Comparative pathogenicity of Fusarium graminearum isolates from China revealed by wheat coleoptile and floret inoculations

Fusarium head blight (FHB) or scab caused by Fusarium species is an economically important disease on small grain cereal crops worldwide. Accurate assessments of the pathogenicity of fungal isolates is a key obstacle toward a better understanding of the Fusarium-wheat scab system. In this study, a new laboratory method for inoculation of wheat coleoptiles was developed, which consists of cutting off the coleoptile apex, covering the cut apex with a piece of filter paper soaked in conidial suspension, and measuring the lengths of brown lesions 7 days post inoculation. After coleoptile inoculation, distinct brown lesions in the diseased stems were observed, in which the presence of the fungus was verified by PCR amplification with F.␣graminearum Schwable-specific primers. Coleoptile inoculation of six wheat varieties indicated that a highly susceptible wheat variety was more suitable as a differentiating host for the pathogenicity assay. Analysis of the coleoptiles inoculated with a set of 58 different isolates of F. graminearum showed a significant difference in the lengths of the lesions, forming the basis by which pathogenicity of the isolates was assessed. Field inoculation of florets of three wheat varieties over 2 years revealed significant differences in pathogenicity among the 58 isolates, and that the highly resistant and highly susceptible wheat varieties were more appropriate and stable for pathogenicity assessment in field trials. Comparative analyses of eight inoculation experiments of wheat with 58 F. graminearum isolates showed significant direct linear correlations (P<0.001) between coleoptile and floret inoculations. These results indicate that the wheat coleoptile inoculation is a simple, rapid and reliable method for pathogenicity studies of F.␣graminearum in wheat.     

Pathogenicity of Fusarium graminearum Schwabe isolates from Poland towards seedlings of cereal species

Ten isolates ofFusarium graminearum Schwabe originating from diseased cereal plants and kernels were tested for pathogenicity to various cultivars of wheat, rye, triticale and oats. The isolates varied greatly in their pathogenicity to the seedlings of the species, and were most pathogenic to rye and triticale, less pathogenic to barley and wheat and least pathogenic to oats.     

Functional analysis of the kinome of the wheat scab fungus Fusarium graminearum

As in other eukaryotes, protein kinases play major regulatory roles in filamentous fungi. Although the genomes of many plant pathogenic fungi have been sequenced, systematic characterization of their kinomes has not been reported. The wheat scab fungus Fusarium graminearum has 116 protein kinases (PK) genes. Although twenty of them appeared to be essential, we generated deletion mutants for the other 96 PK genes, including 12 orthologs of essential genes in yeast. All of the PK mutants were assayed for changes in 17 phenotypes, including growth, conidiation, pathogenesis, stress responses, and sexual reproduction. Overall, deletion of 64 PK genes resulted in at least one of the phenotypes examined, including three mutants blocked in conidiation and five mutants with increased tolerance to hyperosmotic stress. In total, 42 PK mutants were significantly reduced in virulence or non-pathogenic, including mutants deleted of key components of the cAMP signaling and three MAPK pathways. A number of these PK genes, including Fg03146 and Fg04770 that are unique to filamentous fungi, are dispensable for hyphal growth and likely encode novel fungal virulence factors. Ascospores play a critical role in the initiation of wheat scab. Twenty-six PK mutants were blocked in perithecia formation or aborted in ascosporogenesis. Additional 19 mutants were defective in ascospore release or morphology. Interestingly, F. graminearum contains two aurora kinase genes with distinct functions, which has not been reported in fungi. In addition, we used the interlog approach to predict the PK-PK and PK-protein interaction networks of F. graminearum. Several predicted interactions were verified with yeast two-hybrid or co-immunoprecipitation assays. To our knowledge, this is the first functional characterization of the kinome in plant pathogenic fungi. Protein kinase genes important for various aspects of growth, developmental, and infection processes in F. graminearum were identified in this study.     

Analysis of the Fusarium graminearum species complex from wheat, barley and maize in South Africa provides evidence of species-specific differences in host preference

Species identity and trichothecene toxin potential of 560 members of the Fusarium graminearum species complex (FGSC) collected from diseased wheat, barley and maize in South Africa was determined using a microsphere-based multilocus genotyping assay. Although three trichothecene types (3-ADON, 15-ADON and NIV) were represented among these isolates, strains with the 15-ADON type predominated on all three hosts. A significant difference, however, was identified in the composition of FGSC pathogens associated with Gibberella ear rot (GER) of maize as compared to Fusarium head blight (FHB) of wheat or barley (P<0.001). F. graminearum accounted for more than 85% of the FGSC isolates associated with FHB of wheat and barley (N=425), and was also the dominant species among isolates from maize roots (N=35). However, with the exception of a single isolate identified as an interspecific hybrid between Fusariumboothii and F. graminearum, GER of maize (N=100) was exclusively associated with F. boothii. The predominance of F. graminearum among FHB isolates, and the near exclusivity of F. boothii among GER isolates, was observed across all cultivars, collection dates, and provinces sampled. Because these results suggest a difference in host preference among species of the FGSC, we hypothesize that F. graminearum may be less well adapted to infect maize ears than other members of the FGSC.     

Phylogenetic diversity of Fusarium incarnatum-equiseti species complex isolated from Spanish wheat

Wheat is the most important cereal grown in the European Union and Spain is its fifth largest wheat producer. There is little information about Fusarium species associated with wheat in Spain. Phylogenetic diversity of 51 strains belonging to Fusarium incarnatum-equiseti species complex (FIESC) isolated from Spanish wheat was investigated using partial sequences of the translation elongation factor gene (EF-1α). Maximum-parsimony and Bayesian analysis of aligned DNA sequences resolved 18 haplotypes and 7 phylogenetic species. Strains morphologically identified as F. equiseti belonged to two different phylogenetic species, FIESC-5 and FIESC-14. Some correlation between phylogenetic species and geographical region was found. The present results highlight the potential contribution of FIESC to the mycotoxin contamination of Spanish wheat.     

Quantification of Fusarium graminearum in infected wheat by species specific real-time PCR applying a TaqMan Probe

A new real-time PCR based method was developed for the species-specific detection, identification and quantification of Fusarium graminearum in planta. It utilizes a TaqMan hybridisation probe targeting the beta-tubulin gene and a plasmid standard. The assay is highly specific giving no product with DNA of closely related species. It is very sensitive, detecting down to five gene copies per reaction, and is able to produce reliable quantitative data over a range of six orders of magnitude.     

Differential proteomic analysis of proteins in wheat spikes induced by Fusarium graminearum

Scab, caused by Fusarium graminearum, is a serious spike disease in wheat. To identify proteins in resistant wheat cultivar Wangshuibai induced by F. graminearum infection, proteins extracted from spikes 6, 12 and 24 h after inoculation were separated by 2-DE. Thirty protein spots showing 3-fold change in abundance when compared with treatment without inoculation were characterized by MALDI-TOF MS and matched to proteins by querying the mass spectra in protein databases or the Triticeae EST translation database. Based on their volume profiles, these proteins were classified into four categories. The first one fell off rapidly at the initial inoculation and then rose at 12 or 24 hai, the second one decreased considerably after inoculation and remained at low level, the third one rose at the initial inoculation and then declined at 12 or 24 hai, the forth one showed steady increase after inoculation and maintained at a high level. Many of the proteins identified in the first two categories are related to carbon metabolism and photosynthesis. While most of proteins identified in the last two categories are related to stress defense of plants, indicating that proteins associated with the defense reactions were activated or translated shortly after inoculation.     

Fusarium sinensis sp. nov., a new species from wheat in China

Two strains of Fusarium sinensis sp. nov. were isolated from the seeds and roots of wheat (Triticum aestivum L.) in Shandong Province, China. They are superficially similar to F. dlaminii in producing fusiform-to-reniform and napiform microconidia and chlamydospores in almost the same size range as F. dlaminii. However the colonies and chlamydospores of F. sinensis resemble those of species in sections Roseum, Gibbosum and Discolor. Phylogenetic analysis of partial translation elongation factor 1alpha (EF-1alpha) sequence data indicates that F. sinensis is closely related to but distinct from the F. avenaceum/F. tricinctum/F. acuminatum species complex.     

Multilocus genotyping and molecular phylogenetics resolve a novel head blight pathogen within the Fusarium graminearum species complex from Ethiopia

A survey of Fusarium head blight (FHB)-contaminated wheat in Ethiopia recovered 31 isolates resembling members of the Fusarium graminearum species complex. Results of a multilocus genotyping (MLGT) assay for FHB species and trichothecene chemotype determination suggested that 22 of these isolates might represent a new species within the Fg complex. Phylogenetic analyses of multilocus DNA sequence data resolved the 22 Ethiopian isolates as a novel, phylogenetically distinct species. The new species also appears to be novel in that MLGT probe data and sequence analysis of both ends of the TRI-cluster identified 15ADON and NIV recombination blocks, documenting inter-chemotype recombination involving the chemotype-determining genes near the ends of the TRI-cluster. Results of pathogenicity experiments and analyses of trichothecene mycotoxins demonstrated that this novel Fg complex species could induce FHB on wheat and elaborate 15ADON in planta. Herein the FHB pathogen from Ethiopia is formally described as a novel species.     

An adaptive evolutionary shift in Fusarium head blight pathogen populations is driving the rapid spread of more toxigenic Fusarium graminearum in North America

Analysis of Fusarium head blight (FHB) pathogen diversity revealed that 3ADON producing Fusarium graminearum are prevalent in North America and identified significant population structure associated with trichothecene chemotype differences (F(ST)>0.285; P<0.001). In addition, we identified a trichothecene chemotype cline in Canada and documented a recent and significant shift in FHB pathogen composition by demonstrating that the 3ADON chemotype frequency in western Canada increased more than 14-fold between 1998 and 2004. On average, isolates from 3ADON populations produced significantly (P<0.05) more trichothecene and had significantly (P<0.005) higher fecundity and growth rates than isolates from the 15ADON population. These results indicate that selection is driving the rapid spread of an introduced pathogen population that is more toxigenic and potentially more vigorous. The discovery of this previously unrecognized pathogen diversity has significant implications for food safety and cereal production in North America.     

The transcriptome of Fusarium graminearum during the infection of wheat

Fusarium graminearum causes head blight disease in wheat and barley. To help understand the infection process on wheat, we studied global gene expression of F. graminearum in a time series from 24 to 196 h after inoculation, compared with a noninoculated control. The infection was rapid and, after 48 h, over 4,000 fungal genes were expressed. The number of genes expressed increased over time up to 96 h (>8,000 genes), and then declined at the 144- and 192-h post-inoculation time points. After subtraction of genes found expressed on complete medium, during carbon or nitrogen starvation, and on barley, only 355 were found exclusively expressed in wheat, mostly genes with unknown function (72.6%). These genes were mainly found in single-nucleotide polymorphism-enriched islands on the chromosomes, suggesting a higher evolutionary selection pressure. The annotated genes were enriched in functional groups predicted to be involved in allantoin and allantoate transport, detoxification, nitrogen, sulfur and selenium metabolism, secondary metabolism, carbohydrate metabolism, and degradation of polysaccharides and ester compounds. Several putative secreted virulence factors were also found expressed in wheat.     

Baseline sensitivity of Fusarium graminearum from wheat fields in Henan,China, to metconazole and analysis of cross resistance with carbendazim and phenamacril

The filamentous plant pathogenic fungus Fusarium graminearum is one of the most important pathogens causing Fusarium head blight (FHB) in wheat in the Henan Province of China. Metconazole is among the demethylation inhibitor (DMI) fungicides with a higher inhibitory activity on the mycelial growth of F. graminearum. In 2016 and 2017, 119 single spore isolates of F. graminearum, prior to being exposed to metconazole, were recovered from 52 wheat fields near 11 cities in Henan Province. The inhibitory activity of metconazole on the mycelia of the Henan F. graminearum population was determined, and EC₅₀ values were calculated. The range of EC₅₀ values of the Henan F. graminearum population to metconazole was 0.0103 to 0.0775 μg/ml with an average EC₅₀ value of 0.0293 ± 0.0114 μg/ml. The sensitivity frequency distribution curve presented a single peak in a narrow range. No cross-resistance was found between the DMI fungicide metconazole and the benzimidazole fungicide carbendazim or the cyanoacrylate fungicide phenamacril. Therefore, these sensitivity data could be used as the baseline of F. graminearum susceptibility to metconazole in the Henan Province and provide the basis for monitoring metconazole resistance in this area.     

An adaptive evolutionary shift in Fusarium head blight pathogen populations is driving the rapid spread of more toxigenic Fusarium graminearum in North America.

Analysis of Fusarium head blight (FHB) pathogen diversity revealed that 3ADON producing Fusarium graminearum are prevalent in North America and identified significant population structure associated with trichothecene chemotype differences (F(ST)>0.285; P<0.001). In addition, we identified a trichothecene chemotype cline in Canada and documented a recent and significant shift in FHB pathogen composition by demonstrating that the 3ADON chemotype frequency in western Canada increased more than 14-fold between 1998 and 2004. On average, isolates from 3ADON populations produced significantly (P<0.05) more trichothecene and had significantly (P<0.005) higher fecundity and growth rates than isolates from the 15ADON population. These results indicate that selection is driving the rapid spread of an introduced pathogen population that is more toxigenic and potentially more vigorous. The discovery of this previously unrecognized pathogen diversity has significant implications for food safety and cereal production in North America.     

A real-time qPCR assay to quantify Fusarium graminearum biomass in wheat kernels

Aims: To develop a real‐time PCR assay to quantify Fusarium graminearum biomass in blighted wheat kernels. Methods and Results: Primers designed to amplify a gene in the trichothecene biosynthetic cluster (TRI6) were evaluated for sensitivity and specificity. Primer pair Tri6_10F/Tri6_4R specifically and consistently amplified a 245‐bp DNA fragment from F. graminearum. A workflow was developed and validated to extract DNA from infested grain. The assay detected as little as 10 μg of F. graminearum mycelia in 1 g of ground wheat grain with a high correlation between fungal biomass and cycle threshold values (R² = 0·9912; P = 0·004). In field‐inoculated grain, qPCR measurements of biomass correlated closely with deoxynivalenol levels (R = 0·82, P < 0·0001) and two visual techniques to assess grain quality (R = 0·88, P < 0·0001 and R = 0·81, P < 0·0001). Conclusions: The qPCR assay provided accurate and precise assessments of the amount of F. graminearum biomass in blighted wheat kernels. This method represents a technical advance over other approaches to quantify kernel colonization and real‐time PCR detection methodologies for F. graminearum that do not correlate quantification of fungal genomic DNA to biomass. Significance and Impact of the Study: Quantifying F. graminearum biomass, especially low levels of growth associated with kernels that are visually asymptomatic, represents a new approach to screen for resistance to kernel infection, an understudied yet potentially important avenue to reduce the impact of head blight.     

Rapid identification of Fusarium graminearum species complex using Rolling Circle Amplification (RCA)

Rolling Circle Amplification (RCA) of DNA is a sensitive and cost effective method for the rapid identification of pathogenic fungi without the need for sequencing. Amplification products can be visualized on 1% agarose gel to verify the specificity of probe-template binding or directly by adding fluorescent dyes. Fusarium Head Blight (FHB) is currently the world's largest threat to the production of cereal crops with the production of a range of mycotoxins as an additional risk. We designed sets of RCA padlock probes based on polymorphisms in the elongation factor 1-α (EF-1α) gene to detect the dominant FHB species, comprising lineages of the Fusarium graminearum species complex (FGSC). The method also enabled the identification of species of the Fusarium oxysporum (FOSC), the Fusarium incarnatum-equiseti (FIESC), and the Fusarium tricinctum (FTSC) species complexes, and used strains from the CBS culture collection as reference. Subsequently probes were applied to characterize isolates from wheat and wild grasses, and inoculated wheat kernels. The RCA assays successfully amplified DNA of the target fungi, both in environmental samples and in the contaminated wheat samples, while no cross reactivity was observed with uncontaminated wheat or related Fusarium species. As RCA does not require expensive instrumentation, the technique has a good potential for local and point of care screening for toxigenic Fusarium species in cereals.