Toxin production by Fusarium culmorum IMI 309344 and F. graminearum NRRL 5883 on grain substrates

The production of deoxynivalenol, acetyl deoxynivalenol and zearalenone by Fusarium culmorum and F. graminearum on autoclave-sterilized grain (maize, rice, wheat and barley) was investigated. Fusarium culmorum produced significantly greater levels of toxins than F. graminearum. The four substrates examined differed in their ability to support toxin production. Toxin production on maize and rice was significantly greater than toxin production on barley or wheat.     

Production of culmorin compounds and other secondary metabolites by Fusarium culmorum and F. graminearum strains isolated from Norwegian cereals

Twenty-three Fusarium culmorum and 21 F. graminearumisolates were studied for their ability to produce mycotoxins and other secondary metabolites. The strains were cultivated on rice, and the extracts analysed by gas chromatography mass spectrometry (GC-MS) after derivatization with pentafluoropropionic (PFP) reagent. Two F. culmorum strains formed nivalenol and its acetylated derivatives (chemotype II), while all F. graminearum and the otherF. culmorum isolates produced deoxynivalenol (DON) via 3-acetyldeoxynivalenol (3-acetyl-DON) (chemotype IA). 15-hydroxy-culmorin, followed by 5-hydroxy-culmorin were the main other metabolites produced F. culmorum, while 5-, 12- and an unidentified hydroxy-culmorin, suggested to be 14-hydroxy-culmorin, were the main metabolites of F. graminearum. The hydroxy-culmorin profile was found to be significantly different for the two Fusarium species. Minor amounts of about ten other hydroxy-culmorins, four hydroxy-culmorones and 3,13-dihydroxy-epiapotrichothecene were also detected in most cultures. Traces of sambucinol seemed to be present in some of the isolates, but were not detected in any significant amounts. The precursors in the biosynthetic sequence to 3-acetyldeoxynivalenol,7,8-dihydroxycalonectrin and 15-deacetyl-7,8-dihydroxycalonectrin,were detected in most cultures. We also report the assignment of both the ¹H and¹³C NMR data of 15-deacetyl-7,8-dihydroxycalonectrin, which has only been reported incorrectly before. This revised version was published online in August 2006 with corrections to the Cover Date.     

Expression of Resistance of Wheat to Fusarium graminearum and F. culmorum Under Various Experimental Conditions

The resistance ranking was very similar under different periods of incubation except the 17 and 24 h variants under plastic bags and with the presence or absence of Bayleton treatment. Very similar ranking was recorded also in the tests where four plot replicates were used, e.g. the reaction severity was so close in different plot replicates that one plot per genotype seemed to be enough for serial resistance tests. The significant plot × genotype interaction shows that relative resistance ranking may vary to some extent, but basic changes do not occur. Also similar cultivar reactions were observed at different levels of disease severity resulting from inoculation with isolates differing in pathogenicity. An average reaction to these levels is closer to the genetically determined resistance. Analysis of the behaviour of a cultivar at different disease severity levels helps to predict the field behaviour of a given genotype. The Bayleton treatment was ineffective against scab, although there was some interaction with cultivars.     

PCR detection assays for the trichothecene-producing species Fusarium graminearum, Fusarium culmorum, Fusarium poae, Fusarium equiseti and Fusarium sporotrichioides

Contamination of small-grain cereals with the fungal species Fusarium graminearum, F. culmorum, F. poae, F. sporotrichioides and F. equiseti is an important source of trichothecenes, Zearalenone and other mycotoxins which cause serious diseases in human and animals. Additionally, these species contribute to Fusarium Head Blight, a disease which produces important losses in cereal yield. Early detection and control of these Fusarium species is crucial to prevent toxins entering the food chain and a useful tool in disease management practices. We describe the development of specific PCR assays to F. graminearum, F. culmorum, F. poae, F. sporotrichioides and F. equiseti using DNA from pure fungal cultures as well as from naturally infected wheat seeds, using in this case a rapid and easy protocol for DNA isolation. The specific primers were designed on the basis of IGS sequences (Intergenic Spacer of rDNA), a multicopy region in the genome that permits to enhance the sensitivity of the assay in comparison with PCR assays based on single-copy sequences.     

Comparison of environmental profiles for growth and deoxynivalenol production by Fusarium culmorum and F. graminearum on wheat grain

AIMS: Comparisons were made of the effect of water activity (a(w) 0.99-0.85), temperature (15 and 25 degrees C) and time (40 days) on growth/production of the trichothecene mycotoxin deoxynivalenol (DON) by Fusarium culmorum and Fusarium graminearum on wheat grain. METHODS AND RESULTS: Studies examined colonization of layers of wheat grain for 40 days. Fusarium culmorum grew optimally at 0.98 a(w) and minimally at 0.90 a(w) at 15 and 25 degrees C. Colonization by F. graminearum was optimum at 0.99 a(w) at 25 and 0.98 a(w) at 15 degrees C. Overall, temperature, a(w) and their interactions significantly affected growth of both species. Production of DON occurred over a much narrower range (0.995-0.96 a(w)) than that for growth. Optimum DON was produced at 0.97 and 0.99 a(w) at 15 and 25 degrees C, respectively, by F. culmorum, and at 0.99 a(w) and 15 degrees C and 0.98 a(w) at 25 degrees C for F. graminearum. Statistically, one-, two- and three-way interactions were significant for DON production by both species. CONCLUSIONS: This suggests that the ecological requirements for growth and mycotoxin production by such species differ considerably. The two-dimensional profiles on grain for DON production by these two species have not been examined in detail before. SIGNIFICANCE AND IMPACT OF THE STUDY: This type of information is essential for developing climate-based risk models for determining the potential for contamination of cereal grain with this trichothecene mycotoxin. It will also be useful information for monitoring critical control points in prevention of such toxins entering the wheat production chain.     

Population Genetics of Three Important Head Blight Pathogens Fusarium graminearum, F. pseudograminearum and F. culmorum

Homothallic Fusarium graminearum (teleomorph Gibberella zeae) and anamorphic F. culmorum are destructive pathogens causing Fusarium head blight (FHB) of small‐grain cereals worldwide, while heterothallic F. pseudograminearum (G. coronicola) seems to be restricted to Australia as a FHB pathogen. In a comprehensive treatise of pathogen population genetics, this review summarizes global knowledge of genetic diversity among isolates sampled at various spatial and temporal scales, examines the mechanisms that generate this diversity and explores the implications of pathogen diversity and plasticity to resistance breeding. Despite their different modes of reproduction, there is large variation among isolates of all three species originating from different countries and continents. With a few exceptions, haplotype diversity ranges from 60 to 100% even within populations from individual fields. In F. graminearum, over 90% of the variation is found within populations, even when samples are collected from areas as small as 0.25 m². Variation among populations is low (4–8%) with negligible population subdivision. This indicates a high level of gene flow (N_m = 8–71) with linkage equilibrium for the majority of selectively neutral molecular marker loci analysed. These findings for F. graminearum point to large random mating populations driven by occasional outcrossing, high gene flow across large geographical distances and a relatively low host‐mediated directional selection. Similar conclusions can be drawn for the Canadian population of F. pseudograminearum, but not for populations from Australia, where different pathogen ecology may have reduced the frequency of sexual recombination. Phylogenetic analyses indicate delineation of lineages in F. graminearum, often along geographically separated lines, while the related F. pseudograminearum is a single recombining species with limited or no lineage development. The anamorphic F. culmorum shows no obvious clonal structure in its population as might have been expected. High levels of diversity within fields may have been caused by balancing selection from frequent alternation between saprophytic and parasitical life cycle and/or a hidden or recently extinct teleomorph. Other mechanisms including parasexual cycles or active transposable elements may also be involved but these have not been investigated as yet. Crosses between and among F. graminearum lineages have shown a rather simple, additive inheritance of pathogenicity and aggressiveness with frequent transgressive segregation in crosses among isolates with moderate aggressiveness. This raises the spectre of highly aggressive and/or toxigenic isolates evolving if a limited range of quantitative trait locus for FHB resistance is deployed on a large scale. Combining more than one genetically distinct sources of resistance, possibly with different modes of action against the pathogen, will be necessary to avoid severe FHB outbreaks in the future.     

Competitive Aggressiveness in Binary Mixtures of Fusarium graminearum and F. culmorum Isolates Inoculated on Spring Wheat with Highly Effective Resistance QTL

Fusarium head blight (FHB) caused by Fusarium graminearum and F. culmorum is a devastating disease with high effects on grain yield and quality. We developed spring wheat lines incorporating the highly effective FHB resistance quantitative trait loci (QTL) Fhb1 and Qfhs.ifa‐5A. Whether these QTL lead to competition within Fusarium populations in the field resulting in isolates with higher aggressiveness has not been analysed. The aims of this study were to determine (i) the aggressiveness potential of F. graminearum and F. culmorum isolates, (ii) competition effects of these isolates in binary mixtures and (iii) the stability of resistant hosts. Six F. graminearum, two F. culmorum isolates and seven binary mixtures containing these isolates were tested for their aggressiveness and mycotoxin production at two locations in South Germany in 2007 and 2008. Host lines were four spring wheat lines containing the resistance QTL Fhb1 and/or Qfhs.ifa‐5A or none of them and one standard variety. Re‐isolates were sampled from plots inoculated with the binary mixtures to identify the percentage of each isolate in the mixture by simple sequence repeat markers. Resistant host lines reacted as expected and had a high stability to all isolates and mixtures. Only less important host × mixture interactions were detected. Aggressiveness among isolates and mixtures was significantly different. Type and amount of mycotoxin and high single isolate aggressiveness were not necessarily advantageous in the mixture. However, both F. culmorum isolates outcompeted F. graminearum isolates. Significant deviations from the inoculated 1 : 1 proportions occurred in 34 of 49 cases, illustrating that competition effects appeared in the mixtures. These differences depended mainly on the year and not on the level of host resistance. We conclude that resistance should not be affected by the Fusarium isolates and mixtures.     

Contamination of malt barley and wheat by Fusarium graminearum and Fusarium culmorum from the crop years 2001-2003 in eastern Croatia

This study investigated infection levels with Fusarium graminearum and Fusarium culmorum in malt barley and wheat in eastern Croatia. The contamination was surveyed over three consecutive crop years (2001–2003) on five locations for barley and three wheat cultivating locations. F. graminearum loads reached levels of potentially serious threat for the commercial production of malting raw materials in both cereals (up to 29.1%). On the other hand, the mean percentage of kernels infected with F. culmorum was low to medium (up to 6.1%). The fungal invasions for years and locations were affected by meteorologic and other environmental factors and the pattern seemed to be consistent with species-specific optimal conditions reported by other authors.     

Arabidopsis is susceptible to the cereal ear blight fungal pathogens Fusarium graminearum and Fusarium culmorum

The fungal pathogens Fusarium graminearum and F. culmorum cause ear blight disease on cereal crops worldwide. The disease lowers both grain quality and grain safety. Disease prevalence is increasing due to changes in cropping practices and the difficulties encountered by plant breeders when trying to introgress the polygene-based resistance. The molecular basis of resistance to Fusarium ear blight in cereal species is poorly understood. This is primarily due to the large size of cereal genomes and the expensive resources required to undertake gene function studies in cereals. We therefore explored the possibility of developing various model floral infection systems that would be more amenable to experimental manipulation and high-throughput gene function studies. The floral tissues of tobacco, tomato, soybean and Arabidopsis were inoculated with Fusarium conidia and this resulted in disease symptoms on anthers, anther filaments and petals in each plant species. However, only in Arabidopsis did this initial infection then spread into the developing siliques and seeds. A survey of 236 Arabidopsis ecotypes failed to identify a single genotype that was extremely resistant or susceptible to Fusarium floral infections. Three Arabidopsis floral mutants that failed to develop anthers and/or functional pollen (i.e. agamous-1, apetala1-3 and dad1) were significantly less susceptible to Fusarium floral infection than wild type. Deoxynivalenol (DON) mycotoxin production was also detected in Fusarium-infected flowers at >1 ppm. This novel floral pathosystem for Arabidopsis appears to be highly representative of a serious cereal crop disease.     

Formation of moniliformin by Fusarium sporotrichioides and Fusarium culmorum.

Two strains of Fusarium sporotrichioides and one strain of F. culmorum were shown to produce the mycotoxin moniliformin in rice culture. Identification was by reverse-phase liquid chromatography, thin-layer chromatography, and mass spectrometry.     

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.     

Formation of moniliformin by Fusarium sporotrichioides and Fusarium culmorum

Two strains of Fusarium sporotrichioides and one strain of F. culmorum were shown to produce the mycotoxin moniliformin in rice culture. Identification was by reverse-phase liquid chromatography, thin-layer chromatography, and mass spectrometry.     

Molecular Genetic Diversity and Variation for Aggressiveness in Populations of Fusarium graminearum and Fusarium culmorum Sampled from Wheat Fields in Different Countries

Fusarium graminearum and Fusarium culmorum are the major pathogenic organisms causing head blight in small-grain cereals. Natural epidemics may result in severe yield losses, reduction in quality, and contamination of the grain by mycotoxins. The genetic diversity of four field populations of F. graminearum from Germany, Hungary, and Canada, and one population of F. culmorum from Russia was investigated by polymerase chain reaction (PCR)-based fingerprinting. Additionally, a world-wide collection and two of the F. graminearum populations were analysed for their aggressiveness on young plants of winter rye in the greenhouse. The number of isolates analysed per population varied from 25 to 70. Significant quantitative variation for aggressiveness was observed within each of the individual field populations amounting to the same range as the world-wide collection. Abundant variation within populations was also revealed by DNA markers. The F. graminearum populations from Hungary and Winnipeg displayed the least genotypic diversity, the two German F. graminearum populations and the Russian F. culmorum population were highly diverse. Population diversity, however, followed no spatial pattern among samples within a German field for aggressiveness or molecular markers. For F. graminearum , sexual recombination is the most likely explanation for the large genetic diversity within field populations. Asexual and/or parasexual recombination, and balancing selection caused by the periodic alternation between the saprophytic and parasitic phase might play an additional role and account for the variation within the F. culmorum population. For improving Fusarium resistance, several resistance genes of different sources should be combined to avoid an unspecific adaptation of the genetically variable pathogen to an increased resistance level.     

Assessing non-specificity of resistance in wheat to head blight caused by inoculation with European strains of Fusarium culmorum,F. graminearum and F. nivale using a multiplicative model for interaction

To determine whether resistance to Fusarium head blight in winter wheat is horizontal and non-species specific, 25 genotypes from five European countries were tested at six locations across Europe in the years 1990, 1991, and 1992. The five genotypes from each country had to cover the range from resistant to susceptible. The locations involved were Wageningen, Vienna, Rennes, Hohenheim, Oberer Lindenhof, and Szeged. In total, 17 local strains of Fusarium culmorum, F. graminearum, and F. nivale were used for experimental inoculation. One strain, F. culmorum IPO 39-01, was used at all locations. Best linear unbiased predictions (BLUPs) for the head blight ratings of the genotypes were formed within each particular location for each combination of year and strain. The BLUPs over all locations were collected in a genotype-by environment table in which the genotypic dimension consisted of the 25 genotypes, while the environmental dimension was made up of 59 year-by-strain-by-location combinations. A multiplicative model was fitted to the genotype by-environment interaction in this table. The inverses of the variances of the genotype-by-environment BLUPs were used as weights. Interactions between genotypes and environments were written as sums of products between genotypic scores and environmental scores. After correction for year-by-location influence very little variation in environmental scores could be ascribed to differences between strains. This provided the basis for the conclusion that the resistance to Fusarium head blight in winter wheat was of the horizontal and non-species specific type. There was no indication for any geographical pattern in virulence genes. Any reasonable aggressive strain, a F. culmorum strain for the cool climates and a F. graminearum strain for the warmer humid areas, should be satisfactory for screening purposes.     

Identification by PCR of Fusarium culmorum Strains Producing Large and Small Amounts of Deoxynivalenol

Thirty deoxynivalenol-producing F. culmorum strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13 F. culmorum strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (tri5) was amplified and sequenced. According to the tri5 data, the F. culmorum strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing F. culmorum strains, the tri5-tri6 intergenic region was then sequenced, which confirmed the two separate clusters within the F. culmorum strains. According to the tri5-tri6 sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing F. culmorum strains.     

Identification by PCR of Fusarium culmorum strains producing large and small amounts of deoxynivalenol

Thirty deoxynivalenol-producing F. culmorum strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13 F. culmorum strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (tri5) was amplified and sequenced. According to the tri5 data, the F. culmorum strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing F. culmorum strains, the tri5-tri6 intergenic region was then sequenced, which confirmed the two separate clusters within the F. culmorum strains. According to the tri5-tri6 sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing F. culmorum strains.     

PCR Analysis of the Tri13 gene to Determine the Genetic Potential of Fusarium graminearum Isolates from Iran to Produce Nivalenol and Deoxynivalenol

Fusarium graminearum trichothecene producing isolates can be broadly divided into two chemotypes based on the production of the 8- ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV). Functional Tri13 gene required for the production of NIV and 4- acetyl NIV, whereas in the isolates producing DON and its acetylated derivates, this gene is nonfunctional. In this study, a total of 57 isolates from different fields of Mazandaran province, Iran were identified as F. graminearum using classical methods and species specific primers. In order to assess the potential of isolates to produce NIV or DON, we used PCR to determine whether isolates carried a functional or nonfunctional Tri13 gene. Out of the 57 tested F. graminearum isolates with Tri13 PCR assays, 46 yielded an amplicon similar to the size predicted for nivalenol production, while 11 yielded an amplicon similar to the size predicted for deoxynivalenol production. From regions where more than one F. graminearum isolate was obtained, isolates were not exclusively of a single chemotype. It seems that genetic diversity among the isolates has relation with geographical region and wheat cultivar. The assay can provide information about the distribution of Tri13 haplotype that can be used in tracing of trichothecene contaminated samples.