Trichothecene genotypes and chemotypes in Fusarium graminearum complex strains isolated from maize fields of northwest Argentina

Members of the Fusarium graminearum species complex (Fg complex) cause Gibberella ear rot in maize from northwest Argentina. The potential of these pathogens to contaminate maize grains with type B trichothecenes is a health risk for both humans and animals. We evaluated the reliability of multiplex PCR assays based on TRI3 and TRI12 genes, and single PCR assays based on TRI7 and TRI13 genes to infer trichothecene chemotypes of 112 strains of Fg complex collected from northwest Argentina, checking trichothecene production by chemical analysis. Single and multiplex PCR assays indicated that strains belonging to F. meridionale (87/112) had a NIV genotype. The remainder strains (25/112), which belonged to F. boothii, had a DON genotype (based on single PCR assays) or 15ADON genotype (based on multiplex PCR assays). No strains tested were incorrectly diagnosed with a DON/NIV genotype. Chemical analysis indicated that the F. meridionale strains were NIV producers only (44/87), major NIV producers with unexpected high DON/NIV ratios (36/87), or unexpected major DON producers with minor NIV production (7/87). Strains with atypical DON/NIV production seem to be new phenotypes under a putative NIV genotype, since PCR assays do not provide evidences of a new trichothecene genotype. DON production and absence of its acetylated forms were shown for strains of F. boothii. The inconsistencies between genetic and chemical data highlight the risk of inferring the trichothecenes potentially contaminating food and feedstuffs based only on PCR assays. This study confirms for the first time that strains of Fg complex from maize of northwest Argentina are DON and NIV producers. In addition, dominance of NIV producers in the Fg complex population isolated from maize is unprecedented in Argentina, and of significant concern to food safety and animal production.     

Trichothecene and beauvericin mycotoxin production and genetic variability in Fusarium poae isolated from wheat kernels from northern Italy

The importance and widespread incidence of Fusarium poae as a natural contaminant of wheat in different climatic areas warrants investigation into the genetic diversity and toxin profile of a northern Italy population. Eighty-one strains of F. poae isolated from durum wheat kernels, identified by species-specific polymerase chain reaction and translation elongation factor-1α gene sequence analysis, were genetically characterized by the amplified fragment length polymorphism (AFLP) technique and analysed by high-pressure liquid chromatography for their ability to produce the beauvericin (BEA) and trichothecene mycotoxins. A high level of variability was observed by using AFLP analyses, with the lowest level of genetic similarity among the strains being approximately 61%. Most of the strains, 95%, produced BEA at <2655 µg g^?1; 88% produced the trichothecene nivalenol at <865 µg g^?1 and 76% produced the trichothecene fusarenon-X at <167 µg g^?1. These data show that F. poae can produce high amounts of BEA together with trichothecenes, and can represent a high potential mycotoxin risk in Italy for wheat colonized by this species.     

Toxigenic potential of Fusarium culmorum strains isolated from French wheat

Sixty F. culmorum strains were isolated from wheat grains collected from different wheat-growing areas in France and from different cultivars. The isolates were grown on autoclaved wheat grain to assess their ability to produce trichothecenes and zearalenone. Fungal biomass was evaluated through the ergosterol grain content. All the isolates produced zearalenone (0.39-1660mg kg -1 ). Thirty-five of the 60 F. culmorum produced nivalenol (0.11-11.7mg kg -1 ), 12 of 60 produced fusarenone X (0.05-8.42mg kg -1 ), five of 60 produced 15-acetyldeoxynivalenol (0.48- 27.7mg kg -1 ), 13 of 60 produced 3-acetyldeoxyni-valenol (0.07-21.0mg kg -1 ) and 24 of 60 produced deoxynivalenol (0.92-51.9mg kg -1 ). According to the results, the distribution of the different chemotypes as well as the high and the low mycotoxin-producing Fusarium strains could not be associated to geographical origin.     

Trichothecene and moniliformin production by Fusarium species from western Canadian wheat

Fusarium graminearum, Fusarium culmorum, and Fusarium avenaceum, isolated from Fusarium-damaged wheat harvested in western Canada, were cultured and evaluated for mycotoxin production. Extracts of the culture media were assayed for trichothecenes by gas chromatography-mass spectrometry and for moniliformin by liquid chromatography. Deoxynivalenol (DON) was found in 28 of 42 isolates of F. graminearum and 42 of 42 isolates of F. culmorum at levels ranging from 0.5 to 25.0 microg/g. 15-AcetylDON was found in 28 of 42 isolates of F. graminearum at levels ranging from 1.0 to 7.1 microg/g. 3-AcetylDON was found in 41 of 42 isolates of F. culmorum at levels ranging from 0.8 to 13.0 microg/g. Several other trichothecenes were assayed but not detected in the culture medium. Moniliformin was present in 40 of 42 isolates of F. avenaceum at levels ranging from 1.3 to 138.1 microg/g, but was not present in any of the isolates of F. graminearum or F. culmorum.     

Ecophysiology of Fusarium temperatum isolated from maize in Argentina

The effect of water activity (a_w = 0.95, 0.98 and 0.995), temperature (15, 25 and 30°C), incubation time (7, 14, 21 and 28 days), and their interactions on growth and moniliformin (MON), beauvericin (BEA), fusaproliferin (FUS) and fumonisin B₁ (FB₁) production by two strains of Fusarium temperatum isolated from Argentinean maize were determined in vitro on sterile layers of maize grains. The results showed that there was a wide range of conditions for growth and mycotoxins production by F. temperatum. Both strains were found to grow faster with increasing a_w and at 30°C. In relation to mycotoxin production, the two strains produced more FUS than the other mycotoxins regardless of a_w or temperature evaluated (maximum = 50 000 μg g^?1). For FUS, MON and BEA, the maximum levels were observed at 0.98 a_w and 30°C (50 000, 5000 and 2000 μg g^?1 respectively). The lowest levels for these three mycotoxins were detected at 15°C and 0.95 a_w (1700 and 100 μg g^?1 for FUS and MON respectively), and at 0.98 a_w (400 μg g^?1 for BEA). The maximum levels of FB₁ were produced at 15°C and 0.98 a_w (1000 μg g^?1). At all a_w and temperatures combinations evaluated there was an increase in toxin concentrations with time incubation. The maximum levels were detected at 21 days. Statistical analyses of a_w, temperature, incubation time, and the two- and three-way interactions between them showed significant effects on mycotoxins production by F. temperatum. For its versatility on growth and mycotoxin production, F. temperatum represents a toxicological risk for maize in the field and also during grain storage.     

Species diversity and toxigenic potential of Fusarium graminearum complex isolates from maize fields in northwest Argentina

Members of the Fusarium graminearum species complex (Fg complex) are the causal agents of ear rot in maize and Fusarium head blight of wheat and other small grain cereals. The potential of these pathogens to contaminate cereals with trichothecene mycotoxins is a health risk for both humans and animals. A survey of ear rot isolates from maize collected in northwest Argentina recovered 66 isolates belonging to the Fg complex. A multilocus genotyping (MLGT) assay for determination of Fg complex species and trichothecene chemotypes was used to identify 56 of these isolates as F. meridionale and 10 isolates as F. boothii. F. meridionale was fixed for the nivalenol (NIV) chemotype, and all of the F. boothii isolates had the 15-acetyldeoxynivalenol (15ADON) chemotype. The results of genetic diversity analysis based on nine variable number tandem repeat (VNTR) loci supported the hypothesis of genetic isolation between F. meridionale and F. boothii, and provided little evidence of geographic substructure among populations of the dominant pathogen species, F. meridionale. This is the first study to indicate that F. meridionale and F. boothii may play a substantial role in the infection and trichothecene contamination of maize in Argentina. In addition, dominance of the NIV chemotype among Fg complex isolates from Argentina is unprecedented, and of significant concern to food safety and animal production.     

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.     

Population genetic structure of Gibberella zeae isolated from wheat in Argentina

Gibberella zeae (anamorph Fusarium graminearum) causes Fusarium head blight of wheat. The authors used amplified fragment length polymorphisms (AFLPs) to characterize the genetic structure of two G. zeae populations from commercial wheat fields. The working hypothesis was that sufficient genetic exchange occurs between local populations to prevent significant partitioning of allelic variation. We analysed 216 AFLP loci for 113 isolates collected during the 2002 harvest season. All strains had AFLP profiles typical of G. zeae lineage 7. Both populations were genotypically diverse but genetically similar and potentially part of a larger, randomly mating population, with significant genetic exchange probably occurring between the two subpopulations. Linkage disequilibrium was low, but higher than reported for many other populations of G. zeae, and about 20% of the alleles detected were specific to one of the two subpopulations—results consistent with limited gene exchange between the two subpopulations. This study extends previous work with populations of G. zeae to include those found in Argentina, one of the world's largest wheat growing countries.     

Production of trichothecenes and zearalenone by Fusarium species isolated from wheat

Fusaria isolates from wheat were tested for ability to produce trichothecenes and zearalenone. Four isolates of F. culmorum out of 13 produced vomitoxin (DON) and 3 Ac-DON, one produced diacetoxysirpenol and 12 zearalenone. Particularly high yield of zearalenone was observed in cultures of sever pathogenic isolates. Higher temperature (20 °C) during first week of incubation favoured yield of zearalenone. About 50% of zearalenone was produced by surface mycelium.     

Influence of temperature on zearalenone production by regional strains of Fusarium graminearum and Fusarium oxysporum in culture.

Zearalenone production by Fusarium graminearum and Fusarium oxysporum was studied under two temperature conditions. Incubation at 25 degrees C for 4 weeks enhanced zearalenone synthesis, improving detection of zearalenone-producing strains of Fusarium oxysporum. Zearalenone production was either totally or partially inhibited when temperature was lowered to 12-14 degrees C during the last 2 weeks of incubation.     

Temperature and water activity effects on growth and temporal deoxynivalenol production by two Argentinean strains of Fusarium graminearum on irradiated wheat grain

The ability of six strains of Pichia anomala, four strains of Pichia kluyveri and two strains of Hanseniaspora uvarum predominant during coffee processing to produce polygalacturonase (PG), pectin esterase (PE) and pectin lyase (PL) in yeast polygalacturonic acid medium (YPA) and in coffee broth (CB) was studied. For comparison, a reference strain of Kluyveromyces marxianus CCT 3172 isolated from cocoa and reported to produce high amount of PG was included.

Initial screening of PG activity using YPA medium showed that K. marxianus CCT 3172, P. anomala S16 and P. kluyveri S13Y4 had the strongest activity. Enzymatic assays showed that the four yeast species secreted PG, but none of the yeasts investigated was found to produce PE or PL. P. anomala S16 and P. kluyveri S13Y4 were found to produce higher amounts of PG when grown in CB than in YPA. When K. marxianus CCT 3172, P. anomala S16 and P. kluyveri S13Y4 were grown in YPA broth adjusted to pH of 3.0–8.0 and incubated at temperatures of 15–40 °C, the three yeast species secreted the highest amount of PG at pH 6.0 and at 30 °C. For PG secreted by K. marxianus CCT 3172 and P. anomala S16, the optimum pH and temperature for the enzymatic activity were 5.5 and 40 °C, respectively. On the other hand, PG produced by P. kluyveri S13Y4 showed the highest activity at pH 5.0 and 50 °C.

Significant differences in the extracellular activity of PG were found between the yeasts species as well as between strains within same species. High amounts of PG were produced by two strains of P. anomala and P. kluyveri. It is therefore likely that strains of those two species may be involved in the degradation of pectin during coffee fermentation.     

Mycotoxin production by Alternaria strains isolated from Argentinean wheat

The toxigenic potential of Alternaria strains isolated from Argentinean wheat was investigated. A total of 123 strains were assayed for the production of tenuazonic acid (TA), alternariol (AOH) and alternariol monomethyl ether (AME). All but one of the isolates were able to produce at least one of the three mycotoxins. TA was produced by 72% of the strains (1-14782 mg/kg), AOH by 87% (4-622 mg/kg) and AME by 91% (7-2625 mg/kg). The average level of TA detected for all strains (1757 mg/kg) was higher than the average level of both alternariols (162 mg/kg for AOH and 620 mg/kg for AME). TA was the toxin produced at the highest concentration but in lower frequency. Most of the strains were able to synthesize more than one toxin: 74 isolates (60%) were positive for all three toxins, 30 (24%) for both AOH and AME, 5 (4%) for both TA and AME, and 2 (2%) for TA and AOH. The widespread occurrence of Alternaria in wheat and its ability to produce mycotoxins suggests the possible occurrence of its toxins in wheat naturally infected with this fungus.     

Growth inhibition of Fusarium graminearum and reduction of deoxynivalenol production in wheat grain by bacillomycin D

Fusarium graminearum causes Fusarium head blight, which leads to the quality and yield loss of wheat. In the present work, the inhibition effects of bacillomycin D (BD) on the growth of F. graminearum and reduction of deoxynivalenol (DON) production in wheat grain storage were investigated. The hyphal growth and sporulation of F. graminearum were restrained dramatically in the presence of 75 μg/mL BD, and the inhibition rate reached to 94.6% and 97.5%, respectively. Ultrastructural observation of the hyphae showed BD caused stripping of F. graminearum hyphal surface and destroying of the cellular structure. Furthermore, BD could lower the free fatty acid value and total antioxidant capacity and delay the decline of wheat quality, then exhibit an effective protection for wheat infection by F. graminearum. BD could effectively inhibit mold growth and DON production during wheat seeds storage, thus enhancing the quality and shelf life of the kernel. The addition of BD (75 μg/g wheat) remarkably inhibited the amount of F. graminearum growth, and reduced the DON production to 47.5–71.5%. These results indicated that BD might be a promising natural and effective fungicide, and would have potential for reducing mycotoxins in food and feed.     

禾谷镰刀菌发酵液中玉米赤霉烯酮毒素的提取及纯化研究

本研究旨在通过不同萃取剂和层析柱的使用,从禾谷镰刀菌发酵液中提取、纯化玉米赤霉烯酮（zearalenone,ZEN）毒素,以期获得较高纯度的毒素。采用超高效液相色谱串联质谱（ultra-performance liquid chromatography-mass spectrometry,UPLC-MS）分析方法对其效果进行检测。研究结果表明:在单一的萃取剂中,苯的萃取效果最佳,其得率可达到73.20%;采用Daisogel反相层析柱进行纯化,得率可达到69.30%,且样品中的杂峰数目明显减少,由此证明该方法对毒素纯化效果较好。本研究对发展高纯度ZEN毒素的制备技术具有一定的指导意义和参考价值。      

Analysis of deoxynivalenol and deoxynivalenol-3-glucosides content in Canadian spring wheat cultivars inoculated with Fusarium graminearum

Contamination of wheat grains with Fusarium mycotoxins and their modified forms is an important issue in wheat industry. The objective of this study was to analyse the deoxynivalenol (DON) and deoxynivalenol-3-glucosides (D3G) content in Canadian spring wheat cultivars grown in two locations, inoculated with a mixture of 3-acetyldeoxynivalenol (3-ADON)-producing Fusarium graminearum strains and a mixture of 15-acetlyldeoxynivalenol (15-ADON)-producing F. graminearum strains. According to the analysis of variance, significant differences were observed among the cultivars for Fusarium head blight (FHB) disease index, Fusarium-damaged kernel percentage (%FDK), DON content and D3G content. When the effect of chemotype was considered, significant differences were observed for FHB disease index, FDK percentage and DON content. The D3G content and D3G/DON ratio were not significantly different between the chemotypes, except for D3G content at the Winnipeg location. The Pearson correlation coefficient between DON and D3G was 0.84 and 0.77 at Winnipeg and Carman respectively. The highest D3G/DON ratio was observed in cultivars Carberry (44%) in Carman and CDC Kernen (63.8%) in Winnipeg. The susceptible cultivars showed lower D3G/DON ratio compared with the cultivars rated as moderately resistant and intermediate. The current study indicated that Canadian spring cultivars produce D3G upon Fusarium infection.     

Effect of fungicides on the development of Fusarium head blight,yield and deoxynivalenol accumulation in wheat inoculated under field conditions with Fusarium graminearum and Fusarium culmorum

The effect of different fungicide treatments on Fusarium head blight (FHB) development, grain yields and deoxynivalenol (DON) accumulation in wheat was evaluated after artificial inoculation under field conditions with a mixture of Fusarium graminearum and F culmorum. The trials were carried out using commercially available products on five different cultivars of soft and durum wheat (Serio, Genio, Bracco, Duilio and Orobel) in two separate experimental fields located in the North of Italy. Treatments with Tiptor^® S (cyproconazole plus prochloraz) and a mixture of Horizon^® 250 EW (tebuconazole) plus Amistar^® 250 SC (azoxystrobin) significantly reduced the FHB disease severity (by between 25 and 77%) and DON content (by between 32 and 89%) in the grain as compared with the inoculated control. Yields (tonne ha^?1) and thousand grain weight (TGW) were higher in plots subjected to fungicide treatments. Tetraconazole (Eminent^® 40 EW) showed a markedly reduced effectiveness compared with the other treatments. Regression analysis showed a strong correlation of disease severity with DON levels (positive correlation) and with yields or TGW (negative correlation) for individual cultivars and locations. Fusarium graminearum, F culmorum and F poae were the species most commonly isolated from all trials, including inoculated and non‐inoculated control plots. Copyright © 2004 Society of Chemical Industry     

Assessment of trichothecene chemotypes of Fusarium culmorum occurring in Europe

Fusarium trichothecenes are a group of fungal toxic metabolites whose synthesis requires the action of gene products from three different genetic loci. We evaluated, both chemically and by PCR assays, 55 isolates of Fusarium culmorum from eight European countries and different host plants for their ability to produce trichothecenes. Specific sequences in the Tri6-Tri5 intergenic region were associated with deoxynivalenol production. Sequences in the Tri3 gene were also associated with deoxynivalenol production and specific primer sets were selected from these sequences to identify 3-acetyl-deoxynivalenol or 15-acetyl-deoxynivalenol chemotypes. Specific sequences in the Tri5 and Tri7 genes were associated with the nivalenol chemotype but not with the deoxynivalenol chemotype. Two chemotypes were identified by chemical analysis and confirmed by PCR. Strains of the nivalenol chemotype produced nivalenol (up to 260 µg g^-1) and 4-acetyl-nivalenol (up to 60 µg g^-1), strains with the 3-acetyl-deoxynivalenol chemotype produced deoxynivalenol (up to 1700 µg g^-1) and 3-acetyl-deoxynivalenol (up to 600 µg g^-1). Three strains of F. culmorum from France, previously reported as 15-acetyl-deoxynivalenol producers, had the 3-acetyl-deoxynivalenol chemotype. The results are consistent with data from other European countries on the occurrence of the nivalenol and 3-acetyl-deoxynivalenol chemotypes and provide support for the hypothesis that European isolates of F. culmorum producing deoxynivalenol belong only to the 3-acetyl-deoxynivalenol chemotype. The production of trichothecenes from F. culmorum isolates from walnut (3-acetyl-deoxynivalenol chemotype) and leek (nivalenol chemotype) is reported for the first time.     

Analysis of deoxynivalenol, masked deoxynivalenol, and Fusarium graminearum pigment in wheat samples, using liquid chromatography-UV-mass spectrometry

Tolerable limits set for deoxynivalenol (DON) do not consider DON conjugates such as DON-3-glucoside. Conjugates may be metabolized in vivo to DON. Such masked mycotoxins and the potentially toxic Fusarium pigment are not routinely analyzed in cereals. We quantified DON, DON-3-glucoside, and a red Fusarium pigment in hard red spring wheat, using a new liquid chromatography-mass spectrometry method. Extraction protocols using centrifugation and shaking, and methanol-methylene chloride (50:50 [vol/vol]) or acetonitrile-water (84:16 [vol/vol]) were assessed. Purposively and randomly selected hard spring wheat samples were extracted with solvent filtered through a C18 column and analyzed using liquid chromatography-UV-mass spectrometry. Isocratic mobile phase (70% methanol) was used. Recoveries were 96.4% (DON) and 70.0% (DON-3-glucoside), while limits of detection were 1 microg/kg (MS) and 10 microg/kg (UV), and limits of quantification were 1 microg/kg (UV) and 0.5 microg/kg (MS), respectively. The pigment limits of quantification and limits of detection on the MS were 4.3 and 0.0005 microg/kg, respectively. The purposively selected samples had DON, DON-3-glucoside, and pigment averages of 3.4 +/- 4.0 microg/g, 3.8 +/- 8.3 microg/g, and 0.31 +/- 3.71 g/kg, respectively. The randomly selected spring wheat had lower mean levels of DON (1.4 +/- 2.3 microg/g), DON-3-glucoside (0.2 +/- 1.0 microg/g), and pigment (147.93 +/- 247.84 microg/g). Analytical tools such as this new liquid chromatography-UV-mass spectrometry method can be used to quantify masked and parent mycotoxins, plus a potentially toxic pigment for risk assessment.