Changes in fungi and mycotoxins in pearl millet under controlled storage conditions

Pearl millet is increasingly being grown as a premium-value grain for the recreational wildlife and poultry industries in the southern US. We conducted three experiments to assess grain mold development in storage conditions typically encountered in the region of production. Variables included production year, temperature, relative humidity, atmosphere, and grain moisture content. In the first experiment, grain was stored for 9 weeks at 20 or 25°C and maintained at 86% or 91% relative humidity (r.h.). In the second experiment, grain was stored for 9 weeks at 20 or 25°C in either air (aerobic) or N₂ (anaerobic), and maintained at 100% r.h. In the third experiment, high-moisture grain was stored for 3 weeks at 20 or 25°C and maintained at 100% r.h. Grain was sampled at weekly intervals and plated to determine changes in fungal frequency. Fungi isolated included Fusarium chlamydosporum (19% of grain), Curvularia spp. (14%), F. semitectum (16%), Alternaria spp. (9%), Aspergillus flavus (8%), “Helminthosporium”-type spp. (6%), and F. moniliforme sensu lato (3%). Year of grain production significantly affected isolation frequency of fungi. Isolation frequencies from low-moisture grain were rarely affected by temperature, relative humidity, or atmosphere treatments, but was affected by storage duration for some fungi. Changes in isolation of toxigenic fungi occurred in high-moisture grain. Isolation frequency of F. chlamydosporum increased in grain stored at 86% and 91% r.h. Incidence of A. flavus increased in high-moisture grain treatments, particularly at 25°C. Incidence of deoxynivalenol was not affected by storage treatment. Low concentrations of nivalenol were detected in most grain incubated at 100% r.h. Zearalenone was detected only when grain moisture content was 20–22%. Aflatoxin contamination averaged 174 ng g⁻¹ over all treatments, and increased up to 798 ng g⁻¹ in high-moisture grain at stored at 25°C.     

Ear-rot fungi and mycotoxins in South African corn of the 1989 crop exported to Taiwan

A shipment of South African corn (1989) exported to Taiwan, was analyzed for various ear-rot fungi andFusarium mycotoxins. Two sets of samples, one from the points of origin in South Africa prior to shipment, and the other from the end-point distributors in Taiwan, were studied. Surface-sterilized kernels were plated onto two different agar media and the fungal colonies identified. High Performance Liquid Chromatography was used to analyze mycotoxin levels. The predominant ear-rot fungi, in decreasing order of isolation frequency, wereFusarium subglutinans, F. moniliforme, Diplodia maydis andF. graminearum. Aspergillus flavus andA. parasiticus were not isolated from samples prior to export, but a small number ofA. flavus isolates were found after shipment. The predominant mycotoxins were fumonisins B₁ (0–865 ng/g) and B₂ (0–250 ng/g). Low levels of moniliformin (390 ng/g) were detected in some samples before shipment. Zearalenone (25 ng/g), and nivalenol (120 ng/g) were detected in two out of 32 samples taken in Taiwan. The samples contained no detectable levels of either aflatoxins (>0.5 ng/g) or deoxynivalenol (>100 ng/g) before or after shipment.Abbreviations RSA South Africa(n) - FB₁ fumonisin B₁ - FB₂ fumonisin B₂ - ETVL eastern Transvaal - WTVL western Transvaal     

Fungi associated with food and feed commodities from Ecuador

Freshly harvested soybean, rice and corn from farms and corn-based pelleted feeds were collected from ranches from the coastal and mountain regions in Ecuador during 1998, and assessed for fungal contamination. The most prevalent fungi on pelleted feed were Aspergillus flavus and Fusarium graminearum. The prevalent fungi recovered from soybean were F. verticillioides, F. semitectum, Aspergillus flavus and A. ochraceus. In rice, F. oxysporum was the most prevalent toxigenic fungal species recorded, followed by F. verticillioides and A. flavus. In corn, F. verticillioides was the most prevalent fungus isolated in both the coastal and mountain regions, with high isolation frequencies of A. flavus and A. parasiticus at the coast. Based on the toxigenic species recovered, ochratoxin A may pose a contamination risk for soybean. A higher probability of aflatoxin contamination of corn was found in the coastal samples compared to those of the mountain region, while a risk of fumonisin contamination of corn exists in both regions.This revised version was published online in October 2005 with corrections to the Cover Date.     

Fungal utilization of organophosphate pesticides and their degradation byAspergillus flavus andA. sydowii in soil

Fungal species were isolated which utilize organophosphate pesticides,viz. phosphorothioic (pirimiphos-methyl and pyrazophos), phosphorodithioic (dimethoate and malathion), phosphonic (lancer) and phosphoric (profenfos) acid derivatives. Pesticide degradation was studiedin vitro andin vivo (soil).Aspergillus flavus, A. fumigatus, A. niger, A. sydowii, A. terreus, Emericella nidulans, Fusarium oxysporum andPenicillium chrysogenum were isolated from pesticide-treated wheat straw. The number ofA. sydowii colonies was significantly promoted by 1 mmol/L pirimiphos-methyl, pyrazophos, lancer, dimethoate and malathion when used as phosphorus sources and by pirimiphos-methyl and pyrazophos when used as carbon sources. The number ofA. flavus colonies increased with 0.5 mmol/L lancer and malathion used as the only carbon sources.A. sydowii, A. niger, A. flavus, E. nidulans andF. oxysporum grew on, and utilized, 5 pesticides as phosphorus source and showed more than 50% mass growth.A. sydowii, A. flavus andF. oxysporum phosphatase hydrolyzed the pesticides suggesting that these species are important pesticide degraders.A. sydowii produced higher amounts of the phosphatase thanA. flavus andF. oxysporum. The enzyme was highly active against pyrazophos, lancer and malathion used as the only sources of organic phosphate.A. flavus andA. sydowii phosphatases efficiently hydrolyzed pesticides at 300 ppm in soil, the degradation at 1000 ppm was lower. Mineralization of 1000 ppm pesticides in soil amended with wheat straw was higher than in nonamended soil. All added pesticides except profenfos were degraded within 3 weeks. Lyophilized adapted biomass ofA. flavus andA. sydowii could thus be used for field biodegradation of these pesticides.     

Influence of formic acid on fungal flora of barley and on aflatoxin production inAspergillus flavus link

In recent yearsAspergillus flavus and aflatoxin production have been noted on several occasions in grain preserved with formic acid. Samples of mouldy barley treated with formic acid and stored in an open bin were investigated for the presence of fungi. In the lower part of the bin there was a clear dominance ofFusarium sporotrichioides, and deoxynivalenol and neosolaniol were detected.A. flavus andA. fumigatus were also present.Paecilomyces variotii occurred, almost as a pure culture, in the upper part of the bin, but no patulin was found. Cultivation of four fungal isolates from these genera on laboratory substrates containing formic acid showedP. variotii to be the most tolerant to formic acid, withstanding 150 mM, but still without patulin production.F. sporotrichioides andA. fumigatus tolerated only 6 mM formic acid. The growth ofA. flavus was reduced and atypical at 60 mM formic acid. Pretreatment ofA. flavus spores with formic acid increased aflatoxin production about 800 times.     

Preharvest seed infection byAspergillus flavus group fungi and subsequent aflatoxin contamination in groundnuts in relation to soil types

Preharvest seed infection byAspergillus flavus and aflatoxin contamination in selected groundnut genotypes (fourA. flavus-resistant and fourA. flavus-susceptible) were examined in different soil types at several locations in India in 1985–1990. Undamaged mature pods were sampled at harvest and seed examined forA. flavus infection and aflatoxin content in two or more trials at ICRISAT Center on light sandy soils and red sandy loam soils (Alfisols), and on Vertisols, at Anantapur on light sandy soils, and at Dharwad and Parbhani on Vertisols. Rainy season trials (1985–1989) were all rainfed. Post-rainy season trials were irrigated; late-season drought stress (90 days after sowing (DAS) until harvest at 125 DAS) was imposed in the 1987/88 and 1989/90 seasons.A. flavus infection and aflatoxin contamination levels were much lower in seed of all genotypes from Vertisols than in seed from Alfisols across locations and seasons. Vertisols also had significantly lower populations ofA. flavus than Alfisols. There were no marked differences between light sandy soils and red sandy loam soils (Alfisols) in respect of seed infection byA. flavus and aflatoxin contamination. Significant interactions between genotypes and soil types were evident, especially in theA. flavus-susceptible genotypes. Irrespective of soil types,A. flavus-resistant genotypes showed lower levels of seed infection byA. flavus and other fungi than didA. flavus-susceptible genotypes. The significance of the low preharvest aflatoxin risk in groundnuts grown on Vertisols is highlighted.ICRISAT Journal Article No. JA 1122     

Preharvest aflatoxin contamination of maize inoculated withAspergillus flavus andFusarium moniliforme

A two-year factorial experiment was utilized to test plants field-inoculated singly and in combination withAspergillus flavus andFusarium moniliforme. Pinbar inoculations were made through the husks with conidial suspensions, and 10-ear maize samples were harvested at 60 days post-silking for aflatoxin determinations. When ears were inoculated with both fungi simultaneously,F. moniliforme reduced aflatoxin formation byA. flavus isolate NRRL 3357 by approximately two-thirds.F. moniliforme had no significant effect on naturally occurring aflatoxin contamination byA. flavus. This may be due to the timing of infection by both fungi in the field. In nature,A. flavus andF. moniliforme respond differently to the environment, offering one explanation of whyF. moniliforme did not measurably affect the other fungus.     

Fungicide inhibition of aflatoxins,diacetoxyscirpenol and zearalenone production

The effect of fungicides on the production of aflatoxin byAspergillus flavus IMI 89717, diacetoxyscirpenol and zearalenone byFusarium graminearum was studied. In a yeast extract - sucrose medium, dicloran, iprodione and vinclozolin fungicides significantly inhibited mycelial growth ofA. flavus at 250 ppm and significantly decreased aflatoxin production at 100, 250 and 500 ppm, respectively. In potato — dextrose broth, these fungicides diminished the mycelial growth ofF. graminearum and production of diacetoxyscirpenol and zearalenone at 100 ppm. Sensitivity of toxigenic mycelia to fungicides increased approximately five-fold in a yeast extract-starch medium with an appreciable reduction in sugar uptake andα-amylase activity.     

Mycotoxins in developing countries: A case study of maize in Nepal

Maize (Zea mays) is an important food crop in the foothills of the Nepal Himalaya Mountains. Surveys have found that maize in Nepal is contaminated withFusarium species, mainlyF. verticillioides andF. proliferatum, which produce fumonisins, andF. graminearum, which produces trichothecenes, mainly nivalenol and 4-deoxynivalenol. Maize from smallholder farms and markets is often contaminated with fumonisins and trichothecenes above 1000 ng/g, a level of concern for human health. These mycotoxins were not eliminated by traditional fermentation for producing maize beer, but Nepalese women were able to detoxify contaminated maize by hand-sorting visibly disease kernels. An integrated approach to reduce mycotoxins risks in maize in Nepal and other developing countries should include plant breeding to produce ear rot resistant cultivars, along with education in mycotoxins risks and in agricultural and grain storage practices to reduce mycotoxin contamination. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005     

Natural occurrence of aflatoxin andAlternaria mycotoxins in oilseed rape from Catalonia (Spain): Incidence of toxigenic strains

During an investigation of the mycoflora on oilseed rape, the predominant fungal species present in 20 samples collected from Catalonia (Spain) wereAlternaria alternata (Fries) Keissler,Penicillium spp. andAspergillus flavus. None of the 20 samples analyzed presented contamination byAlternaria mycotoxins (tenuazonic acid, alternariol, alternariol methyl ether, altertoxin I and altertoxin II). Only aflatoxin B₁ was detected in 1 of the 20 samples analyzed, with a concentration of 0.25 ppb. Of the 40Aspergillus flavus strains isolated from oilseed rape samples, only 3 revealed aflatoxigenic capacity. None of thePenicillium spp. isolated from oilseed rape samples revealed mycotoxigenic capacity (citreoviridin, griseofulvin, citrinin, patulin and penicillic acid).     

Comparison of four media for the isolation ofAspergillus flavus group fungi

Four agar media used to isolate aflatoxin producing fungi were compared for utility in isolating fungi in theAspergillus flavus group from agricultural soils collected in 15 fields and four states in the southern United States. The four media wereAspergillus flavus andparasiticus Agar (AFPA, 14), the rose bengal agar described by Bell and Crawford (BCRB; 3), a modified rose bengal agar (M-RB), and Czapek's-Dox Agar supplemented with the antibiotics in BC-RB (CZ-RB). M-RB was the most useful for studying the population biology of this group because it permitted both identification of the greatest number ofA. flavus group strains and growth of the fewest competing fungi. M-RB supported an average of 12% moreA. flavus group colonies than the original rose bengal medium while reducing the number of mucorales colonies and the number of total fungi by 99% and 70%, respectively. M-RB was successfully employed to isolate all three aflatoxin producing species,A. flavus, A. parasiticus andA. nomius, and both the S and L strains ofA. flavus. M-RB is a defined medium without complex nitrogen and carbon sources (e.g. peptone and yeast extract) present in BC-RB. M-RB should be useful for studies on the population biology of theA. flavus group.Abbreviations M-RB Modified Rose Bengal Agar - CZ-RB Czapeks Rose Bengal Agar - BC-RB Bell and Crawford's Rose Bengal Agar - AFPA Aspergillus flavus andparasiticus agar     

A rapid identification method for aflatoxin-producing strains ofAspergillus flavus andA. parasiticus by ammonia vapor

The colony reverse of aflatoxin (AF)-producing strains ofAspergillus flavus andA. parasiticus turned pink when their cultures were exposed to ammonia vapor. The color change was visible for colonies grown on media suitable for AF production such as potato dextrose, coconut, and yeast extract sucrose agars after 2 d incubation at 25°C. Of the 120 strains ofA. flavus, A. parasiticus, and two related species inA. flavus group:A. oryzae andA. sojae tested in this study, only the AF-producing strains ofA. flavus andA. parasiticus showed the pink pigmentation. The color change occurred immediately after the colony was contacted with ammonia vapor. This method was useful for rapid screening the AF-producing strains ofA. flavus andA. parasiticus.     

Evaluation of selected geocarposphere bacteria for biological control of Aspergillus flavus in peanut

Selected bacterial strains isolated from the region of peanut pod development (geocarposphere) and two additional bacterial strains were screened as potential biological control agents against Aspergillus flavus invasion and subsequent aflatoxin contamination of peanut in laboratory, greenhouse, and field trials. All 17 geocarposphere strains tested delayed invasion of young roots and reduced colonization by the fungus in a root-radicle assay used as a rapid laboratory prescreen. In a greenhouse study, seven bacterial strains significantly reduced pod colonization by A. flavus compared to the control. In a field trial, conducted similarly to the greenhouse assay, pods sampled at mid-peg from plants seed-treated with suspensions of either 91A-539 or 91A-550 were not colonized by A. flavus, and the incidence of pods invaded from plants treated with either 91A-539 or 91A-599 was consistently lower than nonbacterized plants at each of five sampling dates. At harvest, 8 geocarposphere bacterial strains significantly lowered the percentage of pods colonized (> 51%) compared to the control. Levels of seed colonization ranged from 1.3% to 45% and did not appear related to aflatoxin concentrations in the kernels.     

Proof of concept: could snake venoms be a potential source of bioactive compounds for control of mould growth and mycotoxin production

The objective was to screen 10 snake venoms for their efficacy to control growth and mycotoxin production by important mycotoxigenic fungi including Aspergillus flavus, Aspergillus westerdijkiae, Penicillium verrucosum, Fusarium graminearum and F. langsethiae. The Bioscreen C rapid assay system was used. The venoms from the Viperidae snake family delayed growth of some of the test fungi, especially F. graminearum and F. langsethiae and sometimes A. flavus. Some were also able to reduce mycotoxin production. The two most potent crude snake venoms (Naja nigricollis and N. siamensis; 41 and 43 fractions, respectively) were further fractionated and 83/84 of these fractions were able to reduce mycotoxin production by >90% in two of the mycotoxigenic fungi examined. This study suggests that there may be significant potential for the identification of novel fungistatic/fungicidal bioactive compounds as preservatives of raw and processed food commodities post-harvest from such snake venoms.     

Development and applications of a yeast-based bioassay for the mycotoxin zearalenone

Zearalenone (ZON) is a non-steroidal estrogenic mycotoxin produced by plant pathogenic species ofFusarium. As a consequence of infection withF. culmorum andF. graminearum, ZON can be found in cereals and derived food products. Several countries have established monitoring programs and guidelines for ZON levels in grain intended for human consumption and animal feed. We have developed a sensitive yeast bioassay allowing detection of the estrogenic activity of ZON in cereal extracts without requiring further clean up steps. The high sensitivity makes this assay suitable for low cost monitoring of contamination of small grain cereals with estrogenicFusarium mycotoxins, but also attractive as a tool for basic research. We have successfully used yeast indicator strains to screen for mutants ofF. graminearum which no longer produce detectable amounts of ZON, and have identified a plant cDNA encoding a ZON detoxification enzyme. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Examination of fungal growth and aflatoxin production on marihuana

Under favorable growth conditions,Aspergillus flavus andA. parasiticus produced aflatoxins on marihuana. Cultures ofA. flavus ATCC 15548 produced both aflat oxin B₁(AFB₁) and G₁(AFG₁). The production of AFG₁ was substantially greater than that of AFB₁. Cultures ofA. flavus NRRL 3251 andA. parasiticus NRRL 2999 produced only AFB₁. All natural flora cultures tested negative for aflatoxins. NoAspergilli sporulations were observed in these cultures. In the cultures inoculated with known toxigenic fungi, the highest mean level for total aflatoxins was 8.7 g/g of medium. Marihuana appears not to yield large quantities of these mycotoxins but sufficient levels are present to be a potential health hazard for both the user and the forensic analyst who is in daily contact with such plant material. Careful processing, storage, and sanitation procedures should be maintained with marihuana. If these conditions are disregarded due to the illicit status of marihuana, the potential for mycotoxin contamination must be considered.     

Mycotoxigenic infestation in samples of cereal straw from Bihar, India

A survey of different types of cereal straw samples viz. paddy, maize and wheat, from Bihar State, India, was conducted in order to examine the mould flora and mycotoxin contamination. Out of 170 samples examined for mould flora,Aspergillus flavus group of fungi had highest level of incidence followed byA niger. Isolates ofA flavus, A ochraceus, Fusarium verticillioides andPenicillium citrinum were screened for their mycotoxins producing abilities. Out of 75, 63 and 68 isolates ofA flavus group obtained from stored straw of paddy, maize and wheat samples, respectively, 27 (36%), 14 (22%) and 24 (35%) were found to be toxigenic which produced different combinations of aflatoxins in different concentrations. The percentage toxigenicity was comparatively lower in the isolates of other mycotoxigenic fungi from all types of samples. Out of 222 samples of straw analysed for natural occurrence of different mycotoxins, besides the aflatoxins present, zearalenone, ochratoxin A and citrinin were also recorded alone or as co-contaminants. A conducive climate together with the socioeconomic conditions of this region are important determinants for the high incidence of mycotoxins in cereal straw samples.     

Genetic variation, real-time PCR, metabolites and mycotoxins ofFusarium avenaceum and related species

In this paper the latest studies dealing with genetic variation and mycotoxins ofF. avenaceum and related species are reviewed and compared to the data from chromatographic image analyses. Forty-three European strains ofFusarium avenaceum and related species were classified by chromatographic image analysis on full chromatographic matrices. The results were in most cases in agreement with those from morphological and molecular analyses and supported the separation betweenF. avenaceum, F. arthrosporioides andF. tricinctum and betweenF. avenaceum groups I and II. The mycotoxin profiles of the FinnishF. avenaceum, F. arthrosporioides andF tricinctum strains were very similar to each other. Moniliformin and enniatins were the main mycotoxins produced. A fluorogenic TaqMan PCR assay (qPCR) was used for the detection ofF. avenaceum/ F. arthrosporioides DNA in Finnish barley and wheat. The qPCR results obtained from grain samples were compared to mycotoxin levels. A correlation was found betweenF. avenaceum/F. arthrosporioides DNA and moniliformin (MON) and enniatin (ENNs) levels in barley. A correlation was also found between the combinedF. avenaceum/F. arthrosporioides/F. tricinctum contamination and MON and ENNs levels in barley in 2002, but not in 2003. This was probably due to the higher MON and ENNs levels in 2002 than in 2003. It was possible to use the DNA levels ofF. avenaceum/F. arthrosporioides to distinguish between most barley samples containing high amounts of MON and ENNs from those containing low levels of the mycotoxins. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: Grants from the National Technology Agency of Finland (No. 40168/03) and the Academy of Finland (No. 52104); travel grants from NorFA and the European Commission to the Laboratory of Dr. Ulf Thrane     

Isolation and characterization of new chlamydosporol related metabolites ofFusarium chlamydosporum andFusarium tricinctum

Fusarium chlamydosporum strain T-826 isolated from corn in the USA produced chlamydosporol and two analogs which have been identified by various spectroscopic techniques as: 7,8-dihydro-5-hydroxy-4-methoxy-trans-7,8-dimethyl-2H,5H-pyrano(4,3-b)pyran-2-2-one (or isochlamydosporol) and 4-methoxy-5-hydroxymethyl-6-(3-butan-2-ol)-2H-pyran-2-one (or chlamydospordiol). Chlamydosporol (compounda+b) chlamydospordiol (compoundc) and isochlamydosporol (compoundd) were produced together (up to 6000 µg/g) by 3 out of 11 isolates ofF. chlamydosporum and by 3 out of 24 isolates ofF. tricinctum from various substrates and geographic origin. Three isolates ofF. chlamydosporum and one isolate ofF. tricinctum produced only chlamydospordiol and 2 isolates ofF. tricinctum produced chlamydosporol (a+b), and chlamydospordiol (c)PRC Publication, No. 1518     

Host-specific Variation in Infection by Toxigenic Fungi and Contamination by Mycotoxins in Pearl Millet and Corn

Pearl millet is widely consumed in regions of Africa and Asia, and is increasingly being grown as an alternative grain in drought-prone regions of the United States. Pearl millet and corn were grown in dryland conditions at Tifton, Georgia, USA and grains were compared for pre-harvest infection by potentially toxigenic fungi and contamination by mycotoxins. Corn hybrids Agripro 9909 and Pioneer 3146, and pearl millet Tifgrain 102 were grown in 2000 and 2001; pearl millet HGM 100 was included in the test in 2001. Hybrids were sown on multiple planting dates in each year to induce variation in flowering time. Host species differed in the frequency of isolation of potentially toxigenic fungal species in both years. Across years, corn hybrids were more prone to infection by Aspergillus flavus Link (maximum isolation frequency = 8.8%) and Fusarium moniliforme Sheldon sensu lato (maximum isolation frequency = 72.8%), with corresponding greater concentrations of aflatoxins (maximum concentration = 204.9 μg kg⁻¹) and fumonisins (maximum concentration = 34,039 μg kg⁻¹). Pearl millet was more prone to infection by F. semitectum Berk. & Ravenel (maximum isolation = 74.2%) and F. chlamydosporum Wollenweb & Reinking (maximum isolation = 33.0%), and contamination by moniliformin (maximum contamination = 92.1 μg kg⁻¹). Beauvericin (maximum concentration = 414.6 μg kg⁻¹) was present in both hosts. Planting date of corn affected aflatoxin and beauvericin contamination in 2000, and fumonisin concentration in 2001. The observed differences in mycotoxin contamination of the grains, which are likely due to host-specific differences in susceptibility to pre-harvest mycoflora, may affect food safety when the crops are grown under stress conditions.