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BackgroundThe toxigenic fungal plant pathogen Fusarium graminearum compromises wheat production worldwide. Azole fungicides play a prominent role in controlling this pathogen. Sequencing of its genome stimulated the development of high-throughput technologies to study mechanisms of coping with fungicide stress and adaptation to fungicides at a previously unprecedented precision. DNA-microarrays have been used to analyze genome-wide gene expression patterns and uncovered complex transcriptional responses. A recently developed one-color multiplex array format allowed flexible, effective, and parallel examinations of eight RNA samples.ResultsWe took advantage of the 8 × 15 k Agilent format to design, evaluate, and apply a novel microarray covering the whole F. graminearum genome to analyze transcriptional responses to azole fungicide treatment. Comparative statistical analysis of expression profiles uncovered 1058 genes that were significantly differentially expressed after azole-treatment. Quantitative RT-PCR analysis for 31 selected genes indicated high conformity to results from the microarray hybridization. Among the 596 genes with significantly increased transcript levels, analyses using GeneOntology and FunCat annotations detected the ergosterol-biosynthesis pathway genes as the category most significantly responding, confirming the mode-of-action of azole fungicides. Cyp51A, which is one of the three F. graminearum paralogs of Cyp51 encoding the target of azoles, was the most consistently differentially expressed gene of the entire study. A molecular phylogeny analyzing the relationships of the three CYP51 proteins in the context of 38 fungal genomes belonging to the Pezizomycotina indicated that CYP51C (FGSG_11024) groups with a new clade of CYP51 proteins. The transcriptional profiles for genes encoding ABC transporters and transcription factors suggested several involved in mechanisms alleviating the impact of the fungicide. Comparative analyses with published microarray experiments obtained from two different nutritional stress conditions identified subsets of genes responding to different types of stress. Some of the genes that responded only to tebuconazole treatment appeared to be unique to the F. graminearum genome.ConclusionsThe novel F. graminearum 8 × 15 k microarray is a reliable and efficient high-throughput tool for genome-wide expression profiling experiments in fungicide research, and beyond, as shown by our data obtained for azole responses. The array data contribute to understanding mechanisms of fungicide resistance and allow identifying fungicide targets.

Azole fungicides play a prominent role for reliable plant disease management. However, quantitative azole resistance has been shown to develop in fungal pathogens, including Fusarium graminearum, the causal agent of Fusarium head blight (FHB). Due to widespread application of azole fungicides, resistance may accumulate to higher degrees in fungal field populations over time. Although azole fungicides are prominent components in FHB control, little effort has been made to investigate azole resistance in F. graminearum. We allowed F. graminearum strain NRRL 13383 to adapt to an azole fungicide in vitro, applying a strongly growth-reducing but sublethal dose of tebuconazole. Two morphologically distinguishable azole-resistant phenotypes were recovered that differed with regard to levels of fitness, fungicide resistance, virulence, and mycotoxin production. Isolates of the adapted "phenotype 1" exhibited azole-specific cross-resistance, whereas "phenotype 2" isolates displayed the phenomenon of multidrug resistance because the sensitivity to amine fungicides was also affected. Assessment of individual infected spikelets for mycotoxin contents by high-performance liquid chromatography mass spectrometry and for Fusarium DNA by quantitative polymerase chain reaction indicated that some of the adapted isolates produced significantly higher levels of nivalenol per fungal biomass than the NRRL 13383 strain.

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BackgroundPenetration attempts of the hemibiotroph Colletotrichum graminicola may activate PAMP-triggered immunity (PTI) on different cultivars of Zea mays to different extent. However, in most events, this does not prevent the establishment of a compatible pathogenic interaction. In this study, we investigate the extent to which the host variety influences PTI. Furthermore, we assess whether visual disease symptoms occurring on different maize varieties reliably reflect fungal biomass development in planta as determined by qPCR and GFP tracing.ResultsEmploying a set of four maize varieties, which were selected from a panel of 27 varieties, for in-depth assessment of pathogenesis of the wild type strain of C. graminicola, revealed considerable differences in susceptibility as evidenced by symptom severity that decreased from variety Golden Jubilee to Mikado to Farmtop to B73. However, a newly developed qPCR assay and microscopical observation of a GFP-labelled strain showed that disease symptoms are in some instances inconsistent when compared with other indicators of susceptibility. Of the four varieties assessed, either Golden Jubilee, Mikado and B73, or Golden Jubilee, Farmtop and B73 showed a direct correlation between symptom and fungal biomass development. In a pairwise comparison, however, Mikado and Farmtop showed an inverse correlation for these features.ConclusionsThe genotype of maize contributes to the severity of symptoms resulting from an infection with C. graminicola. Partially, this may be attributed to the extent of PTI activated in different varieties, as reflected by papilla formation. Furthermore, when evaluating the susceptibility of a variety, it should be considered that symptom severity must not have to reflect the extent of fungal growth in the infected tissue.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0709-4) contains supplementary material, which is available to authorized users.