Tapani Yli-Mattila

A European Database of Fusarium graminearum and F. culmorum Trichothecene Genotypes

Fusarium species, particularly Fusarium graminearum and F. culmorum, are the main cause of trichothecene type B contamination in cereals. Data on the distribution of Fusarium trichothecene genotypes in cereals in Europe are scattered in time and space. Furthermore, a common core set of related variables (sampling method, host cultivar, previous crop, etc.) that would allow more effective analysis of factors influencing the spatial and temporal population distribution, is lacking. Consequently, based on the available data, it is difficult to identify factors influencing chemotype distribution and spread at the European level. Here we describe the results of a collaborative integrated work which aims (1) to characterize the trichothecene genotypes of strains from three Fusarium species, collected over the period 2000–2013 and (2) to enhance the standardization of epidemiological data collection. Information on host plant, country of origin, sampling location, year of sampling and previous crop of 1147 F. graminearum, 479 F. culmorum, and 3 F. cortaderiae strains obtained from 17 European countries was compiled and a map of trichothecene type B genotype distribution was plotted for each species. All information on the strains was collected in a freely accessible and updatable database (www.catalogueeu.luxmcc.lu), which will serve as a starting point for epidemiological analysis of potential spatial and temporal trichothecene genotype shifts in Europe. The analysis of the currently available European dataset showed that in F. graminearum, the predominant genotype was 15-acetyldeoxynivalenol (15-ADON) (82.9%), followed by 3-acetyldeoxynivalenol (3-ADON) (13.6%), and nivalenol (NIV) (3.5%). In F. culmorum, the prevalent genotype was 3-ADON (59.9%), while the NIV genotype accounted for the remaining 40.1%. Both, geographical and temporal patterns of trichothecene genotypes distribution were identified.

Presence and concentrations of the Fusarium-related mycotoxins beauvericin, enniatins and moniliformin in finnish grain samples.

Fusarium mycotoxins beauvericin, enniatins (A, A1, B, B1) and moniliformin were analysed in 38 Finnish grain samples (14 wheat, 22 barley, one rye, one oats) harvested in 2001–02. The contaminating Fusarium species were identified with the primer-specific polymerase chain reaction as well as with morphological studies. All the studied mycotoxins were found in the samples. Enniatins B and B1 were detected in all samples, and enniatin A, enniatin A1, beauvericin and moniliformin in 74, 95, 95 and 74% of the samples, respectively. There were higher concentrations of the mycotoxins analysed in 2001 compared with 2002. The highest levels of mycotoxins were detected in samples harvested late in the autumn after a long rainy period. Fusarium avenaceum was the most abundant Fusarium species in Finland during both years (0–29.5%) measured as infected kernels. A significant correlation was found between F. avenaceum contamination level and the concentration levels of enniatins B and B1, as well as moniliformin.

A novel Asian clade within the Fusarium graminearum species complex includes a newly discovered cereal head blight pathogen from the Russian Far East

We investigated Fusarium graminearum complex (Fg complex) species diversity and toxin potential in European and Asian regions of the Russian Federation and adjoining regions northwest to Finland and south near Harbin, Heilongjiang Province, China, to expand our knowledge of the host range and geographic distribution of these economically devastating cereal head blight pathogens. Results of a recently described multilocus genotyping (MLGT) assay revealed that F. graminearum was the sole Fg complex pathogen in northern Europe and the predominant one in Asia (90.5%). Even though isolates of F. graminearum were segregating for 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON) chemotype in nearly equal frequencies in the regions sampled on both continents, significant differences in the geographic distribution of isolates producing these acetyl ester derivatives of deoxynivalenol (DON) were observed in Europe. While 93.5% of the isolates in southern Russia (n = 43 of 46) possessed the 15ADON chemotype, isolates of F. graminearum recovered in Finland and northwestern Russia (n = 40) were exclusively 3ADON producers. Based on results of the MLGT assay, species identity of 10 genetically novel Fg complex isolates from the Russian Far East was investigated further via molecular phylogenetic analyses of multi-locus DNA sequence data. Results of these analyses resolved these isolates as a phylogenetically distinct, reciprocally monophyletic sister lineage of F. asiaticum, which together with F. vorosii form a newly discovered Asian clade within the Fg complex. Because this novel lineage fulfills the highly conservative criterion of genealogical exclusivity under phylogenetic species recognition it is formally described herein as F. ussurianum. In addition to morphologically characterizing isolates of F. ussurianum, experiments were conducted to assess pathogenicity to wheat and trichothecene toxin potential in planta.

One fungus, one name

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice.

One fungus, one name: defining the genus Fusarium in a scientifically robust way that preserves longstanding use.

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice.

Molecular and morphological diversity of Fusarium species in Finland and northwestern Russia

In 2001 the range of the total Fusarium contamination percentage of infected seeds was between 0% and 44%, while in 2002 the contamination level was 2–25% in naturally infected Finnish samples and 5–14.5% in six samples from northwestern Russia. The most common Fusarium species in barley were F. avenaceum, F. arthrosporioides, F. sporotrichioides and F. culmorum, while in spring wheat the most common Fusarium species were F. avenaceum, F. arthrosporioides, F. culmorum, F. sporotrichioides and F. graminearum. In most cases, molecular identification with species-specific primers corresponded to the morphological analyses and allowed the identification of degenerated and otherwise morphologically difficult cultures. It was even possible to separate most of the F. arthrosporioides isolates from Finland from the closely-related F. avenaceum isolates. In the phylogenetic analysis of combined β-tubulin, IGS and ITS sequences most European F. arthrosporioides formed a separate clade from most isolates of F. avenaceum and from all isolates of F. tricinctum. Most of the species-specific primers also amplified DNA extracted from grain samples. It was, for instance, possible to detect F. avenaceum in all barley samples with contamination levels higher than 1% and in all spring wheat samples with contamination levels higher than 3%. The detection level for F. graminearum was at a contamination level of 3–5% and that for F. culmorum at a contamination level of 1–5%. In addition, the first Finnish F. langsethiae isolate was found by means of species-specific primers.

Real-time PCR detection and quantification of Fusarium poae, F. graminearum, F. sporotrichioides and F. langsethiae in cereal grains in Finland and Russia

Abstract TaqMan real-time quantitative PCR assays were developed for the accurate detection and quantification of DNA from Fusarium poae and F. graminearum species, which are able to produce trichothecenes. These and other PCR assays were used for the quantification of trichothecene-producing Fusarium fungi in cereal grains. A correlation was found between the levels of F. poae DNA and nivalenol and enniatins in barley and between the levels of F. graminearum DNA and deoxynivalenol in oats. The correlations between F. poae DNA and nivalenol and F. graminearum DNA and deoxynivalenol levels were higher than those between these mycotoxins and morphologically determined F. poae and F. graminearum/F. culmorum contamination levels. The use of F. poae specific primers and probe together with F. sporotrichioides/F. langsethiae specific primers and probe in a multiplex qPCR assay yielded results in accordance with those obtained using these primers and probes separately.

Real-time PCR for Quantification of Toxigenic Fusarium Species in Barley and Malt

A real-time PCR technique was applied for the quantification of trichothecene-producing Fusarium species (TMTRI assay) as well as the highly toxigenic Fusarium graminearum (TMFg12 assay) present in barley grain and malt. PCR results were compared to the amounts of trichothecenes detected in the samples to find out if the PCR assays can be used for trichothecene screening instead of expensive and laborious chemical analyses. DNA was extracted from ground kernels using a commercial DNA extraction kit and analysed in a LightCycler® system using specific primers and fluorogenic TaqMan probes. Both naturally and artificially contaminated grains were analysed. The TMTRI assay and the TMFg12 assay enabled the quantification of trichothecene-producing Fusarium DNA and F. graminearum DNA present in barley grain and malt samples, respectively. Both TaqMan assays were considered to be sensitive and reproducible. Linearity of the assays was 4–5 log units when pure Fusarium DNAs were tested. The amount of Fusarium DNA analysed with the TMTRI-trichothecene assay could be used for estimation of the deoxynivalenol (DON) content in barley grain. Furthermore, the TMFg12 assay for F. graminearum gave a good estimation of the DON content in north American barley and malt samples, whilst the correlation was poor among Finnish samples. DON content and the level of F. graminearum DNA were found to be naturally low in Finnish barleys.

Molecular, morphological and phylogenetic analysis of the Fusarium avenaceum/F. arthrosporioides/F. tricinctum species complex – a polyphasic approach

Differences in morphology, ITS, IGS, mtSSU and β-tubulin sequences and UP-PCR hybridization were compared between morphologically identified F. avenaceum, F. arthrosporioides, F. anguioides, F. tricinctum, F. graminum and F. acuminatum strains. According to the combined β-tubulin, IGS and ITS tree, the strains of the Fusarium avenaceum/F. arthrosporioides/F. tricinctum species complex species can be divided into seven clusters supported by bootstrap values higher than 50%. The two main groups of European F. avenaceum , which cannot be distinguished by morphology, were separated in the tree based on β-tubulin sequences and less clearly in trees based on IGS and ITS sequences. MtSSU sequences were identical in all F. avenaceum and F. tricinctum strains studied. The European F. avenaceum strains of main group II had identical β-tubulin sequences with one American F. avenaceum strain and four European F. arthrosporioides strains, while F. avenaceum strains of main group I were closely related to two European F. arthrosporioides strains and to one Japanese F. anguioides strain. According to the combined β-tubulin/IGS/ITS sequence tree, European F. arthrosporioides strains were divided into four groups; F. tricinctum strains formed a wellsupported cluster, in which two European clusters were separated from one African isolate. In the IGS sequence tree two European F. acuminatum strains together with one American F. acuminatum strain formed a cluster, which was separate from another American F. acuminatum strain. The F. acuminatum cluster was nested within the large F. tricinctum cluster together with one F. reticulatum strain in the combined IGS/β-tubulin tree. Several strains may be intermediates between the F. avenaceum/F. arthrosporioides/F. anguioides and F. tricinctum clusters and represent their own species. These results are partially supported by the results of UP-PCR hybridization analysis. Thus the molecular results may be helpful in future revision in the taxonomy of these species.

In vitro and in vivo mycotoxin production of Fusarium species isolated from Finnish grains

Abstract The capabilities of seven Finnish Fusarium species to produce mycotoxins under controlled laboratory (in vitro) conditions as well as on barley and wheat in the field (in vivo) were studied using mass spectrometric methods. In addition, the contaminating Fusarium species of the in vivo samples were identified and quantified with morphological and real-time polymerase chain reaction techniques. All investigated isolates (representing the species F. avenaceum, F. arthrosporioides, F. tricinctum, F. poae, F. culmorum, F. graminearum and F. sporotrichioides) produced mycotoxins on rice cultures and their production capabilities were generally in accordance with the available literature. The main mycotoxins produced were deoxynivalenol, 3-acetyldeoxynivalenol and zearalenone by F. culmorum and F. graminearum, beauvericin, diacetoxyscirpenol and HT-2/T-2 – toxins by F. sporotrichioides, beauvericin, enniatins, fusarenon-X, diacetoxyscirpenol and nivalenol by F. poae and enniatins and moniliformin by F. avenaceum/F. arthrosporioides and F. tricinctum. Most mycotoxins that were produced in vitro were also produced in vivo when concerning F. culmorum, F. graminearum and F. avenaceum. Statistical significance was recorded between the production of mycotoxins in vitro and in vivo for HT-2 and T-2 toxins in barley and for deoxynivalenol and T-2 toxin in wheat.