Characterization of Fusarium verticillioides strains isolated from maize in Italy: fumonisin production, pathogenicity and genetic variability

Fusarium verticillioides (teleomorph Gibberella moniliformis) is the main fungal agent of ear and kernel rot of maize (Zea mays L.) worldwide, including Italy. F.verticillioides is a highly toxigenic species since it is able to produce the carcinogenic mycotoxins fumonisins. In this study, 25 F. verticillioides strains, isolated from maize in different regions of Italy were analyzed for their ability to produce fumonisins, their pathogenicity and their genetic variability. A further referenced strain of G. moniliformis isolated from maize in USA was also used as outgroup. The fumonisins B₁, B₂, and B₃ were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Pathogenicity tests were carried out by symptom observation and determination of growth parameters after inoculation of maize seeds, seedlings and wounded detached leaves. Total genomic DNA was used for Amplified Fragment Length Polymorphism (AFLP) analysis. About 20% of the analyzed strains were unable to produce fumonisins in in vitro experiments on inoculated maize flour, while, among fumonisin producers, a great variability was observed, with values ranging from 1 to 115 mg kg⁻¹. The different analyzed strains showed a wide range of pathogenicity in terms of effect on seed germination, seedling development and of symptoms produced on detached leaves, which were not correlated with the different in vitro fumonisin production. AFLP analysis indicated the presence of genetic diversity not only between the Italian strains and the American reference but also among the Italian isolates.     

Analysis of potential fumonisin‐producing Fusarium species in corn products from three main maize‐producing areas in eastern China

BACKGROUND: Fusarium species are common fungal contaminants of maize and a number of them can produce mycotoxin fumonisins. China is one of the largest maize producers in the world. This study investigated the contamination of maize samples from three areas in eastern China by Fusarium and fumonisin‐producing fungi as well as their fumonisin‐producing potential. RESULTS: A total of 22 Fusarium strains were isolated, 19 of which were able to produce fumonisin. Among the 19 strains, 16 belonged to F. verticillioides, two to F. subglutinans and one to F. proliferatum. The majority (17/19) of the fumonisin‐forming strains were high FB₁ producers, which is a potential health risk for the population in these areas. Fusarium contamination in samples from the mideastern area was the most serious (11 Fusarium strains, with nine producing fumonisin, isolated from 24 samples), followed by the northeastern area (nine Fusarium strains, with all nine producing fumonisin, isolated from 21 samples) and the southeastern area (two Fusarium strains, with one producing fumonisin, isolated from 19 samples). CONCLUSION: Although the overall levels of FBs and contamination by fumonisin‐producing fungi in corn samples were not serious, the contaminating Fusarium strains possessed fairly strong toxicogenic ability and potential risk for food safety. © 2012 Society of Chemical Industry     

Evaluating three commonly used growth media for assessing fumonisin analogues FB1, FB2 and FB3 production by nine Fusarium verticillioides isolates

ABSTRACT

Maize is most often infected by the fumonisin-producing Fusarium verticillioides. Total fumonisins of natural infected grain is made up of FB₁, FB₂ and FB₃ with FB₁ occurring naturally at higher levels. A maize plant can be infected with more than one F. verticillioides isolate, and finding a reliable method to elucidate the toxigenic potential of these isolates is important to extrapolate the possible fumonisin risk to consumers of grain. It is not clear whether F. verticillioides produces similar fumonisin levels, as well as fumonisin analogue ratios, across media. In this study, nine F. verticillioides isolates were subjected to three methods of fumonisin testing using liquid media, maize patties and a field trial (silk inoculation of grain) in Potchefstroom, South Africa. Spore concentrations of 1 × 10⁶ conidia ml^–1 of each isolate were used to inoculate the different media and levels fumonisin analogues were measured using HPLC. Fumonisin production per isolate was highly variable and was influenced by the two-way interaction of F. verticillioides isolate × growth media. Total fumonisins produced in the liquid medium ranged from 0 to 21.3 ppm, on maize patties fumonisins they ranged from 0 to 21.5 ppm, and in the silk inoculation technique they ranged from 0 to 15.5 ppm. The fumonisin analogue FB₁ occurred at higher levels followed by FB₃ in both in vitro studies. In the silk inoculation technique, fumonisin analogue FB₂ was the second highest occurring analogue after FB₁. Isolate GCI 282 produced higher FB₂ and FB₃ levels than FB₁ in the patties and grain, respectively. In order not to miscalculate the fumonisin and analogue ratio levels per F. verticillioides isolate, the growth medium will have to be optimised for each isolate and more than one growth medium used.     

Recent research on fumonisins: a review

Fumonisins are well known mycotoxins produced by Fusarium verticillioides, F. proliferatum and other Fusarium species. Many new fumonisins and fumonisin-like compounds have been detected by mass spectrometry in cultures of F. verticillioides. Recently, fumonisins B₂ and B₄ were produced by Aspergillus niger isolated from coffee and fumonisin B₂ in A. niger from grapes. Fumonisin B₂ was itself detected in coffee beans, wine and beer, adding to the list of foodstuffs and feedstuffs other than corn (maize) and sorghum in which fumonisins have been found in recent years. Fumonisin B₁ (FB₁) can bind to proteins (PB FB₁) and to other matrix components during food processing involving heat. The occurrence of bound fumonisins in processed corn foods is common. Another type of binding (or association) relates to observed instability of fumonisins in rice flour, corn starch and corn meal at room temperature; this can affect the immunoaffinity column clean-up procedure in analysis of naturally contaminated starch-containing corn foods for fumonisins. The occurrence of N-fatty acylated fumonisin derivatives in retail fried corn foods has also been demonstrated. Bioaccessibility of free FB₁ and total bound FB₁ (TB FB₁) present in corn flakes has been estimated by in vitro digestion experiments. Intentional binding of fumonisins to cholestyramine has been demonstrated in vivo and is a potential means of detoxification of animal feed.     

Exposure,Occurrence, and Chemistry of Fumonisins and their Cryptic Derivatives

Fumonisins are mycotoxins mainly produced by Fusarium proliferatum and Fusarium verticillioides. Because of their wide distribution, the potential health hazard, and economic significance, they are considered one of the most important mycotoxin classes. Epidemiological evidence suggests a relationship between the Fusarium load in corn, exposure to fumonisins, and esophageal cancer. However, mechanisms of actions of fumonisins are not yet fully resolved and epidemiological studies suffer from various confounding factors. Correspondingly, the most relevant congener of the fumonisin family (fumonisin B₁) has been classified as possibly carcinogenic to humans and maximum limits have been set for corn and corn‐based products. However, many non‐corn‐based products are also susceptible to fumonisin contamination. Indeed, some of them contain very high amounts of fumonisins, but enter the market legally. Furthermore, fumonisin exposure of consumers is probably consistently being underestimated because only a fraction of fumonisins can be detected by routine analysis. The bioavailability and toxicity of most nondetectable (cryptic) forms has not been resolved. In this work, we review the developments of cancer research into fumonisins since their discovery in 1988 until today and provide an overview of the contributions of various foodstuffs to fumonisin exposure, including those products that have been largely neglected in the past. In conclusion, (1) corn remains the principal source of fumonisin ingestion, but fumonisins in non‐corn‐based commodities require continuous monitoring; (2) cryptic fumonisins should be included in risk assessment studies; and (3) certain population groups (for example children) may suffer from enhanced exposure and could face increased health risks.     

Genetic variability and Fumonisin production by Fusarium proliferatum

Fusarium proliferatum is together with Fusarium verticillioides the main source of fumonisins, a health risk mycotoxin, contaminating agro-products. Contrary to F. verticillioides, it colonizes a wide range of host plants besides maize, such as wheat or barley among others, in particular in certain regions (Southern Europe). The phylogenetic study performed in this work using a wide sample of isolates from diverse hosts and origins revealed a high variability, while no host preferences could be sustained. A real time RT-PCR assay was also developed specific for F. proliferatum on the basis on fumonisin biosynthetic gene, FUM1, which allowed discrimination from F. verticillioides. FUM1 gene expression showed a high and significant correlation (0.77) with fumonisin production, representing a valuable tool for specific and sensitive diagnosis of metabolically active fumonisin-producing F. proliferatum isolates and for evaluating the influence on environmental conditions on FUM1 gene regulation. The ability to produce fumonisins was also widely distributed indicating that F. proliferatum can represent a risk for health similarly to F. verticillioides. Moreover, the wide range of plants susceptible to colonization by F. proliferatum suggests that the impact of fumonisin risk in a number of commodities might need a revision.     

A PCR-Based Assay for the Detection and Differentiation of Potential Fumonisin-Producing Fusarium verticillioides Isolated from Indian Maize Kernels

One hundred and three Fusarium isolates from maize samples collected from different districts of Karnataka state, India, were analyzed with genus-specific, species-specific, and potential fumonisin specific oligonucleotide primers. One set of genus-specific primers ITS F and ITS R based on a highly conserved ITS region of the genus Fusarium were used to differentiate Fusarium species from closely related genera. All the Fusarium species tested scored positive with the ITS pair of primers. Detection and identification of Fusarium verticillioides species was done by using a newly designed reverse primer VERT-R (5′- CGA CTC ACG GCC AGG AAA CC ?3′) based on an intergenic spacer sequence (IGS) combined with an already designed forward primer VERTF-1 (5′-GCG GGA ATT CAA AAG TGG CC -3′) published previously. Out of 103 Fusarium species tested, 83 isolates of F. verticillioides scored positive for VERTF-1/ VERT-R species-specific pair of primers. Further to discriminate potential fumonisin-producing and nonproducing strains of F. verticillioides, the VERTF-1/VERTF-2 set of primers [VERTF-1 (5′-GCG GGA ATT CAA AAG TGG CC -3′) and VERTF-2 (5′-GAG GGC GCG AAA CGG ATC GG -3′)] were used. 64 isolates of F. verticillioides scored positive for VERTF-1/ VERTF-2 pair of primers. In total, three primers, one forward primer VERTF-1 and two reverse primers VERT-R and VERTF-2, were used for the confirmation of F. verticillioides up to the species level and the second pair of primers were used to confirm the potential for fumonisin production. The developed PCR assay should provide a powerful tool for the detection and differentiation of potential fumonisin-producing F. verticillioides strains in a population.     

Incidence of Fusarium verticillioides and levels of fumonisin B1 and B2 in corn in Turkey

A total of 100 corn samples conforming collected from local farmers and markets from districts of Samsun, Turkey, were analyzed for Fusarium verticillioides, fumonisin B₁ and B₂ contamination. Ninety-three corn samples were found to contain F. verticillioides, 52 samples fumonisin B₁, and 25 samples fumonisin B₂. Fumonisin B₁ contamination ranged from 0.05 to 25.72 mg/kg and B₂ from 0.05 to 5.7 mg/kg, respectively. This figure indicated widespread contamination of fumonisin B₁ and B₂ in maize grown in different areas of Samsun, Turkey.     

Fumonisin detection and analysis of potential fumonisin‐producing Fusarium spp. in asparagus (Asparagus officinalis L.) in Zhejiang Province of China

BACKGROUND: Fumonisins are mycotoxins produced by a number of Fusarium species, including several pathogens of asparagus plants. China is one of the largest asparagus producers in the world. In this study, we analysed the contamination of fumonisins and fumonisin‐producing fungi in asparagus spear samples from Zhejiang Province, the major asparagus production province in China. RESULTS: The asparagus did not contain a detectable level of fumonisins. However, the recovery of Fusarium in asparagus was 72.7%, including F. proliferatum (40.9%), F. oxysporum (22.7%), F. acuminatum (4.55%) and F. equesti (4.55%). A multiplex PCR targeting the internal transcribed spacer sequence (ITS), translation elongation factor 1‐α (TEF), and key biosynthetic genes FUM1 and FUM8, was used to simultaneously determine the identity and the biosynthetic ability of the fungal isolates. Fungal isolates containing the FUM genes also produced fumonisins in cultures, ranging from 28 to 4204 µg g^?1. F. proliferatum was the only fumonisin‐producing Fusarium species in asparagus. CONCLUSION: Although no fumonisin contamination was detected in asparagus in the current survey, we found that the majority of samples contained Fusarium spp. Because F. proliferatum is a high fumonisin‐producing species, potential health risks for human consumption of asparagus exist, if the appropriate environmental conditions are present for this fungus. Copyright © 2010 Society of Chemical Industry     

Mycoflora and fumonisin contamination in Brazilian sorghum from sowing to harvest

BACKGROUND: The aim of this study was to characterise the mycoflora and the presence of fumonisin in sorghum grains, correlating the results with the environment and abiotic factors. RESULTS: Fifty samples (five collections of ten samples each) of sorghum were analysed. All samples were found to be contaminated with fungi, with higher frequencies of Cladosporium spp. (61.8%) and Helminthosporium spp. (33.4%). Fusarium verticillioides was isolated from 15.1% of the samples, with 38% of them being contaminated with fumonisin B₁ (FB₁) at levels ranging from 50 to 368.78 ng g^?1. Regarding abiotic factors, temperature, water activity and rainfall showed a positive correlation with the frequency of F. verticillioides and FB₁ production. There was a significant positive correlation between relative air humidity and FB₁ production. The results obtained from sexual crosses between standard F mating tester strains and the isolated strains confirmed that the strains isolated were F. verticillioides. CONCLUSION: It can be concluded that the decrease in F. verticillioides and fumonisin contamination occurred owing to atypical climatic factors during the period of sorghum cultivation, when there was any occurrence of rain and the level of water activity of grains did not reach 0.58. Copyright © 2010 Society of Chemical Industry     

Fumonisin B2 production by Aspergillus niger in Thai coffee beans

During 2006 and 2007, a total of 64 Thai dried coffee bean samples (Coffea arabica) from two growing sites in Chiangmai Province and 32 Thai dried coffee bean samples (Coffea canephora) from two growing sites in Chumporn Province, Thailand, were collected and assessed for fumonisin contamination by black Aspergilli. No Fusarium species known to produce fumonisin were detected, but black Aspergilli had high incidences on both Arabica and Robusta Thai coffee beans. Liquid chromatography (LC) with high-resolution mass spectrometric (HRMS) detection showed that 67% of Aspergillus niger isolates from coffee beans were capable of producing fumonisins B₂ (FB₂) and B₄ when grown on Czapek Yeast Agar with 5% NaCl. Small amounts (1–9.7 ng g^?1) of FB₂ were detected in seven of 12 selected coffee samples after ion-exchange purification and LC–MS/MS detection. Two samples also contained FB₄. This is the first record of freshly isolated A. niger strains producing fumonisins and the first report on the natural occurrence of FB₂ and FB₄ in coffee.     

Reduction of fumonisin B₁ in corn grits by twin-screw extrusion

Abstract: This study was designed to investigate the fate of fumonisins in flaking corn grits during twin‐screw extrusion by measuring fumonisin B₁ (FB₁) and its analogs with a mass balance approach. Food grade corn grits and 2 batches of grits contaminated with FB₁ at 10 and 50 μg/g by Fusarium verticillioides M‐2552 were processed with or without glucose supplementation (10%, w/w) with a twin‐screw extruder. Extrusion reduced FB₁ in contaminated grits by 64% to 72% without glucose and 89% to 94% with added glucose. In addition, extrusion alone resulted in 26% to 73% reduction in the levels of fumonisin B₂ and fumonisin B₃, while levels of both mycotoxins were reduced by >89% in extruded corn grits containing 10% glucose. Mass balance analysis showed that 38% to 46% of the FB₁ species detected in corn extruded with glucose was N‐(deoxy‐D‐fructos‐1‐yl)‐FB₁, while 23% to 37% of FB₁ species detected in extruded corn grits with and without added glucose was bound to the matrix. It was also found that the hydrolyzed form of FB₁ was a minor species in extruded corn grits with or without added glucose, representing <15% of the total FB₁ species present. Less than 46% of FB₁ originally present in corn grits could be detected in the fumonisin analogues measured in this study. Research is needed to identify the reaction products resulting from extrusion processing of fumonisin‐contaminated corn products. Practical Application: Twin‐screw extrusion is widely used in food industry for its versatility. This technology may reduce the level of fumonisins in corn particularly with added glucose.     

Analysis of fumonisin contamination and the presence of Fusarium in wheat with kernel black point disease in the United States

The ability of the fungus Fusarium proliferatum to cause kernel black point disease in wheat was previously established, but natural contamination of black point wheat with both F. proliferatum and fumonisin mycotoxins has not been studied in the United States. Low levels of fumonisins were detected in nine of 43 wheat samples with kernel black point disease that were obtained from across the United States. A subset of samples was contaminated with F. proliferatum as well as with F. fujikuroi, F. nygamai, F. thapsinum and F. verticillioides, species closely related to F. proliferatum and morphologically similar to it in that they produce chains of asexual spores, or conidia. Nevertheless, of conidial chain-forming fusaria isolated from symptomatic wheat, F. proliferatum dominated. In greenhouse tests, isolates of F. proliferatum and the other species recovered from wheat samples were able to cause symptoms of kernel black point and, in some cases, low levels of fumonisin contamination of wheat. These data add to the understanding of the risk of fumonisin contamination of wheat and the potential for Fusarium species to cause kernel black point disease and fumonisin contamination of wheat. Further, the results of this study indicate that while US-grown wheat can sporadically be contaminated by fumonisins, the natural contamination levels seem to be low. The observations made provide evidence that fumonisins are not likely to be a factor contributing to the ability of Fusarium to cause kernel black point disease.     

Antifungal and antimycotoxigenic effects of Zingiber officinale,Cinnamomum zeylanicum and Cymbopogon martinii essential oils against Fusarium verticillioides

ABSTRACT

There is an increasing demand for fungi control in grains, especially toxigenic. Also, there is growing concern on the use of synthetic fungicides; thus alternatives are needed. The aim of this study was to evaluate the antifungal and antimycotoxigenic action of essential oils (EOs) from Zingiber officinale, Cinnamomum zeylanicum and Cymbopogon martinii against Fusarium verticillioides, a spoilage and toxigenic fungus. Essential oils were first chemically characterised by gas chromatography coupled to mass spectrometry, and their antioxidant potential was measured by the DPPH, ABTS and FRAP methods. Minimum inhibitory concentration (MIC) and disc diffusion were used to assess antifungal activity. Scanning electron microscopy was used to evaluate morphological changes in the fungus. Antimycotoxigenic activity of the EOs against the production of fumonisin B₁ and B₂ by F. verticillioides was evaluated using ultra-high-performance liquid chromatography system. Z. officinale, C. zeylanicum and C. martinii EOs were predominantly composed by zingiberene and geranial; eugenol; and geraniol, respectively. All the EOs had high antioxidant power, especially that from C. zeylanicum. The MICs were 250, 500 and 2,000 µg mL^?1 for C. zeylanicum, C. martinii and Z. officinale EOs, respectively. Mycelial reduction of F. verticillioides was observed when EOs were used, and the lowest activity was detected in the Z. officinale EO. Overall, the tested EOs promoted structural damage to the fungal cell wall, decreased conidia size and mycelial reduction. Antimycotoxigenic evaluation of the EOs evidenced a significant reduction (p < .05) in the production of fumonisins B₁ and B₂ with all the EOs evaluated in the study. These results suggest that especially C. zeylanicum and C. martinii EOs are highly useful for controlling F. verticillioides and fumonisins production.     

Fusariotoxins in asparagus – their biosynthesis and migration

Asparagus is often infected by fungi of the Fusarium genus, a causal agent of crown and root rot, which decreases the quantity and quality of spears. Fusarium oxysporum and Fusarium proliferatum are the most severe asparagus pathogens, well known as mycotoxin producers, mainly fumonisins and moniliformin. The present study was undertaken to estimate fumonisin B₁, moniliformin and ergosterol concentrations in asparagus tissue. Moreover, the possibility of toxin transport to the edible asparagus part during the inoculation by F. oxysporum and F. proliferatum of different plant parts (root, crown and stem base) and the potential risk for consumers were assessed. Our studies showed that the highest capability of producing fumonisin B₁ and moniliformin was demonstrated from isolates of F. proliferatum. The highest level of fumonisin B₁ in edible spears was detected when the asparagus crown was inoculated with F. oxysporum and F. proliferatum. The lowest concentration was found in the case of storage root inoculation, which corresponds with mycelium absence and the long distance from the roots to the stem. Similar results were demonstrated for moniliformin. The mycotoxin content was confirmed even in healthy spears (without disease symptoms and mycelium presence), which might indicate that the transport of mycotoxins is possible from the soil through the root system to the top part of the plants.     

Impact of environment and interactions of Fusarium verticillioides and Fusarium proliferatum with Aspergillus parasiticus on fumonisin B1 and aflatoxins on maize grain

Fusarium verticillioides and F proliferatum isolates were inoculated in mixed cultures with Aspergillus parasiticus on irradiated maize grain at two different inoculum concentrations (2 × 10⁵ and 2 × 10² conidia g^?1 dry maize). The treatments were 0.93–0.98 water activity (a_w) and 15 and 25 °C for 28 days. A complex relationship was found between a_w, temperature, inoculum concentration and the interactions which took place between fumonisin and aflatoxin producers. In general, A parasiticus reduced F verticillioides and F proliferatum populations (by 6–36%) but did not affect fumonisin B₁ production by these species. In contrast, while the Fusarium species were not able to decrease A parasiticus populations, they significantly reduced aflatoxin B₁ accumulation (by 30–93%). © 2001 Society of Chemical Industry     

Contamination of freshly harvested Bambara groundnut (Vigna subterranea) seed from Mpumalanga,South Africa,with mycotoxigenic fungi

Freshly harvested Bambara groundnut (BGN) is occasionally consumed raw and can potentially become infected with mycotoxingenic field fungi. In this study, BGN samples were obtained from 12 farms in three districts of Mpumalanga in South Africa. Eight pooled samples were screened for multi-mycotoxin contamination using Ultra Performance Liquid-Chromatography tandem mass spectrometry (UPLC-MS/MS). To identify mycoflora, 12 samples were screened using conventional and molecular methods. Selected potential mycotoxin producing isolates were screened for mycotoxins using UPLC-MS/MS. No mycotoxins were detected on the freshly harvested BGN samples, but they were infected with various mycotoxin producing fungal species namely Aspergillus flavus (50%), Penicillium citrinum (25%), Penicillium oxalicum (17%), Penicillium citreoviridin (0.8%), and Fusarium verticillioides (0.8%). Following screening of selected fungal cultures, aflatoxin B₁ (0.4, 0.45 and 0.4 ppm) and fumonisin B₁ (0.7 ppm) were detected from A. flavus and F. verticillioides, respectively. Identification of mycotoxigenic fungi on freshly harvested BGN presents a potential health risk.     

Relative populations and toxin production by Aspergillus flavus and Fusarium verticillioides in artificially inoculated corn at various stages of development under field conditions

The presence, development and production of mycotoxins by Aspergillus flavus and Fusarium verticillioides were studied in corn ears under field conditions after artificial contamination of corn silks. The planted area was divided into five treatments: T1, inoculated with A. flavus solution containing 1 × 10⁸ spores, ears covered; T2, inoculated with F. verticillioides solution containing 1 × 10⁸ spores, ears covered; T3, inoculated with F. verticillioides plus A. flavus solution containing 1 × 10⁸ spores of each, ears covered; T4, sprayed with sterile phosphate‐buffered saline, ears covered; T5, non‐sprayed silks, uncovered ears. Soil and air samples were also collected and analysed for the occurrence of fungi. Water activity, relative air humidity, rainfall and temperature were determined to assess the correlation between abiotic factors and the presence of fungi in the samples. Contamination with the inoculated fungus predominated in T1 and T2. In the other treatments, F. verticillioides was the most frequently isolated contaminant irrespective of treatment. Considering the production of mycotoxins, a positive relation between the production of fumonisins B₁ and B₂ and the frequency of F. verticillioides was statistically verified in all treatments. Copyright © 2007 Society of Chemical Industry     

On fumonisin incidence in monoculture maize under no‐till,conventional tillage and two nitrogen fertilisation levels

BACKGROUND: Fusarium ear rot and fumonisin contamination are serious problems for maize growers. The lack of maize genotypes highly resistant to fumonisin contamination emphasises the need for management strategies to prevent contamination by this mycotoxin. There are conflicting reports regarding no‐till and nitrogen (N) fertilisation practices in relation to the incidence of fumonisins. In this study the effect of no‐till compared with conventional tillage and of N fertilisation rates on fumonisin occurrence was investigated over three years in Northern Italy. RESULTS: The average contamination of grain by fumonisins B₁ and B₂ over the three years was significantly different, with a lower value in 2000 (516 µg kg^?1) than in the other years (5846 and 3269 µg kg^?1 in 2001 and 2002 respectively). Conventional tillage and no‐till treatments had no significant effect on the incidence of fumonisins. This finding suggests that above‐ground residues infected by Fusarium would not lead to an increase in fumonisin incidence. However, N fertilisation significantly increased fumonisin levels, by 99 and 70% in 2000 and 2001 respectively. CONCLUSION: Maize monoculture does not show a cumulative effect on the occurrence of fumonisins, while high rates of N fertiliser consistently result in elevated fumonisin levels. Both these effects can be influenced by annual meteorological fluctuations. Copyright © 2008 Society of Chemical Industry