Development of a PCR assay to detect the potential production of nivalenol in Fusarium poae

Fusarium species can produce mycotoxins, which can contaminate cereal-based food producing adverse effects for human and animal health. In recent years, the importance of Fusarium poae has increased within the Fusarium head blight complex. Fusarium poae is known to produce trichothecenes, especially nivalenol, a potent mycotoxin able to cause a variety of toxic effects. In this study, a specific primer pair was designed based on the tri7 gene to detect potential nivalenol-producing F.?poae isolates. A total of 125 F.?poae, four F.?cerealis, two F.?culmorum, one F.?langsethiae, one F.?sporotrichioides and seven F.?graminearum, plus F.?austroamericanum, F.?meridionale, F.?graminearum sensu stricto and F.?cortaderiae from the NRRL collection were analysed, and only F.?poae isolates gave a positive result for the presence of a 296-bp partial tri7 DNA fragment. Moreover, the primer set was tested from cereal seed samples where F.?poae and other Fusarium species with a negative result for the specific reaction ( F.?graminearum, F.?oxysporum, F.?chlamydosporum, F.?sporotrichioides, F.?equiseti and F.?acuminatum) were isolated, and the expected fragment was amplified. We developed a rapid and reliable PCR assay to detect potential nivalenol-producing F.?poae isolates.     

Mycotoxin production and cytotoxicity of Fusarium strains isolated from Norwegian cereals

Thirty-four isolates of the eight most common Fusarium species isolated from Norwegian cereals; F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. poae, F. sporotrichioides, F. torulosum and F. tricinctum were studied for their cytotoxicity and ability to produce mycotoxins. The strains were cultivated on rice, and analysed for trichothecenes (all species), zearalenone (all species), fusarochromanone (F. equiseti), wortmannin (F. torulosum), moniliformin and enniatins (F. avenaceum, F. tricinctum and F. torulosum). The cytotoxicity of the extracts were examined with an (in vitro) MTT-cell culture assay. All F. graminearum and five of seven F. culmorum isolates belonged to chemotype IA, producing deoxynivalenol and 3-acetyl-deoxynivalenol, while the two other F. culmorum strains were nivalenol producers (chemotype II). The F. equiseti isolates and one of the F. poae isolates produced both type A and B trichothecenes, and relatively large quantities of fusarochromanone were detected in the F. equiseti cultures. All Fusarium species studied showed significant cytotoxicity, but with a large variation between species, and also within each species. F. sporotrichioides and F. equiseti showed the highest average cytotoxicity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Microbiological and SYBR green real-time PCR detection of major Fusarium head blight pathogens on wheat ears

Fusarium head blight (FHB) caused by several Fusarium species is one of the most serious diseases affecting wheat throughout the world. The efficiency of microbiological assays and real-time PCRto quantify major FHB pathogens in wheat ears after inoculation with F. graminearum, F. culmorum, F. avenaceum and F. poae undergreenhouse and field conditions were evaluated. The frequency of infected kernel, content of fungal biomass, disease severity and kernel weight were determined. To measure the fungal biomass an improved DNA extraction method and a SYBR Green real-time PCR were developed. The SYBR Green real-time PCR proved to be highly specific for individual detection of the species in a matrix including fungal and plant DNA. The effect of Fusarium infection on visible FHB severity, frequency of infected kernels and thousand-kernel mass (TKM) significantly depended on the Fusarium species/isolate. F. graminearum resulted in highest disease level, frequency of infected kernels, content of fungal biomass, and TKM reduction followed by F. culmorum, EF avenaceum and F. poae, respectively. The comparison of frequency and intensity of kernel colonization proved differences in aggressiveness and development of the fungi in the kernels. Only for F. graminearum, the most aggressive isolate, application of microbiological and real-time PCR assays gave similar results. For the other species, the intensity of kernel colonization was lower than expected from the frequency of infection.     

An oligonucleotide microarray for the identification and differentiation of trichothecene producing and non-producing Fusarium species occurring on cereal grain

Cereal grain may be infected with a number of Fusarium species some of which are producers of highly toxic compounds such as the trichothecenes. Correct identification of these species is essential for risk assessment of cereal grain for human or animal consumption. Most of the available methods for identification are either time consuming or aimed at only one or a few target species. Microarray technology offers parallel analysis of a high number of DNA targets. In this study 57 capture oligonucleotides (CO) were designed based upon Fusarium ITS2 rDNA sequences, and used for microarray production. From this array COs could be selected that were able to hybridise specifically to labelled PCR products from the ITS region of Fusarium graminearum/Fusarium culmorum, Fusarium pseudograminearum, Fusarium poae, Fusarium sporotrichioides, Fusarium equiseti, Fusarium langsethiae and Fusarium tricinctum/Fusarium avenaceum. A few COs showed some cross hybridisation to non-target species. In a preliminary experiment it was shown that this cross hybridisation could be eliminated by increasing hybridisation stringency. The array could be used to detect individual Fusarium species in mixed samples and in environmental samples. This study demonstrates the feasibility of oligonucleotide microarrays for parallel detection of a number of Fusarium species.     

Fusarium head blight: distribution in wheat in Latvia

Fusarium head blight (FHB) of wheat has, in recent years, been a very important worldwide disease in intensive growing of cereal. The objectives of this study were to evaluate the occurrence of FHB in wheat in Latvia and to identify the Fusarium species involved. This paper describes the distribution of Fusarium species that were isolated from samples representing winter and spring wheat varieties in Latvia, identified both by the classical morphological analyses of J. Leslie and B. Summerell (2006) and by PCR. The FHB incidence range in winter wheat was 1-20%, in spring wheat was 1-42%. The most significant factor affecting the incidence of fusarial head blight in wheat in Latvia was heightened temperature at the time of an thesis of wheat. In winter wheat 9 Fusarium species caused FHB: F. culmorum, F. avenaceum, F. graminearum, F. equiseti, F. poae, F. oxysporum, F. cerealis, F. sporotrichoides and F. verticillioides were identified by morphological characterization, and 5 were confirmed by PCR-analysis. After experience of 5 years, it can be concluded that the most frequent in winter wheat were F. poae and F. culmorum. In spring wheat from F. culmorum was dominant among 8 Fusarium species. Among 13 varieties of spring wheat, three were sensitive ('Chamsin', 'W 166', 'Azurite') and one was resistant ('Granny') to FHB in conditions of high natural infection in 2009. The monitoring surveys demonstrate a significant presence of FHB in spring wheat in conditions of heightened temperature at the time of flowering in Latvia.     

Mating type sequences in asexually reproducing Fusarium species

To assess the potential for mating in several Fusarium species with no known sexual stage, we developed degenerate and semidegenerate oligonucleotide primers to identify conserved mating type (MAT) sequences in these fungi. The putative alpha and high-mobility-group (HMG) box sequences from Fusarium avenaceum, F. culmorum, F. poae, and F. semitectum were compared to similar sequences that were described previously for other members of the genus. The DNA sequences of the regions flanking the amplified MAT regions were obtained by inverse PCR. These data were used to develop diagnostic primers suitable for the clear amplification of conserved mating type sequences from any member of the genus Fusarium. By using these diagnostic primers, we identified mating types of 122 strains belonging to 22 species of Fusarium. The alpha box and the HMG box from the mating type genes are transcribed in F. avenaceum, F. culmorum, F. poae, and F. semitectum. The novelty of the PCR-based mating type identification system that we developed is that this method can be used on a wide range of Fusarium species, which have proven or expected teleomorphs in different ascomycetous genera, including Calonectria, Gibberella, and Nectria.     

Differences in susceptibility of winter wheat varieties for Fusarium species under Belgian growing conditions

Fusarium head blight is an important disease of cereal crops caused by Fusarium species. It causes not only a reduction in yield, but most Fusarium species (F. graminearum. F. culmorum, F. avenaceum. F. poae) produce also a range of toxic metabolites such as deoxynivalenol (DON) and zearalenone (ZEA). The evaluation of Fusarium species was followed up under natural infection conditions during the growing seasons 2001--2002 and 2002--2003 in two varietal winter wheat experiments on the experimental farm of the Hogeschool Gent at Bottelare. Disease pressure, DON and ZEA content, different Fusarium species as well as growth and yield parameters were determined. In both years there were significant differences between the varieties concerning the susceptibility to Fusarium and the DON content. ZEA was not found in the kernels. The mean deoxynivalenol (DON) content was in 2002 (1,126 mg/kg) higher than in 2003 (0.879 mg/kg) although the mean disease severity was bigger in 2003 than in 2002 what means that the DON content was not always correlated with the disease severity. The Fusarium species most frequently identified in our two field trials (Bottelare) were F. graminearum and F. culmorum Varietal differences in susceptibility to Fusarium species and DON contamination could be detected.     

FLASH-PCR diagnostics of toxigenic fungi of the genus <Emphasis Type="Italic">Fusarium</Emphasis>

A test system for the diagnostics and identification of seven toxigenic fungi causing Fusarium head blight (Fusarium graminearum, F. culmorum, F. poae, F. sporotrichioides, F. langsethiae, F. avenaceum, and F. tricinctum) was developed using PCR. The identification of pathogen is based on the specific amplification of a DNA fragment of the gene of translation elongation factor 1 alpha (tef-1α) and subsequent detection of the results by the fluorescent amplification-based specific hybridization method. The system was tested on 38 isolates of different fungi of the genus Fusarium.     

Application of an enzyme-linked immunosorbent assay for screening of T-2 toxin-producing Fusarium spp.

Culture filtrates of Fusarium species were subjected without clean-up procedures to an indirect competitive enzyme-linked immunosorbent assay with anti-T-2 toxin monoclonal antibody. Fusarium sporotrichioides, F. poae, F. tricinctum, and F. culmorum strains were positive for T-2 toxin, with a minimum detection limit of 5 pg per assay (100 pg/ml of culture filtrate), and the assay data correlated well with the gas-liquid chromatographic data.     

Application of an enzyme-linked immunosorbent assay for screening of T-2 toxin-producing Fusarium spp

Culture filtrates of Fusarium species were subjected without clean-up procedures to an indirect competitive enzyme-linked immunosorbent assay with anti-T-2 toxin monoclonal antibody. Fusarium sporotrichioides, F. poae, F. tricinctum, and F. culmorum strains were positive for T-2 toxin, with a minimum detection limit of 5 pg per assay (100 pg/ml of culture filtrate), and the assay data correlated well with the gas-liquid chromatographic data.     

Phylogenetic relationships of Fusarium poae based on EF-1 alpha and mtSSU sequences

A molecular phylogenetic analysis of Fusarium poae isolates from South America (Argentina) and Europe (mainly England, Germany, Italy) was performed using 98 F. poae, four Fusarium culmorum, two Fusarium sporotrichioides and one Fusarium langsethiae isolates. Phylogenetic analyses were performed using nuclear (translation elongation factor 1-alpha, EF-1 alpha) and mitochondrial (mitochondrial small subunit rDNA, mtSSU) sequences. Partitioned (each dataset separately) and combined (EF-1 alpha+mtSSU) analyses did not reveal any clear correlations from the inferred branching topology, between the distribution of observed haplotypes and the geographic origin and/or host species. Results from the present study confirmed that isolates from F. poae form a monophyletic group, and the low variability within isolates from a broad geographic range suggests a common lineage history. Among F. poae isolates from Argentina, however, some were found to possess an insert within mtSSU with structural similarities to group IC2 introns. F. poae isolates differing by the presence/absence of a mtSSU insertion were characterized further by analysis of a portion of the Tri5 gene, but this sequence was unable to reveal variability. The presence of this insert only within isolates from Argentina suggests that evolutionary events (insertions/deletions) are probably taking place within the Argentinian F. poae isolates, and that the acquisition of this insert occurred after geographic isolation of the Argentinian and European populations.     

Production of a novel steroid sulfate metabolite [4,4,24-trimethylcholesta-8,14,24(28)-trien-2 alpha,3 beta,11 alpha,12 beta-tetrol 12-acetate, 3-sulfate] by Fusarium species and its biological activity.

A novel steroid sulfate, 4,4,24-trimethylcholesta-8,14-24(28)-trien-2 alpha,3 beta,11 alpha,12 beta- tetrol 12-acetate, 3-sulfate, was discovered in Fusarium spp. Forty Fusarium strains belonging to F. sporotrichioides, F. chlamydosporum, E. equiseti, F. acuminatum, F. sambucinum, F. culmorum, and F. graminearum produced the steroid on white corn grits at 25 degrees C for 20 days. This steroid sulfate is one of the more abundant and easily attainable microbial steroids. At a concentration of 160 micrograms/ml, it inhibited the growth of six fungi, two gram-positive bacteria, and an alga, as well as the germination of both wheat and tomato seeds.     

Production of a novel steroid sulfate metabolite [4,4,24-trimethylcholesta-8,14,24(28)-trien-2 alpha,3 beta,11 alpha,12 beta-tetrol 12-acetate, 3-sulfate] by Fusarium species and its biological activity

A novel steroid sulfate, 4,4,24-trimethylcholesta-8,14-24(28)-trien-2 alpha,3 beta,11 alpha,12 beta- tetrol 12-acetate, 3-sulfate, was discovered in Fusarium spp. Forty Fusarium strains belonging to F. sporotrichioides, F. chlamydosporum, E. equiseti, F. acuminatum, F. sambucinum, F. culmorum, and F. graminearum produced the steroid on white corn grits at 25 degrees C for 20 days. This steroid sulfate is one of the more abundant and easily attainable microbial steroids. At a concentration of 160 micrograms/ml, it inhibited the growth of six fungi, two gram-positive bacteria, and an alga, as well as the germination of both wheat and tomato seeds.     

Multiplex PCR assay for the identification of nivalenol, 3- and 15-acetyl-deoxynivalenol chemotypes in Fusarium

The ability to rapidly distinguish trichothecene chemotypes in a given species/population of the genus Fusarium is important due to significant differences in the toxicity of these secondary metabolites. A multiplex PCR assay, based on primer pairs derived from the Tri3, Tri5 and Tri7 genes of the trichothecene gene cluster was established for the identification of the different chemotypes among Fusarium graminearum, F. culmorum and F. cerealis. Using the selected primers, specific amplification products of 625, 354 and 708 bp were obtained from Fusarium isolates producing nivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol, respectively. Moreover, the multiplex PCR was successfully used to identify the chemotype of the Fusarium species contaminating wheat kernels. Four picograms of fungal DNA were found to be necessary to obtain a visible amplification product.     

Real-time PCR assay to quantify Fusarium graminearum wild-type and recombinant mutant DNA in plant material

Fusarium graminearum (teleomorph, Gibberella zeae) is the predominant causal agent of Fusarium head blight (FHB) of wheat resulting in yearly losses through reduction in grain yield and quality and accumulation of fungal generated toxins in grain. Numerous fungal genes potentially involved in virulence have been identified and studies with deletion mutants to ascertain their role are in progress. Although wheat field trials with wild-type and mutant strains are critical to understand the role these genes may play in the disease process, the interpretation of field trial data is complicated by FHB generated by indigenous species of F. graminearum. This report describes the development of a SYBR green-based real time PCR assay that quantifies the total F. graminearum genomic DNA in a plant sample as well as the total F. graminearum genomic DNA contributed from a strain containing a common fungal selectable marker used to create deletion mutants. We found our method more sensitive, reproducible and accurate than other similar recently described assays and comparable to the more expensive probe-based assays. This assay will allow investigators to correlate the amount of disease observed in wheat field trials to the F. graminearum mutant strains being examined.     

Mycotoxin-producing Fusarium Species Occurring in Winter Wheat in Belgium (Flanders) During 2002–2005

Deoxynivalenol (DON) content and Fusarium spp. ( Fusarium graminearum , Fusarium culmorum , Fusarium avenaceum , Microdochium nivale and Fusarium poae ) of mycotoxin-producing Fusarium species in winter wheat in Belgium (Flanders) were determined. Field trials were set up in the varietal testing network of Flanders Agricultural Centre for Small Grains (Roeselare – Beitem, Belgium) and followed up during growing seasons 2001–2002, 2002–2003, 2003–2004 and 2004–2005. Fusarium infection and DON contamination were mainly influenced by location and environmental parameters. Mean DON levels ranged from 0 to 15 mg/kg. Over the period of four growing seasons cvs Deben, Ordeal and Napier had the highest DON contamination. Seasonal and local weather conditions before and during flowering together with local crop husbandry measures (crop rotation, soil preparation) seemed to be of great importance in explaining the variation in results obtained. At Bottelare a positive correlation between disease index and DON content was found for the growing seasons 2001–2002 and 2002–2003, but not the season 2003–2004. Fusarium graminearum and F. culmorum were in general the most frequently occurring Fusarium spp. in Flanders over the 4 years but the composition of the Fusarium population varied strongly from location to location and from year to year. Fusarium graminearum predominated in areas especially where maize was cultivated, whereas in areas with more small cereals in the crop rotation more F. culmorum was present. Also temperature played a role in the composition of Fusarium spp.     

Bikaverin production by Fusarium species

Production of bikaverin has been examined in 130Fusarium isolates belonging to 21 species. The highest yield of bikaverin was produced on autoclaved rice — up to 2.5g/kg of dry culture. Bikaverin was produced by the following species:F verticillioides, F sacchari varsubglutinans, F proliferatum, F anthophilum, F oxysporum, F dlamini, F nygamai, F napiforme, andF solani. SpeciesF coeruleum, F poae, F sporotrichioides, F tricinctum, F chlamydosporum, F culmorum, F graminearum, F cerealis (F crookwellense), F avenaceum, F acuminatum, andF equiseti did not produce bikaverin. The production of bikaverin determines the colour of the mentionedFusarium species cultures on agar media and on rice. The pigment has indicator properties and changes colour from red in acidic solution to violet-blue in alkaline. The role it plays in fungus metabolism is not elucidated.     

A genetic and biochemical approach to study trichothecene diversity in Fusarium sporotrichioides and Fusarium graminearum

The trichothecenes T-2 toxin and deoxynivalenol (DON) are natural fungal products that are toxic to both animals and plants. Their importance in the pathogenicity of Fusarium spp. on crop plants has inspired efforts to understand the genetic and biochemical mechanisms leading to trichothecene synthesis. In order to better understand T-2 toxin biosynthesis by Fusarium sporotrichioides and DON biosynthesis by F. graminearum, we compared the nucleotide sequence of the 23-kb core trichothecene gene cluster from each organism. This comparative genetic analysis allowed us to predict proteins encoded by two trichothecene genes, TRI9 and TRI10, that had not previously been described from either Fusarium species. Differences in gene structure also were correlated with differences in the types of trichothecenes that the two species produce. Gene disruption experiments showed that F. sporotrichioides TRI7 (FsTRI7) is required for acetylation of the oxygen on C-4 of T-2 toxin. Sequence analysis indicated that F. graminearum TRI7 (FgTRI7) is nonfunctional. This is consistent with the fact that the FgTRI7 product is not required for DON synthesis in F. graminearum because C-4 is not oxygenated.     

Screening for Fusarin C production by European isolates of Fusarium species

A total of 137Fusarium isolates were screened forin vitro production of the mutagenic metabolite fusarin C, using a simple thin layer chromatographic method. It has been proven thatFusarium species (F. culmorum, F. graminearum, F. crookwellense, F. sporotrichioides, F. poae, F. tricinctum, andF. Avenaceum) isolated from European agricultural crops and soils are able to produce fusarin C. No fusarin C production was detected among isolates ofF. arthrosporioides, F. acuminatum, or F. equiseti. Results obtained by High-Performance Liquid Chromatography (HPLC) analyses of fungal extracts show that up to 26 chromatographic peaks having UV spectra similar to that of fusarin C are produced. It is not known if any of these metabolites are as mutagenic as fusarin C.     

Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels

Identification of Fusarium species by traditional methods requires specific skill and experience and there is an increased interest for new molecular methods for identification and quantification of Fusarium from food and feed samples. Real-time PCR with probe technology (Taqman) can be used for the identification and quantification of several species of Fusarium from cereal grain samples. There are several critical steps that need to be considered when establishing a real-time PCR-based method for DNA quantification, including extraction of DNA from the samples. In this study, several DNA extraction methods were evaluated, including the DNeasy Plant Mini Spin Columns (Qiagen), the Bio robot EZ1 (Qiagen) with the DNeasy Blood and Tissue Kit (Qiagen), and the Fast-DNA Spin Kit for Soil (Qbiogene). Parameters such as DNA quality and stability, PCR inhibitors, and PCR efficiency were investigated. Our results showed that all methods gave good PCR efficiency (above 90%) and DNA stability whereas the DNeasy Plant Mini Spin Columns in combination with sonication gave the best results with respect to Fusarium DNA yield. The modified DNeasy Plant Mini Spin protocol was used to analyse 31 wheat samples for the presence of F. graminearum and F. culmorum. The DNA level of F. graminearum could be correlated to the level of DON (r(2) = 0.9) and ZEN (r(2) = 0.6) whereas no correlation was found between F. culmorum and DON/ZEA. This shows that F. graminearum and not F. culmorum, was the main producer of DON in Swedish wheat during 2006.