Molecular analysis of fungal pathogenicity in crown rot disease of wheat caused by Fusarium graminearum

Several Fusarium species can cause Fusarium head blight (FHB) and Fusarium crown rot (FCR) diseases in wheat and these are of economic importance in wheat production systems globally. Fusarium graminearum represents a model pathogen species to study these diseases because it has a sequenced genome, commercially available gene expression arrays and an growing collection of mutants impaired in pathogenicity and virulence, at least for FHB. FCR occurs at the stem base of the wheat plant, causing major reductions in grain yield. FCR has been much less intensively researched than FHB and the infection process of F. graminearum during crown rot disease in wheat has not been studied previously at the molecular level. Fungal biomass estimations by real-time quantitative PCR analysis of DNA from inoculated plants identified three distinct phases of infection during FCR, an initial increase in fungal mass in phase 1 up to 2 days post inoculation (dpi), then a reduction during phase 2 until 14 dpi followed by a large increase thereon in phase 3 that corresponded to symptom development. Histological characterisation of F. graminearum colonisation during these three phases of infection showed that initially the spores germinated on the stem surface at the point of inoculation forming a superficial hyphal mat. This occurred within the first two days of infection. The second phase was characterised by a period of low amounts of fungal tissue present in the infected plants and 14 days following infection hyphae were only observed below the point of inoculation at the stem base of the wheat seedling and had penetrated and colonised the adaxial epidermis of the outer leaf sheath. Following this, the third phase was characterised by a major colonisation of the internal tissues of the crown which corresponded to visible symptom development around 35 days after inoculation. Fungal gene expression during all three phases of infection were examined using the Affymetrix GeneChip system comprised of 22,000 F. graminearum gene probe sets. This analysis showed 1,839 genes were significantly up regulated in planta compared to axenic vegetative mycelia, including some known FHB virulence genes (e.g. those involved in the biosynthesis of trichothecene toxins). Fungal genes differentially regulated between the phases were identified indicating that FCR disease development requires a coordinated process involving distinct fungal gene expression programs. A bioinformatic comparison of global F. graminearum gene expression during FCR of wheat with published data for FHB of barley indicated similarities at very early stages of infection but divergence thereafter. It was decided to functionally test whether F. graminearum utilises the same virulence genes in FCR and FHB diseases. Because no virulence genes have been previously identified from FCR studies a small group of genes were initially selected from the FCR gene expression studies for further functional analysis using gene knock-out technology. Only two of these genes showed a changed phenotype during Fusarium infection of wheat plants and they encoded a probable ABC transporter (FgABC1) and a probable superoxide dismutase (FgSOD1). It was interesting to note that even though both FgABC1 and FgSOD1 exhibited similar transcription profiles during both FCR of wheat and FHB of barley it was found that FgABC1 was specifically required for full FCR disease development on the wheat cultivar Kennedy whereas FgSOD1 was specifically required for FHB disease on the same cultivar. This indicated that F. graminearum virulence genes can show specificity to the infection of different plant tissues and that these types of genes cannot be predicted based only on their transcription profiles. It is suggested that F. graminearum induces a global set of virulence factors but only some of these may be effective in particular tissues. To test further whether there was tissue specialisation for specific tissues and FCR & FHB diseases, a group of F. graminearum genes that were known virulence factors during FHB were tested to see if they were also virulence factors for FCR. This analysis showed that two genes displayed specificity only for FHB and five were virulence factors for both FHB and FCR. One of the genes that was a virulence factor for both diseases was the Tri5 gene that is necessary for the biosynthesis of trichothecene mycotoxins. This gene and these toxins did not appear to be necessary for symptom development and the induction of host defence responses but were necessary for fungal colonisation of the crown and stem in later stages of infection. Interestingly there were parallels in the role played by the Tri5 gene in FCR and that reported for FHB where it is necessary for colonisation for the spike. This study is the first molecular analysis of any Fusarium species during crown rot of wheat. Importantly, it shows that there may be specialisation towards host tissues for some virulence genes but also suggests that some factors may be non-specifically required for infection and it is these factors that will represent attractive targets for future control measures of both diseases