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Printed in the Netherlands. 113. Mycoflora of Iranian maize harvested in the main production areas in. 2000. Seyed Amir Ghiasian1,3, Parivash Kord-Bacheh1, ...
Mycopathologia 158: 113–121, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.

113

Mycoflora of Iranian maize harvested in the main production areas in 2000 Seyed Amir Ghiasian1,3 , Parivash Kord-Bacheh1 , Seyed Mahdi Rezayat2 , Amir Hossein Maghsood3 & Heshmatallah Taherkhani3 1 Medical Parasitology and Mycology Department, School of Public Health, Tehran University of Medical Sciences

and Health Services, PO Box 14155-6446, Tehran, Islamic Republic of Iran; 2 Pharmacology Department, School of Medicine, Tehran University of Medical Sciences and Health Services, PO Box 13145-784, Tehran, Islamic Republic of Iran; 3 Medical Parasitology and Mycology Department, School of Medicine, Hamedan University of Medical Sciences and Health Services, PO Box 65155-518 Hamedan, Islamic Republic of Iran Received 26 March 2003; accepted in final form 9 September 2003

Abstract Maize is one of the most important cereals produced in, and imported into, Iran. The incidences of seed-borne fungi were determined in Iranian maize harvested in 2000 from four major production areas with different climatic conditions, namely Fars, Khuzestan, Kermanshah and Mazandaran provinces. This is the first study to compare the mycoflora of maize in the aforementioned areas. Mycological analyses showed a predominance of Fusarium species (38.5%), followed by Aspergillus species (8.7%), Rhizopus species (4.8%), Penicillium species (4.5%), Mucor species (1.1%), and four other fungal genera. Fusarium verticillioides was the most prevalent species (83% of Fusarium isolates and 52% of the total isolations), with the highest incidence in Mazandaran (59%), a region of Iran with the highest rainfall and relative humidity, high rate of esophageal cancer (EC) and high levels of fumonisins in maize. Aspergillus flavus was the most widely recovered Aspergillus species and 38% of samples were contaminated with this potentially aflatoxigenic fungus. The incidence of A. flavus was highest in Kermanshah, the province with lowest mean minimum temperature. Penicillium species were seen in all the samples and Fars had the highest incidence, with highly significant differences when compared to the other three provinces. Diplodia species were not isolated from any of the samples examined. Key words: Aspergillus, fumonisins, Fusarium, Iran, maize

Introduction The prevalence of seed-borne fungi and contamination by mycotoxin producing species in maize (Zea mays L.) is important in assessing the risk of mycotoxin contamination. A number of fungal species associated with maize, mainly belonging to the genera Fusarium and Aspergillus, have been reported to produce mycotoxins that cause mycotoxicoses in domestic animals and man [1, 2]. In some maize producing countries, information is available regarding the mycoflora of freshly harvested maize grain, including Argentina [3]; United States [4, 5]; South Africa [6–9]; Canada [10]; and some other countries [11, 12]. In Iran, maize is one of the most

important cereals produced and imported. However, little information is available on the fungal species associated with maize grain, their geographical distribution in the main production areas, and the isolation frequency of toxigenic species. Recently, samples of field-trial maize from Iran have been shown to be contaminated with the mycotoxins fumonisins, aflatoxins and ochratoxin A [13–16]. Shephard et al. [13] showed for the first time that maize grain of Mazandaran province can be highly contaminated with fumonisins B1 , B2 and B3 . Yazdanpanah et al. [16], Tayebi and Esavi [15] have shown that maize samples collected from the southern Caspian littoral were contaminated with aflatoxins and ochratoxin A. Surveys have shown

114 natural occurrence of aflatoxins in bread [17] as well as ochratoxin A in red wheat [18]. Previous analyses of farm rice, wheat and maize grains showed the presence of Fusarium verticillioides (Sacc.) Nirenberg and F. proliferatum (Matsushima) Nirenberg as the most common seed-borne fungi in the Mazandaran province in Iran [18–20]. Another study has shown that after Aspergillus species, F. verticillioides and F. proliferatum, were most often isolated from maize in northern parts of Iran [21]. Studies on fungal species isolated from Iranian cereals have demonstrated their ability to produce aflatoxins, [18], moniliformin [22], trichothecene [23] and fumonisins [Ghiasian et al., unpublished data]. In this paper the significance of the internal mycoflora of freshly harvested maize grain intended for human and animal consumption is considered. The objectives of this study were to identify the fungi associated with maize grain harvested in an extensive region of the main maize production areas in Iran in 2000, to determine the species distribution of genera of mycotoxicological interest, and to compare the mycoflora at different locations, especially in Mazandaran, an endemic area of esophageal cancer [24].

Table 1. Provincial locations, number of samples and meteorological stations recorded for the 2000 maize season in Iran Provinces

Locations

No. of samples

Meteorological stations

Fars

Marvdasht Shiraz Sepidan Darab Boanat Estahban

13 5 3 10 1 5

Fasa Shiraz Neyriz Lar Abadeh

Khuzestan

Shoosh Dezful Andimeshk Ahvaz

5 10 2 2

Abadan Bostan Mahshahr Ahvaz Ramhormoz Masjed-Soleyman Omidirh

Kermanshah

Kangavar Kermanshah Sahneh Harsin Eslam-Abad Sarpol-Zahab

Mazandaran

Sari Dashte-naz

Materials and methods

3 3 2 2 2 2 5 17

Kangavar Kermanshah Ravansar Eslam-Abad Sarpol-Zahab

Sari Babolsar Ramsar Sadde-Dorodzan

Maize sampling and procedure A total of 92 samples (1.5–5 kg) of maize grain harvested in 2000 were collected at 18 locations in the four provinces, Fars, Khuzestan, Kermanshah and Mazandaran, in the main maize producing areas in Iran (Figure 1, Table 1). These locations were selected based on land size and one sample was taken for every 1300 ha. of maize planted. All the samples were taken from freshly harvested lots before drying and were dried in the sun for two days directly after harvesting. Most of the samples were intended for animal consumption, some were intended for human consumption and none had visible signs of mold contamination. Maize cultivar Yellow SC-704 was sampled in all provinces. Meteorological characteristics The four regions represent a range of climatological conditions from warm in Khuzestan, moderate in Fars, relatively cool and humid in Kermanshah to moderate and highly humid in Mazandaran (Table 2). The meteorological data given in Table 1 were obtained

from 21 meteorological stations. The meteorological characteristics of minimum temperature (◦ C), maximum temperature (◦ C), mean temperature (◦ C), relative humidity (%) and rainfall (mm) were recorded as monthly averages from 1981 up to the season of sampling (2000) in four regions (Table 2). Mycological analysis Subsamples of grain (each ca. 100 g) from each sample were surface disinfected for 1 min with a 1% sodium hypochlorite solution, rinsed twice in sterile distilled water and dried in a laminar flow cabinet. One hundred grains (5 per plate) per subsample were plated onto Nash-Snyder Agar containing 500 mg/l of chloramphenicol, to suppress the growth of bacteria [25]. Nash and Snyder culture medium was used because it contains pentachloronitrobenzene to reduce the growth of the Zygomycetes to prevent colony mixing.

115

Figure 1. Map of Iran, showing relative positions of the four provinces from which samples were collected.

The plates were incubated at 28 ◦ C in the dark for 5–7 days. The developing fungal colonies were counted directly and where several fungi were isolated from a single grain, all the colonies were recorded, and different species subcultured onto potato dextrose agar (PDA). Due to the close vicinity of some colonies to each other, and the possibility of mixed cultures, isolates were purified by preparing single-spore cultures as follows: a suspension of conidia in 10 ml sterile distilled water was prepared by scraping conidia from PDA to obtain a concentration of 1–10 conidia in a drop viewed under the low power objective (10×) of a microscope. A droplet of suspension was streaked on the PDA plates and incubated at 25 ◦ C for 24 h. The plates were examined using a stereomicroscope and single germinated conidia removed on a small piece of agar using a scalpel and transferred to PDA slants.

for Aspergillus and Fusarium species. The fungi of primary interest were Fusarium and Aspergillus species because of their known ability to produce mycotoxins and their relationship with human and animal diseases. The isolation frequency (Fq), relative density (Rd) [29] and the incidence of genera and species isolated were calculated as follows: Frequency(%) = Number of samples in which a genus/species occurred Total number of samples ×100 Relative density(%) =

Identification of fungi Identification of Fusarium species subcultures were made on SNA (Spezieller Nahrstoffarmer Agar) [26] and PDA incubated for 7 days at 25 ◦ C. Final identifications were made following Nelson et al [25] for Fusarium species and Raper and Fennell [27] for Aspergillus species as well as Pitt and Hocking [28]

Number of isolates of a genus/species × 100 Total number of fungi/genus isolates Incidence(%)

=

Number of grains infected by a genus/species ×100 Total number of grains

116 Table 2. Meteorological data1 of four provinces in Iran, 1981–2000 Provinces

Mean minimum temperature (◦ C)

Mean maximum temperature (◦ C)

Mean temperature (◦ C)

Mean relative humidity (%)

Mean rainfall (mm)

Range altitude2 (m)

Fars Khuzestan Kermanshah Mazandaran

8.2 ± 3.5 a3 16.7 ± 1.8 b 4.8 ± 4 c 10.2 ± 1.9 a

34.4 ± 8 ab 35.4 ± 7.6 b 34.1 ± 9.8 ab 28.6 ± 7 a

19.3 ± 3.3 b 24.9 ± 0.4 c 15.2 ± 2.5 a 16.7 ± 2.2 a

37.8 ± 5.4 a 43.5 ± 1.9 a 45.3 ± 3.9 a 68.8 ± 17.3 b

202.4 ± 140 a 224.8 ± 35 a 444.6 ± 103 b 672.6 ± 323 c

792–2100 3–320 545–1460 –20–1620

Values shown are the mean ± standard deviation. 1 Data supplied by the IR of Iranian Meteorological Organization (IRIMO). 2 Minimum and maximum of altitude based on the meteorological stations of each province. 3 Within columns, means followed by the same letter indicate no significant difference by Tukey’s test (p > 0.05).

Statistical analysis Statistical analyses were performed using SPSS version 9.0 (SPSS Inc. Chicago, Illinois). Analyses of variance (ANOVA) and Tukey’s multiple comparison test at 5% significance level test were used to compare the means of fungal incidence and different meteorological characteristics of the four provinces. Pearson correlations were computed to investigate relationships between Fusarium, Aspergillus and Penicillium species as well as meteorological conditions of the provinces. The mycological analyses were undertaken on Log (x +1) transformed data, but results are presented in the original (untransformed) state. The analyses and results of meteorological characteristics are also shown untransformed. Results and discussion Fungi associated with maize grain A total of 5584 fungal isolates were recovered from 92 maize samples collected in the four main maize production provinces in Iran during 2000. Based on means of incidences Fusarium species were the most frequent (38.5%), followed by Aspergillus species (8.7%), Penicillium species (4.5%), Rhizopus species (4.8%), and Mucor species (1.1%) (Table 3). The four genera Alternaria, Cladosporium and Geotrichum, and Acremonium zeae were isolated at low frequencies, whereas Diplodia species and A. parasiticus were not isolated at all. The specific frequencies of the fungi identified are given in Table 4. Based on frequency as well as relative density, F. verticillioides was the predominant Fusarium species (52% of the total isolations) with the highest

incidence in Mazandaran (59%) and the lowest in Kermanshah (15.4%). After F. verticillioides, F. proliferatum was the most prevalent Fusarium species recorded, with an incidence of 7.7 and 4.5% in Fars and Khuzestan, respectively (Table 4). The highest prevalence of Fusarium species found in the present investigation is in accordance with worldwide reports on these fungi in most maize-producing areas [6, 7, 30], and their high prevalence in Mazandaran is in accordance with the data previously reported from Iran [20–22]. The prevalence of F. verticillioides as a predominant seed-borne fungus in Iranian maize grain was similar to the other maize-producing countries such as South Africa [8] United States [31] Brazil [30] and Argentina [3]. The frequency of F. verticillioides was 90.2%, accounting for 52% of fungal isolates recovered from maize grain. The distribution of this species has implications for human and animal health due to its ability to produce fumonisins, a group of toxic and carcinogenic metabolites that the International Agency for Research on Cancer (IARC) has designated as Group 2B carcinogens, that is, ‘possibly carcinogenic to humans’ [32–34]. The incidence of F. verticillioides in Mazandaran was significantly higher (p < 0.002) than in Kermanshah and Khuzestan but no significant difference was found between Mazandaran and Fars (p > 0.05) (Table 5). The high frequency of F. verticillioides in Mazandaran is the reverse to that cited by Norred [35], namely that the incidence of F. moniliforme infection in the high-EC region of Iran was low in 1982. In contrast, the high incidence of F. verticillioides in Mazandaran supports the reports on high levels of fumonisins in maize from this province [13, 14]. At the same time, the highest frequency, relative density and incidence of Fusarium species in Mazandaran province (Table 4) are in accordance

117 Table 3. Incidence percentage of fungal genera from four provinces in Iran Provinces

Fusarium

Aspergillus

Penicillium

Rhizopus

Mucor

Acremonium

Geotrichum

Alternaria

Cladosporium

Fars Khuzestan Mazandaran Kermanshah

37.2 26.3 66.3 20.6

7.1 3.4 3 24

11.6 1 0.5 1.6

4.9 4.2 1 10

2.9 Nd 0.6 Nd

0.02 Nd 0.05 0.1

0.5 0.5 0.9 Nd

0.5 2.8 0.1 0.2

1.2 0.3 Nd Nd

Overall mean

38.5

8.7

4.5

4.8

1.1

0.04

0.5

0.9

0.5

Nd = not detected.

with the climatic conditions of this province, which from 1981 to 2000 (Table 5) has the highest mean rainfall (672.6 mm; p < 0.001), highest mean relative humidity (68.8%; p < 0.001) as well as the lowest minimum altitude of the four provinces (Table 2). All these conditions were suitable for grain infection by F. verticillioides. It is not uncommon to find lots of shelled maize with 100% infection and in this study 8 samples (36%) of Mazandaran province, 5 samples (13.5%) of Fars province and 3 samples (15.8%) of Khuzestan province had 100% infection with F. verticillioides and F. proliferatum. F. proliferatum was the second most predominant Fusarium species. The frequency and relative density of F. proliferatum was 56.5 and 11%, respectively, and no significant incidence differences (p = 0.07) were detected between provinces (Tables 4, 5). Several studies on the distribution of F. proliferatum on hosts (corn, wheat, rye, barley, sorghum, rice and pearl millet), its similar pathogenic association, fumonisin production and toxicity on corn suggest that this species should behave like that of F. verticillioides [32]. Furthermore, fumonisin production has been demonstrated by all F. proliferatum strains isolated from four different provinces in Iran (Ghiasian et al., unpublished data). Other Fusarium species that were present at low incidence levels were F. acuminatum, F. scirpi, F. equiseti, F. semitectum, F. nygamai (also a fumonisin producer) [36] and F. culmorum. Within the genus Aspergillus, the potentially toxigenic A. flavus was the most widely recovered. This species was isolated from 5.9% of maize grain in 38% of all samples. The other Aspergillus sp. identified was A. niger, which was isolated from 1% of maize grain with a frequency of 19.6%. In North Carolina, USA, A. flavus and A. niger have been reported to infect 31.8 and 10.9% of grain, respectively [4], and

1.35 and 0.24% of grain in Argentina, respectively [3]. The incidence of A. flavus in Kermanshah was significantly higher than in maize from Mazandaran, Fars and Khuzestan (p < 0.01) (Table 5). In Kermanshah province two samples (14%) were 100% infected with A. flavus. This fungus initially colonizes the plant and predisposes the agriculture commodities to mycotoxin contamination after harvest [37]. The potential risk of A. flavus in foodstuff due to aflatoxins B1 and B2 production is considerable. Aflatoxins are highly toxic, mutagenic, carcinogenic and teratogenic metabolites produced mainly by A. flavus and have been implicated as causative agents in human hepatic and extrahepatic carcinomas. Aflatoxin B1 has been evaluated as a Group 1 carcinogen, that is, ‘Carcinogenic to humans’ [34]. The potential risk of A. niger in foodstuff due to mycotoxin production should also be considered, because studies have shown that occasional isolates of A. niger can produce ochratoxin A [38]. According to Marin et al. [39] when A. flavus, A. niger and A. ochraceus (Bainier) Thom are members of competing microflora, they may inhibit the synthesis of fumonisins by Fusarium isolates, or they may degrade the mycotoxin as soon as it is produced. It has been shown that the production of fumonisins by F. verticillioides and F. proliferatum could be altered by other maize fungi. Furthermore, fumonisin production appears to be enhanced by the presence of A. flavus, A. niger and A. ochraceus under certain environmental conditions, but under most conditions of temperature and water activity (aw ) encountered, production is variable or diminished. In the present study the cooccurrence incidence of A. flavus and F. verticillioides was 4.3% and no significant correlation were found between A. flavus and F. verticillioides (p > 0.05). Similar results have been found in Colombia [40] and the United States [41, 42]. However, two of these studies did not obtain any correlation between the incidence of A. flavus and F. verticillioides, and aflatoxin

29.3 7.7 0.08 0.03 0.03 Nd Nd Nd 1.3 3.9 1.8 4.9 2.9 0.03 0.5 11.6 0.5 1.2

In (%) 84.2 63.2 Nd 5.3 10.5 10.5 15.8 5.3 15.8 42.1 10.5 47.4 Nd Nd 5.3 31.6 36.8 10.5

19

741

55.9 11.6 Nd 0.1 0.3 0.3 0.4 0.1 0.7 7.6 0.4 10.8 Nd Nd 1.4 2.6 7.3 0.7

Khuzestan Fq RD (%) (%)

Fq = frequency; Rd = relative density; In = incidence; Nd = not detected.

37

Total samples

44.5 11.8 0.1 0.04 0.04 Nd Nd Nd 2 6 2.7 7.4 4.4 0.04 0.7 17.7 0.8 1.8 2435

91.9 59.5 8.1 2.7 2.7 Nd Nd Nd 24.3 24.3 27 37.8 18.9 2.7 5.4 75.7 13.5 16.2

Fusarium verticillioides F. proliferatum F. acuminatum F. scirpi F. equiseti F. semitectum F. culmorum F. nygamai Aspergillus species A. flavus A. niger Rhizopus species Mucor species Acremonium zeae Geotrichum species Penicillium species Alternaria species Cladosporium species

RD (%)

Total isolates

Fars Fq (%)

Fungal isolates

21.8 4.5 Nd 0.05 0.1 0.1 0.2 0.05 0.3 2.9 0.2 4.2 Nd Nd 0.5 1 2.8 0.3

In (%) 95.5 45.5 Nd Nd Nd Nd Nd Nd 36.4 50 Nd 41 27.3 4.5 18.1 13.6 9.1 Nd

22

1596

81.4 16.7 Nd Nd Nd Nd Nd Nd 0.6 0.5 Nd 0.2 0.1 0.1 0.3 0.1 0.1 Nd

Mazandaran Fq RD (%) (%)

Table 4. Frequency, relative density and incidence of fungal species in maize from four provinces in Iran

59 7.3 Nd Nd Nd Nd Nd Nd 1.5 1.5 Nd 1 0.6 0.05 0.9 0.5 0.1 Nd

In (%) 85.7 57 Nd Nd Nd Nd Nd Nd 43 50 43 43 Nd 14 Nd 50 7 Nd

14

812

26.6 8.9 Nd Nd Nd Nd Nd Nd 3.9 37.4 2.5 17.2 Nd 0.3 Nd 2.8 0.4 Nd

Kermanshah Fq RD (%) (%) 15.4 5.1 Nd Nd Nd Nd Nd Nd 2.3 21.7 1.4 10 Nd 0.1 Nd 1.6 0.2 Nd

In (%)

90.2 56.5 3.3 2.3 3.3 2.3 3.3 1.1 28 38 19.6 41.3 14 4.3 7.6 47.8 16.3 8.7

92

5584

52 11 0.1 0.04 0.3 0.3 0.4 0.1 2.1 9.7 1.6 7.6 2.1 0.1 0.8 8.6 1.4 0.9

Overall mean Fq RD (%) (%)

31.4 6.2 0.02 0.02 0.03 0.02 0.05 0.01 1.4 7.5 0.9 5 0.9 0.05 0.5 3.7 0.9 0.4

In (%)

118

119 Table 5. Comparison between incidences (%) of mycotoxigenic fungi recovered from maize grain in four provinces in Iran Provinces F. verticillioides Fars Khuzestan Kermanshah Mazandaran

29.3 ab∗ 21.8 b 15.4 b 59 a

Mycotoxigenic fungi F. proliferatum A. flavus

Penicillium spp

7.7 a 4.5 a 5.1 a 7.3 a

11.6 a 1b 1.6 b 0.5 b

3.9 b 2.9 b 21.7 a 1.5 b

∗ Within columns, means followed by the same letter do not differ significantly (p > 0.05).

production. For one study [42], F. verticillioides was isolated from maize grain about three times more than A. flavus, but there was no indication of antagonism with A. flavus or of any effect on the levels of aflatoxin found. It is possible that there could be synergism between these two species, whereby, metabolites of F. verticillioides stimulate the metabolism of A. flavus and so increase aflatoxin production. Among the Zygomycetes, fungi of the genera Rhizopus and Mucor were found at a incidence levels of 4.6 and 1.3%, respectively. Bojari and Ershad [21] isolated Mucor cf. indicus and Rhizopus oryza from all maize samples they tested form Mazandaran and Karaj in Iran. Penicillium species were isolated from maize grain and were seen in 47.8% of samples. The highest and lowest incidence of Penicillium species occurred in Fars and Mazandaran provinces (11.6%) and (0.5%), respectively. Statistical comparisons of the Penicillium species isolated in the four regions were made and significant differences (p < 0.0001) were observed between Fars and the other three provinces (Table 5). Hesseltine et al. [4] found that 28.6% of samples of maize grain from in North Carolina, USA, were infected by Penicillium species, while Gonzalez et al. [3] found only 2.16% of maize grains to be contaminated by Penicillium species in Argentina. To establish the potential toxigenic risk of these fungi on maize, it will be necessary to identify the Penicillium cultures isolated to species level. There was no significant correlation between the incidence of F. verticillioides and meteorological characteristics (p > 0.05). Furthermore, the correlation between the incidences of F. verticillioides and Aspergillus flavus and Penicillium species gave significant negative correlations (respectively: p < 0.05; r = – 0.205, and p < 0.01; r = –0.250). According to Zummo et al. [31] when maize ears were inoculated simul-

taneously with F. verticillioides and A. flavus or with A. flavus alone, significantly fewer grains were infected with A. flavus in ears inoculated with both fungi, than grains from ears inoculated with A. flavus alone. Zummo et al. concluded that F. verticillioides can inhibit grain infection by A. flavus in inoculated maize ears and reduce aflatoxin contamination in these grains. Hill et al. [43] also found that F. verticillioides occurred more abundantly than members of the A. flavus group and a negative correlation between the A. flavus group and F. verticillioides occurred in both years of their studies. Diplodia species were not isolated from any of the samples examined. In addition, in a previous study it had been noted that Diplodia species are not important as ear-rot pathogens in the main production areas of Iran [21]. The dominance of F. verticillioides in Iranian maize is in accordance with other reports that F. verticillioides is regarded as the most common seed-borne fungus of maize and is associated with ear-rot of maize in maize-producing countries worldwide [32, 44, 45]. The importance of F. verticillioides is due not only to its high incidence as a seed-borne fungus, often causing symptomless infections in maize grains, but also because of its potential toxigenicity [1, 32, 34]. Knowledge about grain mycoflora on the farm and during storage can predict the post-harvest deterioration, changes Fungal Flora and further mycotoxin hazards. Both fungal and mycotoxin contamination are a primary concern in the effort to reduce economic losses and minimize potential risks to human and animal health [46]. In conclusion, although the detection of toxigenic fungi in a substrate does not necessarily indicate that mycotoxins are naturally occurring in the field, it highlights the potential risk of contamination [47]. This survey of freshly harvested maize grain in the main

120 production areas, as well as a high-EC region, in Iran where there are significant differences in mean temperature, mean rainfall and mean relative humidity, indicated that the maize grain is infected with important potentially toxigenic species, including F. verticillioides, F. proliferatum and A. flavus. The high frequency of these three mycotoxin-producing species in this study emphasizes the importance of continued research on fumonisin and aflatoxin in Iranian maize.

Acknowledgements The authors acknowledge with great thanks K. Zabihi of the Herbal Forage of Ministry of Agriculture, Tehran, Iran for providing facilities for the collection of maize; D. Ershad, Plant Pests & Diseases Research Institute, Tehran Iran, and W. Gams, Centeraal Bureau Voor Schimmelcultures, The Netherlands, for assisting with fungal identification, and A. Roohbakhsh for preparation of meteorological data. The authors also are indebted to WFO Marasas, JP Rheeder and HF Vismer, Medical Research Council, PROMEC Unit, Tygerberg, South Africa for reviewing the manuscript.

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