB1) and B2 (FB2) according to the

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water and the toxin eluted with 2 ml of a 70:30 mixture of acetonitrile/water. The ®nal extract was collected in Eppendorf tubes and kept atÿ208C until use.

Two hundred microliters of the ®nal extract were derivatized with 50 ml of a solution of o -phthaldialdehyde (OPA) (40 mg of OPA dissolved in 1 ml of methanol and diluted in 5 ml of 0.1 M sodium tetraborate containing 50 ml of mercaptoethanol). The product of this reaction was analyzed by a reverse-phase isocratic HPLC system consisting of a Shimadzu SCL-6B pump, an RF55 ¯uorescent detector (Shimadzu; excitation and emission wavelength of 355 and 440 nm, respectively) and a 1504.6 mm C18 column (5ODS-20, Phenomenex). The eluent

was methanol/sodium acetate buer (77:23), pH 3.6. Calibration of the apparatus was undertaken using stock solutions of standard fumonisins (Sigma) at the concentrations 0.0125, 0.025 and 0.05 mg/ml for FB1 and 0.005, 0.01 and 0.02 mg/ml for FB2. A recovery experiment

was carried out in quadruplicate with toxin levels that varied from 4±24 ng of FB1 and 8±56

ng of FB2 per gram of ground maize. The recoveries of FB1 and FB2 were 88% and 94%,

respectively. The detection limit was 50 ng/g for both FB1and FB2.

2.5. Climatic data

During storage, the climatic conditions relating to temperature (8C) and relative humidity (%) were recorded as monthly averages.

2.6. Statistical analysis

The experimental design used for each hybrid was totally randomized, with 13 storage time periods analyzed as orthogonal polynomials to obtain the regression equations. The dependent variables (CFU/g of Aspergillusspp, Fusarium spp, Penicillium spp and fumonisins B1, and B2)

were transformed into loge (x+ 1) (Steel and Torrie, 1960). Stepwise multiple regression and

studentized residuals were used to obtain the regression models.

Table 1

Absolute and relative (%) frequencies of fungi recovered from 195 samples of maize grains (hybrids Br 201, C 125 and Cx 322) collected over a one-year period in Brazila

Br 201 C 125 Cx 322

Aspergillus 22.5 (34.6) 20.0 (30.8) 13.0 (20.0)

Fusarium 55.0 (84.6) 60.0 (92.3) 56.0 (86.1)

Penicillium 50.5 (77.7) 47.0 (72.3) 33.5 (51.5)

Trichosporon 16.0 (24.6) 13.5 (20.8) 7.5 (11.5)

Cladosporium 29.0 (44.6) 20.0 (30.8) 31.5 (48.5)

N. S. F.b 2.5 (3.8) 3.0 (4.6) 4.0 (6.1)

a

For each hybrid, 65 samples were analyzed.

b

Non-sporulated fungi.

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3. Results and discussion

The fungi identi®ed in the 195 samples of maize analyzed (hybrids Br 201, C 125 and Cx 322) showed that Fusarium spp were the most frequent molds, followed by Penicillium spp,

Aspergillus spp, Trichosporon spp, Cladosporium spp, and non-sporulated fungi (Table 1). Another 8 genera of fungi were isolated at very low frequencies: Epicoccum spp, Mucor spp,

Rhizopus spp, Neurospora spp, Rhizoctonia spp, Curvularia spp, Trichoderma spp, and

Nigrospora spp. Fusarium moniliforme was the predominant Fusarium species and, within the genus Aspergillus, A. alutaceus Berkeley and Curtis (A. ochraceus) had the greatest frequency of recovery.Penicilliumisolates were not identi®ed to species.

The majority of the fungal isolates were recovered at aw values of 0.7±0.79 (Table 2). The

speci®c frequencies of the total species identi®ed were: F. moniliforme 59.2%, F. proliferatum

36.1%, F. anthophilum A. Braun 4.6%, A. alutaceus 10.8%, A. chrysellus Kwon and Fennell 3.8%, A. tamani Kita 3.8%, A. niger Van Tieghem 3.1%, A. ornatus Raper, Fennell and Tresner 2.3%,A. cremeus Kwon and Fennell 2.3%, A. nidulans Eidam 1.5%, A. candidus Link 1.5% andA. ¯avusLink 0.8% for hybrid Br 201; F. moniliforme55.4%, F. proliferatum42.3%,

F. anthophilum 9.2%, A. alutaceus 10.8%, A. tamarii 5.4%, A. chrysellus 4.6%, A. cremeus

3.1%,A. niger 2.3%, A. ¯avus 2.3%,A. glaucus Link 2.3%, and A. versicolor Tiraboschi 0.8% for hybrid C 125; and F. moniliforme 69.2%, F. proliferatum 38.5%, F. anthophilum 15.4%, F. clamydosporumWollenw 0.8%,F. nygamai Burgess 0.8%,A. alutaceus8.5%, A. ornatus3.1 %,

A. ¯avus 1.5%, A. chrysellus 0.8%, A. cremeus 0.8%, A. glaucus 0.8%, A. niger 0.8% and A. raperi Stolk 0.8%. for hybrid Cx 322. The greater prevalence of Fusarium spp found in the present investigation agrees with the data reported by other authors (Pozzi et al., 1995; Lillehoj and Zuber, 1988; Asevedo et al., 1994; Castro et al., 1995) who also describe this genus as that

Table 2

Absolute and relative (%) frequencies of the generaFusarium, Aspergillus andPenicilliumin 195 samples of maize grains (hybrids Br 201, C 125 and Cx 322) and range of water activity (aw) values recorded over a one-year period

in Brazil. For each hybrid, 65 samples were analyzed; AF=Absolute frequency; RF=Relative frequency; nd=not detected at dilution 10ÿ3

Hybrids Range ofaw Samples in each awgroup

Br 201a 0.79±0.70 7 26(20.0) 52(40.0) 51 (39.2)

0.69±0.67 3 10(7.7) 28(21.5) 23(17.7)

0.90±0.80 3 nd a A 20(15.4) a B 18 (13.8) a B C 125b _0.79±0.70 ₇ _{23 (17.7) b A} _{66(50.8) b B} _{50 (38.5) b B}

0.69±0.66 3 17 (13.0) b A 34(26.2) b B 26 (20.0) b B

0.90±0.80 1 nd 10(7.7) 8(6.2)

Cx 322a 0.79±0.70 11 23(17.7) 93(71.5) 56(43.0)

0.69±0.67 1 3(2.3) 9(6.9) 3(2.3)

a

(P> 0.05).

b

Frequencies followed by dierent lower case letters denote dierences in the rows. Frequencies followed by dier-ent capital letters denote dierences in the columns. Analysis among hybrids byw-square.

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Table 3

Fumonisin levels and number of colony forming units (CFU/g) of the fungi isolated from 195 freshly harvested and stored maize grains (hybrids Br 201, C 125 and Cx 322) collected over a one-year period in Brazila

Fumonisin levels (mg/g)b

Br 201 C 125 Cx 322 Br 201 C 125 Cx 322

Samples Genera FB1 FB2 FB1 FB2 FB1 FB2

Aspergillus ndd nd nd

FHc Fusarium 594.6 460.3 669.8 24.0 15.0 13.1 9.8 12.1 7.2

Penicillium 6.9 18.7 14.9

Aspergillus 1 nd 1

S1e Fusarium 1096 62.5 1415 17.1 11.7 17.2 8.4 7.5 6.5

Penicillium 276 104 41

S3 Fusarium 2616 1523 677 8.0 7.2 11.0 7.1 9.1 6.0

Aspergillus 90 55 31

S4 Fusarium 5340 286 170 14.1 9.8 6.8 10.1 3.3 7.9

S5 Fusarium 872 2790 2940 13.0 7.3 26.0 9.2 18.0 8.7

S6 Fusarium 460 550 490 3.6 6.5 4.1 7.5 6.0 7.2

S7 Fusarium 360 290 160 11.0 7.1 10.1 7.5 11.2 6.8

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Table 3 (continued)

Aspergillus 59.3/281.3 31.1/240.2 19.4/232.7

Meanf/SDg Fusarium 938.3/21442.3 573.7/2785.1 589.1/2789.6 10.5/25.9 8.1/22.5 10.5/26.1 7.8/21.4 8.0/23.9 6.8/20.9

Penicillium 681.8/2734.5 160.6/2183.4 55.7/267.6

a_{For each hybrid, 65 samples were analyzed.} b

Mean of 5 samples.

c

Freshly harvested.

d

Not detected at dilution 10ÿ3

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most frequently associated with Brazilian maize grains. The predominance of F. moniliforme

had already been observed in Brazil, as well as the occurrence ofF. proliferatum as the second most frequent species in maize (Pozzi et al., 1995). However, our results show higher frequencies of these fungi in the maize hybrids studied.

The three most important fungal genera in terms of toxigenicity produced the greatest numbers of colony forming units (CFU/g) in hybrid Br 201, varying from 11104 to 5340104 with Fusarium spp, from 0 to 280104 with Aspergillus spp and from 2104 to

2096104 with Penicillium spp. The values recorded for these fungal genera in the other two

hybrids were 18104 to 2790104 Fusarium spp, 0 to 140104 Aspergillus spp and 10104

to 620104 Penicillium spp for hybrid C 125 and 25104 to 2940104 Fusarium spp, 0 to

120104 Aspergillus spp and 2104 to 260104 Penicillium spp for hybrid Cx 322 (Table 3). These numbers exceed the tolerance limits set by the International Commission on Microbiological Speci®cation for Foods (Elliott, 1980), which are of the order of 102 to 104 CFU/g.

The genus Aspergillus was absent from the myco¯ora of the 15 samples of freshly harvested grains for the three hybrids studied (Table 3). In the remaining samples analyzed, species of

Aspergillus were found in grains with moisture content of 10.2±16.4%, the greater number of recoveries occurring between 12.4±16.4% and at water activities of 0.7±0.79 (Tables 2 and 4). The linear correlation analysis showed a signi®cant positive correlation between the

Table 4

Moisture content (MC) and water activity (aw) values of 195 samples of maize grains (hybrids Br 201, C 125 and Cx

322) listed with the climatic data recorded over a one-year period in Brazil

Mean of 5 samples per month Average per month

Samples Br 201 MC % ±aw C 125 MC % ±aw Cx 322 MC % ±aw Relative humidity

(%)

Mean temperature (8C)

FHa 17.0±0.91 16.8±0.87 16.0±0.83 86.9 20.2

S1b 16.4±0.82 16.8±0.82 15.8±0.78 82.0 19.8

S2 16.2±0.81 16.6±0.79 15.6±0.78 82.3 18.8

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populations of Aspergillus spp and those of Penicillium spp (P< 0.05; 0.34) and signi®cant negative correlations betweenAspergillus spp and both relative humidity (P< 0.05; ÿ0.35) and aw (P< 0.05; ÿ0.34). Signi®cant negative correlations were also found between the genus

Penicillium and both mean temperature (P< 0.01; _ÿ0.41) and relative humidity (P< 0.01;

ÿ0.49). The regression analysis of number of CFU/g versus storage time showed a statistically

signi®cant correlation in the three hybrids analyzed for the genera Aspergillus and Penicillium

(P< 0.01).

The stepwise multiple regression analysis for number of CFU/g, storage time, aw, moisture

content, mean temperature and relative humidity revealed the most important variables in each model: for the genusAspergillus they were storage time in Br 201 (P< 0.05) and storage time, relative humidity, mean temperature and mean temperature2 in C 125 (P< 0.05); for the genus

Penicillium they were time of storage and aw in Br 201 (P< 0.01) and mean temperature

(P< 0.05) in Cx 322.

The highest numbers of CFU/g of the genus Fusarium were recovered during the ®rst ®ve storage months (Table 3) at mean temperature values of 18.8±21.48C, grain moisture contents of 12.6±16.8%, relative humidity of 80.6±83.2% (Table 4). Fusarium spp showed signi®cant negative correlations with mean temperature (P< 0.01; ÿ0.67) and relative humidity

(P< 0.01; _ÿ0.57) and positive correlations with grain moisture content (P< 0.01; 0.52). These results corroborate the ®nding that this mold, despite being able to survive on desiccated grains, thrives in more humid conditions. The regression analysis of number of CFU/g versus storage time showed a statistically signi®cant correlation (P< 0.01) for Fusarium spp. The stepwise multiple regression analysis for number of CFU/g, storage time,aw, moisture content,

mean temperature, and relative humidity revealed the most important variables for each model of the genus Fusarium: mean temperature and mean temperature2 for Br 201 (P< 0.01); aw

and relative humidity for C 125 (P< 0.05); storage time for Cx 322 (P< 0.01).

The lowest frequencies of occurrence were observed in hybrid Br 201 for Fusarium spp and in hybrid Cx 322 for Aspergillus spp and Penicillium spp (Table 1). Hybrids Br 201 and CX 322 did not show statistically signi®cant dierences of Aspergillus, Fusarium and Penicillium

contamination at the aw values detected. However, in hybrid C 125, the higher numbers of

CFU/g forAspergillus spp were statistically signi®cant when compared to contamination levels for the other two genera (P< 0.05) at aw values of 0.90±0.80 (Table 2).

Tables 5 and 6 show the occurrence of fumonisins in grains of the Brazilian maize hybrids analyzed. Out of a total of 195 samples (65 belonging to each of the three hybrids), 176

Table 5

Fumonisin levels in 195 samples of freshly harvested and stored maize grains (hybrids Br 201, C 125 and Cx 322) from Brazil

BR 201 C 125 Cx 322

FB1 FB2 FB1 FB2 FB1 FB2

Positive samples 62(93.38%) 64(98.46%) 56(86.15%) 63(96.92%) 58 (89.2%) 63(96.92%) Mean concentration 10.54 8.17 10.57 7.80 8.05 6.83 Concentration range (mg/g) 0.90±46.39 2.51±29.16 0.98±49.31 3.1±20.65 0.87±37.14 1.96±11.74

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(90.2%) were contaminated with FB1 at concentrations that ranged from 0.87±49.31 mg/g and

190 (97.4%) were contaminated with FB2 at concentrations that ranged from 1.96±29.16 mg/g.

Studies on fumonisin contaminants in maize and maize-based products from several countries (Shephard et al., 1996; Bullerman, 1996) revealed levels of contamination lower than those presently reported. A similar ®nding was observed when comparing our results with data on FB1 and FB2 contamination in maize hybrids or inbred lines grown in countries from three

continents. Of these, Argentina showed the highest concentration of fumonisins (37mg/g) in 51 samples analyzed (Visconti, 1996). In Brazil, Hirooka et al. (1996) reported maximum levels of 18.5mg/g for FB1and 19.3 mg/g for FB2 in 48 samples of maize grains.

With respect to possible dierences in fumonisin contamination between the maize hybrids studied, their pro®les are compared in Tables 5 and 6. The lowest contamination occurred in hybrid CX 322. This applied to both FB1 and FB2 mean levels (8.05 and 6.83 mg/g,

respectively) and range of concentrations (0.87±37.14 mg/g for FB1 and 1.96±11.74 mg/g for

FB2). The highest FB1 levels were found in 140 day-storage samples of the C 125 hybrid

(49.31 mg/g) and in freshly harvested samples of the BR 201 and CX 322 hybrids (46.59 and 37.14 mg/g, respectively). A more uniform distribution of mean concentrations of FB2 as

compared to FB1 occurred in all the hybrids.

After 140 days of storage, an overall decrease in the number of CFU/g for Fusarium spp together with a tendency towards decreased FB1 mean concentrations was observed in the

three hybrids analyzed, although FB1 levels showed some variability. The diversi®ed myco¯ora

of these hybrids, demonstrated by the recovery of 13 distinct fungal genera, presupposes a competition for nutrients. According to Horn and Wicklow (1983), each fungus carries a factor that determines its growth and ability to compete with other fungal species. In this respect, it

Table 6

Distribution of FB1and FB2levels in 195 samples of freshly harvested and stored maize grains (hybrids BR 201, C

125 and Cx 322) in Brazil

BR 201 C 125 CX 322 Total % of samples

Fumonisins levels (mg/g) FB1 FB2 FB1 FB2 FB1 FB2 FB1 FB2 FB1 FB2

< 1.0 nda nd 1 nd 1 nd 2 ± 1.0 ±

1.0±3.0 4 2 6 nd 6 2 16 4 8.2 2

3.1±5.0 20 nd 12 3 14 1 46 4 23.6 2

5.1±8.0 15 43 7 33 16 45 38 121 19.5 62

8.1±10.0 3 5 5 16 3 10 11 31 5.6 15.9

10.1±13.0 5 8 6 7 5 4 16 19 8.2 9.7

13.1±15.0 2 2 2 3 3 1 7 6 3.6 3.1

15.1±18.0 nd nd 2 1 2 nd 4 1 2.0 0.5

18.1±20.0 3 1 3 nd 2 nd 8 1 4.1 0.5

20.1±23.0 4 2 4 nd 3 nd 11 2 5.6 1.0

23.1±25.0 1 nd 1 nd 1 nd 3 ± 1.5 ±

25.1±28.0 1 nd nd nd nd nd 1 ± 0.5 ±

28.1±30.0 nd 1 2 nd nd nd 2 1 1.0 0.5

30.1±50.0 5 nd 5 nd 1 nd 11 ± 5.6 ±

a_{nd=not detected.}

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should be mentioned that studies carried out by Marin et al. (1998) indicated that fungal interactions could act as an additional factor in the control of Fusarium spp contaminants in maize and lead to decreased contamination by this genus, mainly at lower aw. On the other

hand, sample size should also be considered as an in¯uencing factor when trying to account for the decrease and variability of fumonisin levels recorded during our investigation (Whitaker et al., 1998).

The statistical analysis showed a negative correlation between fumonisins B1 and relative

humidity (P< 0.001; ÿ0.68769). Negative correlations were also found between the fumonisin

B2 and relative humidity (P< 0.001; ÿ0.64384) and mean temperature (P< 0.001; ÿ0.48662).

FB2correlated positively with grain moisture content (P< 0.001; 0.56913).

To conclude, the signi®cant levels of contamination with Fusarium spp reported herein, both in terms of prevalence and number of CFU/g, and the detection of fumonisins in freshly harvested and stored maize grains highlights the relevance of studies on the occurrence of fusariotoxins in maize and its by-products in the tropics.

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