Investigations on Fusarium spp. and their mycotoxins causing Fusarium ear rot of maize in Kosovo

After wheat, maize (Zea mays L.) is the second most important cereal crop in Kosovo and a major component of animal feed. The purpose of this study was to analyse the incidence and identity of the Fusarium species isolated from naturally infected maize kernels in Kosovo in 2009 and 2010, as well as the mycotoxin contamination. The disease incidence of Fusarium ear rot (from 0.7% to 40% diseased ears) on maize in Kosovo is high. The most frequently Fusarium spp. identified on maize kernels were Fusarium subglutinans, F. verticillioides/F. proliferatum and F. graminearum. Maize kernel samples were analysed by LC-MS/MS and found to be contaminated with deoxynivalenol (DON), DON-3-glucoside, 3-acetyl-DON, 15-acetyl-DON, zearalenone, zearalenone-14-sulphate, moniliformin, fumonisin B₁ and fumonisin B₂. This is the first report on the incidence and identification of Fusarium species isolated from naturally infected maize as well as the mycotoxin contamination in Kosovo.     

Trichothecenes, ochratoxin A and zearalenone contamination and Fusarium infection in Finnish cereal samples in 1998

The occurrences and concentrations of trichothecenes, ochratoxin A and zearalenone in Finnish cereal samples are presented in this study. Furthermore, infections by moulds, especially Fusarium contamination of grains in the same samples, are reported. In total 68 cereal samples, including 43 rye, 4 wheat, 15 barley and 6 oats samples, were collected after a cool and very rainy growing season in 1998. A gas chromatograph combined with a mass spectrometric detector was used for determination of seven different trichothecenes. A high performance liquid chromatograph with a fluorescence detector was used for ochratoxin A and zearalenone determination. For the identification of moulds, the grain samples were incubated and the moulds were isolated and identified by microscopy. The analytical methods were validated for mycotoxin analysis and they were found to be adequately reliable and sensitive. Heavy rainfalls in the summer and autumn of 1998 caused abundant Fusarium mould infection in Finnish cereals, particularly in rye. Fusarium avenaceum was the most common Fusarium species found in cereals. However, the mycotoxin concentrations were very low and only deoxynivalenol, nivalenol and HT-2 toxin were detected. Deoxynivalenol was detected in 54 samples in the concentration range 5-111 µg/kg. Nivalenol and HT-2 toxin were detected in three and two samples, respectively, in the concentration range 10-20 µg/kg.     

Contamination of barley seeds with Fusarium species and their toxins in Spain: an integrated approach

Fusarium is a globally distributed fungal genus that includes different species pathogenic to cereals among others crops. Some of these Fusarium species can also produce toxic compounds towards animals and humans. In this work, the presence of the most important Fusarium toxins was determined in barley seeds from Spain, sampled according to European Union requirements. The results obtained were compared with the presence of mycotoxigenic species considered responsible for their synthesis by using species-specific polymerase chain reaction protocols. Fumonisins B₁ and B₂, zearalenone, trichothecenes type A (T-2 and HT-2) and trichothecenes type B (deoxynivalenol and nivalenol) were analysed by using high-performance liquid chromatography. Deoxynivalenol and zearalenone were detected in 72% and 38% of the barley samples, respectively, at levels below European Union limits in all cases. However, the co-occurrence of both toxins in 34% of the samples suggested that synergistic activity of these two mycotoxins should be evaluated. Nivalenol and HT-2/T-2 were detected at low levels in 17% and 10% of the samples, respectively. Fumonisins occurred in 34% of the samples at levels up to 300?µg/kg. This suggested that they might represent a risk in Spanish barley, and to our knowledge, this is the first report on the presence of fumonisins in barley in this country. The species-specific polymerase chain reaction assays to detect mycotoxin-producing Fusarium species showed a very consistent correlation between F. verticillioides detection and fumonisin contamination as well as F. graminearum presence and zearalenone, deoxynivalenol and nivalenol contamination in barley samples. The approach used in this study provided information of mycotoxin contamination of barley together with the identification of the fungal species responsible for their production. Detection of the species with the current polymerase chain reaction assay strategy may be considered predictive of the potential mycotoxin risk in this matrix.     

Contents of Fusarium toxins in Finnish and imported grains and feeds

The aim of the present study was to investigate the levels of trichothecenes in representative samples of Finnish and imported cereals and feeds from the 1987 and 1988 crops. Trichothecene concentrations were determined in samples as trimethylsilylether derivatives by a gas chromatograph equipped with a mass selective detector (GC-MSD) using selected ion monitoring (SIM) which proved to be a reliable and sufficiently sensitive technique. Representative samples of Finnish and imported oats, wheat, barley, rye, maize gluten, soy granules, rapeseed, turnip rapeseed, fish meal, poultry feed and pig feed were analysed employing 19-nortestosterone as an internal standard. Almost all grain and feed samples contained deoxynivalenol (DON) from 7 to 300 micrograms/kg and smaller amounts (13-120 micrograms/kg) of 3-acetyldeoxynivalenol (3-AcDON). The most toxic trichothecenes, T-2, HT-2 toxins and nivalenol (NIV) and also zeralenone (ZEN) were found at low concentrations in some samples. Six lots of oats containing 1.3-2.6 mg/kg of DON and 0.2-0.6 mg/kg of 3-AcDON were found in the Finnish grain samples. Silo samples of almost all imported grains contained DON but at levels below 50 micrograms/kg. Imported grains contained no other trichothecenes. DON, 3-AcDON and ZEN were found in imported feedstuffs at low concentrations.     

Fusarium species and fumonisins in subsistence maize in the former Transkei region,South Africa: a multi-year study in rural villages

Fumonisin occurrence was investigated in subsistence maize in four rural villages in each of Mbizana and Centane areas, South Africa. Samples (total 211) were analysed morphologically for Fusarium species and by high performance liquid chromatography for fumonisins. The mean incidence levels of Fusarium verticillioides in Centane good maize were 16% for both 1997 and 2000, but increased to 32% in 2003, whereas Mbizana good maize contained levels of 17% and 11% (2000 and 2003 seasons, respectively). The mean total fumonisin level in good maize in Centane for 1997 and 2000 was 575 and 975 µg/kg and 2150 µg/kg in 2003. In Mbizana, the mean total fumonisin level in good maize for 2000 was 950 µg/kg, but decreased to 610 µg/kg in 2003. The 2003 drought conditions led to a substantial increase in fumonisin levels in dry subhumid Centane, compared to humid subtropical Mbizana. This study emphasises the seasonal fluctuation in fumonisin levels.     

Detection and discrimination of cereal and leguminous species in chestnut flour by duplex PCR

Chestnut and chestnut-derived products are quite expensive and thus a possible target for fraudulent labeling. To obtain a French Label of Origin, chestnut producers need to certify the purity of their products. Chestnut-derived products are consumed by people who may suffer from celiac disease or are allergic to cereals. For these reasons, we developed a qualitative PCR-based method to detect cereal and leguminous species in chestnut flour. The presence of common wheat and barley was determined by amplifying part of the puroindoline-a gene, the presence of rye by amplifying part of the secaloindoline-a gene, the presence of durum wheat, rice, maize and chickpea by amplifying part of lipid transfer protein genes, the presence of oat by amplifying part of a thionin gene, the presence of kidney bean by amplifying part of a late embryogenesis-abundant protein, the presence of soybean by amplifying part of a lectin gene and the presence of fava bean by amplifying part of a nodulin gene. A chestnut thaumatine gene was used as control. PCR conditions were optimized to detect 1% of adulteration. Duplex PCR and specially designed sets of primers that allow amplification of several related species limited the number and cost of analyses required. Interpretation of the results is facilitated by using a decision tree.     

Natural occurrence of Fusarium toxins in peanut,sorghum and maize from Mysore (India)

Peanut, sorghum and maize samples were collected from the wholesale market in Mysore, India, over a period of one year (October 1984 to September 1985). The samples were analysed for the natural occurrence of T-2 toxin (T-2), diacetoxyscirpenol (DAS) and zearalenone by thin-layer chromatography, dermal toxicity test and gas chromatography. Of the total number of peanut samples analysed, 6.9% were positive for the toxic trichothecene(s) (T-2, up to 38.89 mg kg^?1; DAS, up to 2.03 mg kg^?1); 4.8% of total sorghum samples analysed contained T-2 up to 15 mg kg^?1. Zearalenone was not found in any of the samples tested, and no toxins were detected in any of the maize samples. Samples marketed during winter and summer periods were contaminated with the toxins. All the toxin-positive samples except one peanut sample were found in produce stored for more than a week.     

The effects of cereal substrate and temperature on production of beauvericin, moniliformin and fusaproliferin by Fusarium subglutinans ITEM-1434

One strain of Fusarium subglutinans (ITEM-1434) isolated from maize ear rot in Poland was tested for the ability to synthesize moniliformin (MON), beauvericin (BEA) and fusaproliferin (FP) on six cereal substrates (wheat, rye, barley, oat, maize and rice kernels) for 3 weeks at 25oC and on rice at three different temperatures (20, 25 and 30oC). Most MON (497 mug/g) was produced on rice; most BEA (704 mug/g) on wheat or rice, and most FP (422 mug/g) on rye. When cultured on rice, F. subglutinans produced the highest levels of BEA and FP at 20-25oC, while MON production was best at 30oC.     

Distribution of deoxynivalenol and 3‐acetyldeoxynivalenol in naturally contaminated and Fusarium culmorum infected triticale samples

Eight triticale genotypes, naturally contaminated (C) and collected from heads inoculated with two separate isolates Fusarium culmorum (I₁ and I₂) kernels were analysed for accumulation ofFusarium toxins. Triticale kernels were contaminated with deoxynivalenol (DON) and 3‐acetyldeoxynivalenol (3‐AcDON). Statistical analysis revealed significant differences between naturally contaminated samples (C) and those inoculated with isolate I₁ and I₂. The following accumulation of toxins in kernels (mean mg/kg) was found: C, I₁, I₂ – DON 0.06, 14.45, 8.09; 3‐AcDON – 0.01, 0.20, 0.49. Distribution of DON and 3‐AcDON was studied in four size fractions of kernels (> 2.8 mm; ≤ 2.8–2.5 mm, < 2.5–2.2 mm and < 2.2 mm).The highest concentration of DON and 3‐AcDON was found in the smallest fraction of damaged kernels. The percentage distribution of DON and 3‐AcDON in fraction < 2.2 mm was (C) 66%, 77%, (I₁) 38%, 27%, (I₂) 46%, 40%; and in fraction < 2.5 mm: (C) 85%, 94%, (I₁) 57%, 51%, (I₂) 80%, 76%. The naturally contaminated kernel fraction < 2.2 mm (although only 2% of all) contained 33% DON and 59% 3‐AcDON, for more toxigenic isolate I₁ 10%, 15%, 14%, less toxigenic isolate I₂ 4%, 13%, 11%, respectively, calculated on dry matter basis. No significant correlation between examined characteristics was found for either I₁ or I₂ isolate. The biosynthesis mechanism of toxic metabolites was to approximate (significantly correlated) to pair objects C/I₂.     

粮食中新兴真菌毒素的污染现状及毒理学研究进展

产毒真菌污染粮食等农产品产生有毒有害的次级代谢产物,称真菌毒素,严重威胁食用和饲用产品的质量安全。较为常见的真菌毒素包括黄曲霉毒素、镰刀菌毒素脱氧雪腐镰刀烯醇、玉米赤霉烯酮等,国内外针对这些主要污染毒素制定了相应的限量标准。近年来,随着真菌毒素研究的发展和检测技术的更新,一些新型真菌毒素不断被发现。将目前尚未被列入常规风险筛查和监管、也尚无相关限量标准的真菌毒素种类,称谓新兴毒素（Emerging toxins）。目前检出频次高、污染较为严重的新兴毒素主要由镰刀菌属（Fusarium spp.）和链格孢属（Alternaria spp.）等产毒真菌产生。这些新兴真菌毒素通常与主要毒素复合污染,本文将依据现有的风险调查报告与数据,综述粮食中常见新兴镰刀菌和链格孢毒素的污染现状和毒理学研究进展,为真菌毒素的全面风险评估和管控技术研发提供依据。     

Impact of cycling temperatures on Fusarium verticillioides and Fusarium graminearum growth and mycotoxins production in soybean

BACKGROUND: Fusarium graminearum and F. verticillioides are two very important mycotoxigenic species as they cause diverse diseases in crops. The effects of constant and cycling temperatures on growth and mycotoxin production of these species were studied on soybean based medium and on irradiated soya beans. RESULTS: F. graminearum grew better when was incubated at 15, 20 and 15–20 °C (isothermal or cycling temperature) during 21 days of incubation. Maximum levels of zearalenone and deoxynivalenol (39.25 and 1040.4 µg g^?1, respectively) were detected on soya beans after 15 days of incubation and the optimal temperature for mycotoxin production was 15 °C for zearalenone and 20 °C for deoxynivalenol. F. verticillioides grew better at 25 °C in culture medium and at 15/20 °C and 15/25 °C on soybean seeds. Fumonisin B₁ was produced only in culture medium, and the maximum level (7.38 µg g^?1) was found at 15 °C after 7 days of incubation. CONCLUSION: When growth and mycotoxin production under cycling temperatures were predicted from the results under constant conditions, observed values were different from calculated for both species and substrate medium. Therefore, care should be taken if data at constant temperature conditions are to be extrapolated to real field conditions. Copyright © 2012 Society of Chemical Industry     

Molecular survey of trichothecene genotypes of Fusarium graminearum species complex from barley in southern Brazil

Fusarium head blight is a disease of primary concern to small-grain cereals of Brazil, including barley. Its main causal agent, Fusarium graminearum species complex (Fg complex)¸ is able to produce mycotoxins, especially deoxynivalenol (DON) and nivalenol (NIV), that usually contaminate grain. Strains that produce DON may also produce its acetylated derivatives: 3-acetyl-DON (3-ADON) and 15-acetyl-DON (15-ADON). Ninety two isolates were obtained from samplings of barley grain during three years (2007, 2008 and 2009) from several fields in both southern and northern production regions of Rio Grande do Sul state, Brazil. These isolates were examined for polymerase chain-reaction-based (PCR) trichothecene genotype based on the amplification of portions of Tri3 and Tri12. There was no effect of year or region on the proportion of trichothecene genotypes. Overall, 66% of the strains (61/92) were 15-ADON, 4.4% (4/92) were 3-ADON and 29.3% (27/92) were NIV. The overall NIV/DON ratio estimated (0.41) was five times higher than that found in previous studies with strains from wheat grown in the same region. Species identification of nine strains representing the trichothecene genotypes, based on comparisons of DNA sequences of portions of the PHO, RED and URA genes with sequences from curated reference isolates of Fusarium from GenBank, revealed that they belong to F. graminearum sensu stricto (four 15-ADON and one 3-ADON strain), F. meridionale (three NIV strains) and F. austroamericanum (one 3-ADON strain). These results add to the current regional knowledge of trichothecene genotypes and species within the Fg complex affecting barley in the region.     

Molecular strategies for detection and quantification of mycotoxin‐producing Fusarium species: a review

Fusarium contamination is considered a major agricultural problem, which could not only significantly reduce yield and quality of agricultural products, but produce mycotoxins that are virulence factors responsible for many diseases of humans and farm animals. One strategy to identify toxigenic Fusarium species is the use of modern molecular methods, which include the analysis of DNA target regions for differentiation of the Fusarium species, particularly the mycotoxin‐producing Fusarium species such as F. verticillioides and F. graminearum. Additionally, polymerase chain reaction assays are used to determine the genes involved in the biosynthesis of the toxins in order to facilitate a qualitative and quantitative detection of Fusarium‐producing mycotoxins. Also, it is worth mentioning that some factors that modulate the biosynthesis of mycotoxins are not only determined by their biosynthetic gene clusters, but also by environmental conditions. Therefore, all of the aforementioned factors which may affect the molecular diagnosis of mycotoxins will be reviewed and discussed in this paper. © 2014 Society of Chemical Industry     

Fusarium species identification and fumonisin production in maize kernels from Shandong Province,China, from 2012 to 2014

A total of 225 maize kernel samples were collected from Shandong Province in China from 2012 to 2014 and analysed for contamination with Fusarium spp. and fumonisins (FBs) using molecular methods and high-performance liquid chromatography with fluorescence detection. The results showed that the average incidences of Fusarium spp. in 2012, 2013 and 2014 were 23.3%, 37.1% and 36.5%, respectively, Fusarium verticillioides being the predominant species. In 2012, the average contamination level of FBs was 3071 ng g^?1, which was higher than that in 2014 (2913 ng g^?1) and 2013 (2072 ng g^?1). Of all samples, 13% and 19% had FB contamination levels higher than 2000 and 4000 ng g^?1, which are the maximum limits as set by the Food and Drug Administration of the United States and the European Commission, respectively. Therefore, efforts should be taken to minimise the potential risk of FBs to the health of humans and animals.     

Effect of fungal strain and cereal substrate on in vitro mycotoxin production by Fusarium poae and Fusarium avenaceum

Single conidia strains of Fusarium poae and Fusarium avenaceum were investigated for their ability to synthesise mycotoxins in vitro. In a first experiment, rice was inoculated with three strains of F. poae and three strains of F. avenaceum. In a second experiment, the same strains were cultivated on four different cereal substrates. For both experiments, the colonised substrates were analysed using liquid chromatography coupled with tandem mass spectrometric detection (LC-MS/MS) for the content of Fusarium mycotoxins. On rice, a strong effect of strains on mycotoxin content was found. With the analyses of different cereal types, substantial substrate effects were observed. For F. poae, these effects were highly dependent on the strain. The results of this study are of benefit for risk assessment concerning naturally infected grain samples since substrate effects could also be triggered by different cereal species or varieties, and thus could lead to variable mycotoxin contamination in cereal products intended for human consumption.     

Relationship between Fusarium spp. diversity and mycotoxin contents of mature grains in southern Belgium

Over a 4-year period (2010–13), a survey aiming at determining the occurrence of Fusarium spp. and their relations to mycotoxins in mature grains took place in southern Belgium. The most prevalent species were F. graminearum, F. avenaceum, F. poae and F. culmorum, with large variations between years and locations. An even proportion of mating type found for F. avenaceum, F. culmorum, F. cerealis and F. tricinctum is usually a sign of ongoing sexual recombination. In contrast, an unbalanced proportion of mating type was found for F. poae and no MAT1-2 allele was present in the F. langsethiae population. Genetic chemotyping indicates a majority of deoxynivalenol (DON)-producing strains in F. culmorum (78%, all 3-ADON producers) and F. graminearum (95%, mostly 15-ADON producers), while all F. cerealis strains belong to the nivalenol (NIV) chemotype. Between 2011 and 2013, DON, NIV, enniatins (ENNs) and moniliformin (MON) were found in each field in various concentrations. By comparison, beauvericin (BEA) was scarcely detected and T-2 toxin, zearalenone and α- and β-zearalenols were never detected. Principal component analysis revealed correlations of DON with F. graminearum, ENNs and MON with F. avenaceum and NIV with F. culmorum, F. cerealis and F. poae. BEA was associated with the presence of F. tricinctum and, to a lesser extent, with the presence of F. poae. The use of genetic chemotype data revealed that DON concentrations were mostly influenced by DON-producing strains of F. graminearum and F. culmorum, whereas the concentrations of NIV were influenced by the number of NIV-producing strains of both species added to the number of F. cerealis and F. poae strains. This study emphasises the need to pay attention to less-studied Fusarium spp. for future Fusarium head blight management strategies, as they commonly co-occur in the field and are associated with a broad spectrum of mycotoxins.     

The effect of light and temperature on glucosinolate concentration in the leaves and roots of cabbage seedlings

In previous studies it was shown that the concentration of total and individual glucosinolates in brassicaceous plants can vary significantly over a 24-h period grown either in the field or under controlled conditions. The present study shows total and individual glucosinolate variation during a single day. Seedlings of cabbage grown under controlled conditions and at 14 and 15 days after emergence were moved to 20°C (Exp A) and 30°C (Exp B), with a constant photosynthetic photon flux density of 480 μmol m⁻² s⁻¹ and 75% relative humidity, over a 2-day period, during which time aerial parts and roots were sampled at regular intervals. Whilst the glucosinolate patterns of the aerial part of the plant and of the roots remained the same, the levels of major glucosinolates in the aerial part, averaged over all sampling times and 2 days, were 233 ± 60 μmol 100 g⁻¹ DW for 3-methylsulphinylpropyl and 72 ± 22 for 2-propenyl; in the roots, 2-phenylethyl and 1-methoxyindol-3-ylmethyl showed the highest average concentrations, with 678 ± 355 μmol 100 g⁻¹ DW and 411 ± 122, respectively. Total and individual glucosinolate levels showed very high significant differences between the two plant parts. Despite the constant temperature, light and relative humidity, glucosinolates varied within a 24-h period, showing ultradian rhythms that are common to several metabolic processes in plants. The results confirm previous observations that at a temperature of 20°C, close to the optimum for growth and development, the diurnal variation in glucosinolate concentration, was smaller than at 30°C. © 1998 Society of Chemical Industry     

Molecular characterization and fumonisin production by Fusarium verticillioides isolated from corn grains of different geographic origins in Brazil

Gibberella moniliformis is most commonly associated with maize worldwide and produces high levels of fumonisins, some of the most agriculturally important mycotoxins. Studies demonstrate that molecular methods can be helpful for a rapid identification of Fusarium species and their levels of toxin production. The purpose of this research was to apply molecular methods (AFLP, TEF-1α partial gene sequencing and PCR based on MAT alleles) for the identification of Fusarium species isolated from Brazilian corn and to verify if real time RT-PCR technique based on FUM1 and FUM19 genes is appropriated to estimate fumonisins B₁ and B₂ production levels. Among the isolated strains, 96 were identified as Fusarium verticillioides, and four as other Fusarium species. Concordant phylogenies were obtained by AFLP and TEF-1α sequencing, permitting the classification of the different species into distinct clades. Concerning MAT alleles, 70% of the F. verticillioides isolates carried the MAT-1 and 30% MAT-2. A significant correlation was observed between the expression of the genes and toxin production r = 0.95 and r = 0.79 (correlation of FUM1 with FB₁ and FB₂, respectively, P < 0.0001); r = 0.93 and r = 0.78 (correlation of FUM19 with FB₁ and FB₂, respectively, P < 0.0001). Molecular methods used in this study were found to be useful for the rapid identification of Fusarium species. The high and significant correlation between FUM1 and FUM19 expression and fumonisins production suggests that real time RT-PCR is suitable for studies considering the influence of abiotic and biotic factors on expression of these genes. This is the first report concerning the expression of fumonisin biosynthetic genes in Fusarium strains isolated from Brazilian agricultural commodity.