Wheat Root-dip Inoculation with Fusarium graminearum and Assessment of Root Rot Disease Severity

Background

The Fusarium root rot (FRR) bioassay uses root-dipping for inoculation in combination with different measurements of disease severity parameter. This protocol is principally also applicable to investigations of other root-fungus interactions. The presented root-dip inoculation proved to be an effective and reliable method to investigate wheat root-Fusariuminteractions (phenotypically and histologically) and to screen wheat genotypes for their response to root infection (Wang et al., 2015). Using the described protocol, genetic, molecular, and metabolomic aspects of the FRR disease have meanwhile been examined with reliable results in terms of biological repetitions and consistent observations across different research approaches. This corresponds with observations made in a study on the Verticillium wilt disease, which characterised root-dipping as superior to the pot immersion or soil infestation method in terms of effectiveness and reliability (Trapero et al., 2013). Growing wheat seedlings in F. graminearum contaminated (root zone) soil led to FRR-genotype responses similar to root dip inoculation (Wang et al., 2015), but the infection conditions are comparatively less controlled in terms of root specificity and time of infection. This might be a restriction for investigations that require time-based analyses. The use of Petri dishes to germinate and inoculate roots via mycelial agar plugs is a method that has been applied to F. culmorum infection in wheat seedlings (Beccari et al., 2011). However, in comparison to root dip inoculation, this method is not applicable to adult plant root infection, as was done in our lab to study plant age-related effects on FRR disease progress and wheat responses.

In the described protocol, disease severity can be assessed by percentage reductions of diseased root biomass, root and shoot length as well as by rates of visible root necrosis. FRR significantly inhibits root biomass production of wheat seedlings and adult plants (Wang et al., 2015), which can be measured by quantitative real-time PCR (qPCR). Disease severity and progress in terms of fungal growth can be monitored by measuring the relative amount of F. graminearum DNA in the host tissue by qPCR. This also enables detection and monitoring of infection during or in case of symptom-free disease periods. The F. graminearum spread into the lower stem internode is a crucial event as it initiates the colonisation of upper stem internodes, leaves, further tillers and even spikes (Wang et al., 2015) and can be readily evaluated by appearance time and rate of visual necrosis. For the FRR disease progress over time, a good agreement was found between the quantified relative F. graminearum biomass in roots and the measured impacts on seedling growth or the rated visible symptoms (Wang et al., 2015). Briefly, seedlings with the lowest level of F. graminearum accumulation measured displayed relative minor root necrosis and reductions in root biomass and length, while relatively moderate and maximum levels of pathogen accumulation each led to correspondingly moderated and maximum disease impacts and symptoms. Finally, Fusarium resistance is quantitative or partial. Therefore, the combination of classical, subjective tools such as symptom rating with the sensitive, non-subjective qPCR diagnosis of pathogen and/or root biomass proved to be advantageous, in terms of an improved assessment of disease dynamics and genotype performances.