Mycopathologia 138: 77–89, 1997. 77c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Handling and aflatoxin contamination of white maize in Costa Rica

Miguel Mora1 & John Lacey2

1C.I.G.R.A.S., Universidad de Costa Rica, Costa Rica2IACR – Rothamsted, Harpenden, Herts AL5 2JQ, England

Received 21 October 1996: accepted 28 July 1997

Abstract

Projects funded by International Development Research Centre (IDRC) of Canada and the European Commissionhave enabled the examination of more than 3000 samples of maize collected from all regions of Costa Rica atdifferent stages, from the growing crop through storage to final sale, and at different water contents. Contaminationwith Aspergillus flavus was frequent and about 80% of samples contained more than 20 ng aflatoxins g�1 grain.Average contamination with aflatoxins in the Brunca Region was >274 ng g�1 while that in other regions was< 70 ng g�1. Except in Brunca region, where it averaged 376 ng g�1, contamination of grain from commercialsources was slightly less than of that from farms (615 ng g�1). It appeared that samples kept on the cob afterharvest contained almost no aflatoxin while shelled samples were frequently highly contaminated. Experimentswere therefore done in Brunca and Huetar Atlantic Regions, utilising 34 experimental maize crops to study indetail the development of A. flavus and aflatoxin from before harvest, through postharvest treatment before dryingand through storage for six months. A. flavus was isolated more frequently from maize shelled immediately afterharvest than from that kept on the cob until it could be dried, and from more samples from the Brunca Region thanfrom the Huetar Atlantic Region. Samples harvested with >18% water content often contained >70% of grainsinfected with A. flavus but sometimes there were few grains infected. As found in the initial survey, more aflatoxincontamination developed in shelled maize than in that handled on the cob during the period from harvesting todrying, especially if the delay was more than 5 days, and more in Brunca than in Huetar. Shelled grain contained400–800 ng aflatoxin g�1 in Brunca but<100 ng g�1 in Huetar while grain kept on the cob contained<30 ng g�1,even with >18% water content. Incidence of Fusarium spp. exceeded 50% except where A. flavus colonized morethan 80% of grains.

Key words: Aflatoxin, Aspergillus flavus, Fusarium spp., maize, postharvest treatment, preharvest contamination,storage, Costa Rica

Introduction

Harvesting conditions in tropical countries are oftendifficult with heavy rain and high humidities leading tolarge water contents in harvested seed and heavy lossesduring storage. Under these conditions, the tradition-al division between field and storage fungi, derivedfrom studies of grain stored in temperate climates, canno longer be maintained. Species regarded as storagefungi, especially Aspergillus species, are common inthe field environment and may colonise grain and evenform mycotoxins before harvest. For instance, aflatox-ins have been shown to occur in maize and ground-

nuts prior to harvest in humid sub-tropical parts of theUnited States [3, 4] and Australia [1]. Infection ofthese crops by Aspergillus flavus may be associatedwith insect damage but it is greatly enhanced, evenin undamaged seeds, by heat and drought stress. Ifstored crops are to be free of mycotoxins, it is essentialthat the freshly harvested crop entering storage is freeof contamination and that postharvest handling doesnothing to enhance invasion by mycotoxigenic fungiand the formation of mycotoxins.

Agriculture is one of the most important sectors ofthe Costa Rican economy. About 28% of the popula-tion is employed in this sector and agricultural pro-

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duction contributes about 19% to the Gross NationalIncome. Grain production is an important componentof agriculture, both for food and feed, and it makes animportant economic contribution. The most importantgrain crops in Costa Rica are white maize and rice butsmaller quantities of sorghum and beans are also pro-duced. Rice is produced on large farms and is driedand processed in centralized mills so that there is littleopportunity for fungal and mycotoxin contamination.By contrast, white maize is produced in all parts ofCosta Rica by thousands of small farmers who haveinadequate handling facilities. These allow little quali-ty control and provide many opportunities for contam-ination by fungi. Wheat and yellow maize are mainlyimported and are subject to good quality control.

Because of the potential risk to humans from afla-toxin in the food chain, mould and mycotoxin contam-ination of white maize has been studied in Costa Ricaover several years. A research project funded by theInternational Development Research Centre of Canada(IDRC) enabled a survey of the incidence of aflatoxincontamination in white maize throughout Costa Rica[8]. This showed marked differences in aflatoxin con-tamination between the Brunca and Huetar AtlanticRegions, even though these were similar climatically.A second project, funded by the European Commis-sion, enabled a more detailed study of factors affectingAspergillus flavus and aflatoxin contamination in thesetwo regions and are described in this paper.

Materials and methods

Grain samples

In an initial survey, more than 3000 samples of whitemaize were collected between 1985 and 1988 at dif-ferent handling stages from before harvest to the finalselling point from all regions of Costa Rica. About1000 samples each were collected in Brunca and Hue-tar Regions (Table 2; Fig. 1) and smaller numbers (200–300) from Central and Chorotega Regions where lessmaize is grown. Samples consisted of at least 30 cobsor 5 kg grain and these were assayed for fungal infec-tion, aflatoxin content, mechanical damage, commer-cial quality (as indicated by visible mould and insectdamage), water content (%) and germination.

Experimental studies

Experiments were done on farms in each of the twomain maize producing regions of Costa Rica, the Brun-ca and Huetar Atlantic Regions in each of three years,1989–1992, to study changes in mould and mycotoxincontamination of maize from physiological maturity,through drying and about six months storage. The cli-mate of Costa Rica allows two crops to be planted eachyear (Table 1) and enabled two sets of experimentsannually, in different ambient conditions. Over thethree years, 34 plots were sown with white endospermmaize on different farms, distributed through the tworegions and chosen according to the type of seed usedfor planting and for their accessibility and readiness tocollaborate. Eighteen plots were on farms in the Brun-ca Region and 16 in the Huetar Region. Grain fromhalf of each plot was stored on the cob before dryingand that from the other half was shelled at harvest.

In the first year, 12 plots, each of approximate-ly 3000 m2, were sown and were expected to yield atleast 20 quintals of grain (1 quintal = 46 kg).At harvest,all the cobs were picked by hand and about half wereshelled mechanically immediately after harvest whilethe remainder were left on the cob. Both lots were thenstored in sacks without drying, usually for 5 days. Atthe end of this period, the remaining cobs were shelledand about 5 quintals from each lot were dried to about17% water content, a common level in farm-storedgrain and the remainder were dried to about 13%. Asmall, electrically-heated forced-air sample dryer wasused to dry maize kernels after the postharvest treat-ments. Drying took about 150 minutes and in this timethe grain attained a temperature of about 50�C. Thegrain from each shelling and water content treatmentwas then placed separately in four hermetically closed200 l metal drums and stored for 6 months. In the nexttwo years, 20 larger plots were sown to allow about30 quintals of grain to be harvested at three water con-tents, with targets of 23, 20 and 17% water content.Otherwise the methods used were identical to thosewith the smaller plots.

The effect of delayed drying was evaluated in maizefrom three 1 ha plots which were sampled regular-ly until the water content was about 20% when thecrop was harvested and shelled. The shelled grain wasplaced in sacks and the produce from each plot wasdivided into 12 sub-lots of six sacks (about 240 kg).The sacks were stored on the farm for 5 days under cov-er before individual sublots were dried at 12 h intervals.Samples were dried to about 13% w.c., at about 50�C

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Figure 1. Map of Costa Rica showing the locations of the different geographical regions.

Table 1. Planting and harvesting season, by regions, of maize in differentregions of Costa Rica

Season Central Brunca Huetar Chorotega

Wet Sown Apr–Jun Mar–Apr May–Sep Apr–Aug

Harvested Aug–Oct Jul–Aug Oct–Feb Aug–Nov

Dry Sown Aug–Dec Sep–Nov Nov–Feb Aug–Nov

Harvested Dec–Mar Jan–Apr Mar–Jul Dec–Feb

in a forced air dryer, and grain temperature and watercontent were measured by meter every 30 minutes.

Samples consisted of at least 30 cobs or 5 kg grain.Samples from field experiments for drying and analy-sis were taken from rows selected from random num-ber tables and those from harvested grain were select-ed at random from sacks or from the stream as grainwas removed from storage containers. Cobs were thenshelled and whole grains were assayed for fungal infec-tion, mechanical damage, commercial quality (as indi-cated by visible mould and insect damage), and germi-nation. For aflatoxin analysis and water content deter-mination, 5 kg samples of grain were ground and wellmixed before taking 50 g sub-samples.

Weather data

Weather data were obtained from the National Meteo-rological Institute to aid interpretation of results.

Water content determination

Water contents were determined from samples collect-ed daily from physiological maturity of the kernelsthrough harvest to the end of the postharvest treat-ments and, after drying, from those taken monthlyduring storage. Water contents were determined usinga Motomco 919 moisture meter and confirmed subse-quently by oven drying at 103 �C for 72 h.

Germination

Germination was assessed by incubating seedsbetween layers of damp absorbent paper, as describedby the International Seed Testing Association [6].

Fungal infection

To assess fungal colonisation, 100 grains were platedeither on moist blotters or, after surface sterilizationwith 1% sodium hypochlorite (NaOCl) for about 1 min,onto malt 5% NaCl or DG18 [5] agars. The numbers

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of grains in each sample yielding different fungal taxawere counted after incubation at 25�C for 5 days andthe percentage infected kernels calculated.

Aflatoxin contamination

To assess the frequency and level of aflatoxin contami-nation, 50 g sub-samples from 5 kg ground grain wereextracted with acetone:water (85 : 15) and assayed bymeasuring the fluorescence of the Florisil layer in mini-columns following Velasco’s method [13]. The pres-ence of aflatoxin was confirmed by thin layer chro-matography (TLC) of samples and standards onto alu-minium sheets pre-coated with silica gel 60 (0.2 mmthick). TLC plates were developed using chloroform+ 5% ethyl ether + 5% acetone as solvent and exam-ined for fluorescent spots corresponding to those of thestandards.

Mechanical damage

Mechanical damage to the grain was assessed in sam-ples of 200 kernels by visual evaluation after stainingwith fast green.

Commercial quality

Commercial quality, as indicated by the presence ofdamaged kernels, visible moulding, broken grains andliving and dead insects, was determined using the stan-dard methodologies of screening for insects and brokenkernels and visual examination for damage producedby insects, moulds, heating and other causes.

Results and discussion

Contamination of samples from all parts of CostaRica

Samples collected during the initial survey (Table 2),like those in other surveys [14], were frequently con-taminated with aflatoxins, sometimes in large concen-trations. Only about 30% of samples contained 620ng aflatoxins g�1 and a further 20% 6 100 ng g�1.Mean aflatoxin contamination in the Brunca Regionwas much greater (274 ng g�1) than in other regions(669 ng g�1), even though the largest mean concen-

tration of aflatoxin in any one sample category (510ng g�1) was in grain stored on a farm in the HuetarAtlantic Region. However, the mean aflatoxin conta-mination in all sample categories, except preharvest,was high in the Brunca Region (exceeding 165 ng g�1).Mean contamination at harvest was already 10 ng afla-toxin g�1. In the Huetar Region, mean aflatoxin conta-mination in categories other than on farm storage nev-er exceeded 65 ng g�1. These levels of contaminationgenerally exceed those reported by Viquez et al. [14]in these regions. By contrast with the present study,Viquez et al. [14] report less aflatoxin in maize fromthe Brunca Region than from Huetar Atlantic Region.Maize from Central and Chorotega Regions had meanaflatoxin concentrations of, respectively, 22 and 48ng g�1 maize. Aflatoxin contamination of commercialmaize samples was also greatest in the Brunca Region(376 ng g�1) but in other regions was 615 ng g�1.Contamination was least in samples of grain kept onthe cob until immediately before drying and greatestwhere it had been shelled on farms immediately afterharvest.

Heavy A. flavus infection and aflatoxin contamina-tion have generally been associated with poor storageof grain, particularly with large water contents [2].However, infection of maize before harvest with con-comitant aflatoxin production has also been reported,especially when the crop is drought and heat stressed[3, 4]. In the Brunca Region, although there was somepreharvest contamination with aflatoxin, most devel-opment appeared to occur on farms after harvest beforethe grain was dried and was possibly associated withthe method of postharvest handling. However, conclu-sions were difficult to draw because, although climaticconditions were similar in Brunca and Huetar Regions,there were large differences in water content and inmould and mycotoxin contamination among samplesand among farms. Also, cultivars and postharvest prac-tices differed between the two regions. Although thetwo cultivars are somewhat similar, a local cultivarknown as "maizena" is grown in the Huetar Regionwhile cv Diamantes is grown in the Brunca Region.Maize grain in the Brunca Region is generally removedfrom the cob by shelling immediately after harvestwhile in the Huetar Region it is stored on the cob untiljust before the grain is dried, about five days afterharvest.

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Table 2. Mean aflatoxin content and numbers of samples collected in relation to handling stages andregions.

Region

Place-Stage Brunca Central Chorotega Huetar Total

Preharvest 101 (22)2 0 (29) nd (0) nd (0) 4 (51)

On harvesting 270 (187) 26 (20) 65 (94) 40 (105) 151 (406)

Stored on farm 230 (249) 20 (219) 35 (75) 510 (38) 144 (581)

Arrival at CNP agency 380 (362) nd (0) 35 (52) 65 (476) 191 (890)

Arrival at CNP plant 200 (81) nd (0) nd (0) 20 (57) 126 (138)

After drying at CNP plant 165 (125) nd (0) nd (0) 30 (240) 76 (363)

Stored at CNP plant 185 (20) 45 (46) nd (0) nd (0) 87 (66)

Overall, by regions 274 (1044) 22 (314) 48 (221) 69 (916) 147 (2495)

Commercial sources 376 (181) 14 (264) 12 (39) 15 (30) 141 (514)

Overall by regions (including 239 (1225) 18 (578) 42 (260) 67 (946) 146 (3009)

grain from commercial sources)

1 Mean aflatoxin concentration in maize samples (ng g�1).2 Number of samples examined.nd, not determined.CNP, Consejo Nacional de Produccion.

Climatic features of Brunca and Huetar Regions

Weekly mean temperatures and rainfall in both regions,but especially in the Brunca Region, during the projectperiod were always high enough to favour rapid graindeterioration (Figure 2). Rainfall reached 3.56 m peryear in the Brunca Region and 3.86 m in Huetar. Afairly well defined low rainfall season in the BruncaRegion occurs between November and April but inHuetar rainfall fluctuates widely and, although usuallyheaviest between May and July, may be heavy at anytime.

Contamination of freshly harvested maize

Maize samples from the 34 farms studied could begrouped according to region, method of handling andwater content (Table 3). Before harvest, maize grainchanged little in its characteristics except for loss ofwater as it ripened. Consistent with a recent report byViquez et al. [15], Fusarium spp., mainly F. monili-forme, usually infected>50% of the kernels and oftenalmost all of them. By contrast, A. flavus was usuallyonly recovered from <5% of kernels although it wassometimes isolated from up to 25% and on one occa-sion from 48% of kernels. Aflatoxin contaminationbefore harvest was usually<20 ng g�1 and often nonecould be detected (Figure 3).However, for the first timein Costa Rica, although reported previously from the

USA and Australia [1, 3], four lots of maize harvestedin mid-1991, including three from one farm in Hue-tar Region, were heavily contaminated with aflatoxins.Contamination on this farm 22 days before harvest was280 ng g�1 but then increased to 750 ng g�1 at 7 daysbefore harvest with little further increase to harvest.Differences in aflatoxin content of up to 600 ng afla-toxin g�1 among successive samples from some plotssuggested that contamination had resulted from infec-tion of only a small proportion of cobs. There was noevidence of drought stress nor serious insect damage,which have previously been implicated in pre-harvestaflatoxin occurrence [4, 7, 16], in the heavily conta-minated crops. Nevertheless, Viquez et al. [14] havesuggested that drought stress can sometimes occur.

Aspergillus flavus colonisation and aflatoxincontamination between harvesting and drying

Harvesting at predetermined water contents was diffi-cult because cobs dried at different rates and there wasa high level of variability among sequential samples.Nevertheless, experiments on different farms in thetwo regions enabled conclusions to be drawn regard-ing the patterns of colonisation of white maize grainby A. flavus and of contamination by aflatoxins.

Between harvest and drying (postharvest stage),the most common fungi isolated from surface-sterilisedgrains were Aspergillus flavus and Fusarium spp..

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Figure 2. Weekly average temperature and rainfall in selected weather stations from the Brunca and Huetar regions during the test period. Key:—, temperature,�—�, weekly rainfall.

Table 3. Water content distribution in samples by region, stage and method of handling.

Numbers of samples (%)

Water content Pre-harvest Harvest Postharvest Stored

(%) Unshelled Shelled Unshelled Shelled

Brunca

<14 0 0 4 (5) 11 (14) 73 (53) 55 (40)

14–17 7 (18) 5 (33) 22 (27) 9 (11) 43 (31) 56 (41)

17–20 13 (34) 5 (33) 28 (34) 32 (41) 22 (16) 26 (19)

20–23 7 (18) 4 (27) 18 (22) 22 (28) 0 0

>23 11 (29) 1 (7) 10 (12) 5 (6) 0 0

Total 38 (100) 15 (100) 82 (100) 79 (100) 138 (100) 137 (100)

Huetar

<14 0 0 1 (1) 0 70 (44) 70 (45)

14–17 3 (6) 4 (24) 21 (24) 23 (26) 82 (52) 81 (52)

17–20 13 (28) 9 (53) 47 (55) 50 (57) 6 (4) 6 (4)

20–23 9 (19) 4 (24) 17 (20) 14 (16) 0 0

>23 22 (47) 0 0 0 0 0

Total 47 (100) 17 (100) 86 (100) 87 (100) 158 (100) 157 (100)

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Figure 3. Average aflatoxin in maize by stage of handling, postharvest treatment and water content. Shelled and unshelled refer to postharvesthandling.

Figure 4. Development of aflatoxin contamination in white maize during postharvest treatments and storage. Sampling was daily during thepostharvest period and monthly during storage; shelled and unshelled refer to postharvest handling.

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Figure 5. Aflatoxin contamination and mechanical damage of shelled maize.

However, Aspergillus niger, Drechslera, Penicilliumand Nigrospora spp. were also frequently isolated andPenicillium was sometimes numerous. A. flavus waspresent in nearly all samples and increased markedlyduring postharvest storage of some samples to infectup to 90% within 5 days and 98% in 8 days. More A.flavus was isolated from maize that had been shelledat harvest than from that which had been kept on thecob. Overall, the frequency of isolation of A. flavusfrom shelled samples was more than double that fromkernels stored on the cob. There was also more A.flavus in grain from the Brunca Region than in thatfrom the Huetar Region. The greatest incidence of A.flavus, exceeding 70%, was found in samples withmean water contents exceeding 18%. However, therewere also several lots in which this water content wasassociated with little A. flavus.

Fusarium spp. were isolated from 83.3� 13.3% ofall kernels plated and from all kernels in some samples.This is similar to the 86% infection with F. moniliformewhich was associated with yields of up to 32.4� 103 F.moniliforme colony forming units g�1 grain in samplesfrom Brunca and Huetar Regions [14]. By the fifth

day of the “postharvest” period, Fusarium spp. couldbe isolated from more than 50% of kernels in mostsamples of white maize plated. The only exceptionswere samples where more than 80% of kernels yieldedA. flavus when F. moniliforme was sometimes found infewer than 10% of kernels.

Up to 24% of shelled kernels, mean 7.4 � 6.1%,were stained by malachite green indicating damage,compared to a mean of only 0.3 � 1.5% of kernelsstored on the cob. However, although most infectionsby A. flavus were found in samples with the most heav-ily damaged kernels, there was no significant relation-ship between amounts of damage and infection. Stoloffet al. [11] have reported that kernels remained free ofA. flavus infection unless damaged while Qasem andChristensen [9] and Tuite et al. [12] found that injuriesto the pericarp over the germ and, to a lesser extent,from removal of the pedicel facilitated colonisation bystorage fungi. Shelling was also often associated withloss of germinability on several farms where there washeavy aflatoxin contamination. There was also littlecorrelation between water content and infection.

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Figure 6. Effect of delayed drying on aflatoxin contamination of three lots of maize.

Other commercial quality factors showed no dis-cernible relationship with postharvest treatment. Amean of 78.2 � 12.5% of grains germinated at har-vest and 76.5� 18.2% germinated after 5 days storageof unshelled grain. However, after 5 days storage ofshelled grain, only 63.8 � 22.1% germinated. Therewas generally little insect infestation, with usually few-er than 4 live or dead insects kg�1 and a maximum of16 live and 12 dead insects kg�1 in one sample. Apartfrom about six farms with up to 20% grains classified asinsect damaged, samples only occasionally containedmore than 1% of grains damaged by insects. However,there appeared to be no correlation between insect dam-age and the occurrence of either live or dead insects. Bycontrast, often more than 10% of grains were damagedby moulds and sometimes up to 30%. Sprouting wasrarely recorded in more than 2% of grains and oftenwas not seen in any. Broken grains numbered up to5.6% but were generally61.0%.

As expected from the earlier work, aflatoxin con-tamination was greater in grain sampled between har-vest and drying than was usual before harvest (Figure

3). Aflatoxin contamination of shelled maize increaseddaily during the postharvest stage (Figure 4) and, aswith A. flavus colonisation, aflatoxin contaminationfive days after harvest was much greater in maizeshelled at harvest than in that which had been kepton the cob and was greater in the Brunca Region thanin the Huetar Region, perhaps indicating greater sus-ceptibility to A. flavus colonisation or aflatoxin for-mation in cv Diamantes used in Brunca than in thelocal “maizena” cultivar used in Huetar (Figures 3, 4).Among the maize kernels stored shelled in the BruncaRegion, samples from three farms contained about 400ng aflatoxin g�1 grain while those from another con-tained 800 ng g�1. By contrast, shelled maize in theHuetar Region contained less than 100 ng g �1 whileall maize kept on the cob contained less than 30 ng afla-toxin g�1, even with water contents similar to those inthe shelled maize. Mean aflatoxin concentrations weregreater in samples with larger water contents but evensamples with mean water contents of 14–17% couldbe highly contaminated (Figure 3). Aflatoxin contami-nation, like A. flavus colonization, was greatest among

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Figure 7. Development of aflatoxin during the five days after harvest in shelled maize stored on ten different farms.

samples showing mechanical damage, especially insome of the most heavily damaged grain, but, again,there was no consistent relationship between amountsof damage and contamination (Figure 5).

Delaying drying beyond 5 days postharvest couldresult in large increases in aflatoxin contamination. Forinstance, one lot (lot 1, Figure 6) contained about 50 ngaflatoxin g�1 throughout the 5 day postharvest periodand remained at this level for the next 24 h. However,delaying a further 12 h before drying increased con-tamination to three to five times that after 5 days andanother 12 h to ten times. Another sample (lot 3, Figure6) also showed large changes when drying was delayedbut there were only small changes in a third lot (lot 2,Figure 6). Thus, only a few hours delay in drying cancause large changes in the level of contamination.

Prediction of the amount of aflatoxin contaminationat the end of the five day postharvest’ period was diffi-cult because of differences in the rate of aflatoxin pro-duction, water content and sample variability. A kernelstarts with little or no aflatoxin and, in appropriate con-ditions with a given level of A. flavus colonisation, itstoxin content might be expected to increase at a given

rate so that grain lots stored similarly might be expectedto develop similar contamination. However, data frompostharvest treatment experiments often show unex-pected differences. Figure 7 shows aflatoxin concen-trations in samples from ten different lots of aflatox-in contaminated shelled grain taken at daily intervalsfrom harvest through the five day “postharvest” stage.Most lots showed aflatoxin concentrations increasingfrom the second or third day over the remaining period,with some (lots 19 and 27) showing particularly largeincreases between days 4 and 5, to give concentrationsat the end of the five days ranging from 20 to 400ng g�1. However, there was markedly less aflatoxin inlot 23 on the fourth day postharvest than on the daysbefore or after while lots 1 and 5 reached their great-est concentrations on the fourth day. Lots 21 and 23already contained very large aflatoxin concentrations atharvest while lot 18 yielded large aflatoxin concentra-tions on day 1 of the “postharvest” stage even though nocontamination had been detected at harvest. However,although the overall trend of aflatoxin contaminationin lots 18 and 21 remained upwards, concentrationsin lot 23 increased dramatically to > 1000 ng g�1 in

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Figure 8. Average mould invasion and aflatoxin contamination on maize samples. Groups ordered by ascending A. flavus count (every 10%).

samples taken on day 1 and then fell to zero on day2 before increasing to give a final concentration littledifferent from that at harvest on day 5. Samples fromsome other lots also showed marked differences fromday to day. Such large differences are likely to be theconsequence of a marked variability in the degree ofcontamination within treatments.

Some differences among lots could have beena consequence of differences in water content ormechanical damage but results were inconsistent. Lotswith no aflatoxin were harvested with 13–19% watercontent and 0–19% mechanical damage, those withless than 100 ng g�1 with 17–23% water content and 1–20% damage and those with more than 100 ng g�1 with16–24% water content and 2–22% damage. However,among the most heavily contaminated samples, all fourwith 18% water or less were already heavily contam-inated with aflatoxin at harvest and all except one ofthose with more than 18% water content were light-ly contaminated. Contamination with 400 and 800 ng

aflatoxin g�1 occurred with little mechanical damage(2%) and little prior contamination when water con-tents were, respectively, 18 and 21% and with 300 and900 ng g�1 with, respectively, 21 and 14% mechanicaldamage and 22 and 18.7% water contents.

Despite the high incidence of Fusarium spp., it wasnot possible in this study to analyse their mycotoxins.However, the widespread occurrence of fumonisin B1

in Costa Rican maize has recently been reported [15].

Colonisation and aflatoxin contamination of storedmaize in Costa Rica

Infection by A. flavus and contamination with aflatox-in continued to increase during the storage of experi-mental white maize in Costa Rica after all postharvesttreatments but especially when the kernels were storedwith high water contents (Figure 3). After 6 months,28% of grains nominally at 13% water content and35% of grains stored at 17% water content carried A.

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flavus. By contrast, Fusarium spp. declined slowlyduring storage, from infecting about 64% of kernelsafter 1 month to 50% after six months, with no signifi-cant relationship with water content. Germination alsodeclined markedly at the higher water content.

Differences in both A. flavus colonisation and afla-toxin contamination found during the postharvest peri-od between grain that was shelled at harvest and thatleft on the cob until immediately before drying persist-ed through the subsequent storage period. A. flavus andaflatoxins continued to be more abundant, even after 6months storage, in grain that had been shelled immedi-ately after harvest (Figures 3, 4) although, at this stage,there were few differences between the postharvestshelled and unshelled treatments in the incidence ofFusarium spp., percentage germination and mechan-ical damage. Greatest contamination with aflatoxin,1600 ng g�1, was found on one farm in grain shelledat harvest and stored with 17.5% water content, equiv-alent to a water activity of about 0.865 aw, still belowthe optimum for aflatoxin production [4, 10].

Overall, the most common fungi isolated frommaize, during the postharvest stage and during stor-age after drying, were A. flavus and Fusarium spp.Figure 8 relates their occurrence to aflatoxin contam-ination. Fusarium spp. were markedly fewer when A.flavus was abundant. Numbers of Fusarium spp. start-ed to decline when A. flavus infected about 40% ofthe grains but the decline was then more uniform inBrunca Region than in Huetar where a sharp decline inFusarium spp. occurred only when A. flavus colonised>60% of the grain. There were slight differences inthe occurrence of A. flavus among regions with 9% ofsamples from the Brunca Region having>50% of ker-nels infected compared to 3% from the Huetar Region.Samples with more abundant A. flavus tended to havemore aflatoxin. This is consistent with earlier reportsthat infection of maize grain by F. moniliforme inhibit-ed subsequent colonization by A. flavus and decreasedaflatoxin contamination [4, 17].

Insect infestation was severe on a few farms butmany samples yielded a few insects after storage for 4months or more. Germination often declined sharplyduring 6 months storage to a mean of 28.6% but some-times to zero, especially in samples with water con-tents >17%. Sprouting was generally less than 1%but reached 4.8% in occasional samples. Insect andmould damage increased in some samples but the over-all amounts differed little from pre-drying levels.

Conclusion

With similar treatments applied to grain in bothregions, patterns of A. flavus colonisation and afla-toxin contamination, during both postharvest handlingand subsequent storage, were similar, regardless ofmaize cultivar, although absolute amounts of aflatox-in were greater in Brunca with cv Diamantes than inHuetar with a local cv, “maizena”. The initial hypoth-esis, that differences in A. flavus and aflatoxin conta-mination were the result of differences in the methodsof postharvest handling, was proven. Thus, providingthere was little contamination before harvest, aflatox-in development during the postharvest stage could becontrolled by delaying shelling until the latest possi-ble time before drying and by minimising the delaybetween harvesting and drying. Drying to less than14% water content is necessary to minimise aflatoxindevelopment in storage.

Acknowledgements

Farms were chosen, with the assistance of employeesof the Consejo Nacional de Produccion (CNP) work-ing in the zone. Construction of the drier used in thisstudy was also made possible by the notable assistanceof the specialized personnel and shop facilities of theCNP. Financial support from the European Commis-sion, under Contract No. TS2-164.M(H), which madethis study possible is gratefully acknowledged

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Address for correspondence: J. Lacey, IACR-Rothamsted, Harpen-den, Herts AL5 2JQ, UKFax: 44-582-760981