Fusarium graminearum Maize Stalk Infection Assay and Associated Microscopic Observation Protocol

Background

Maize (Zea mays) is one of the most important crops across the world. The stalk is the main stem of a maize plant. It is composed of nodes and internodes (Figure 1). The internodes comprise rind and pith, and vascular tissues are scattered in the pith. Figure 1 shows the microscopic images of maize stalk sections under bright field or GFP fluorescence channel. This provides context for maize stalk inoculation and observation.

The ascomycete fungus Fusarium graminearum (previously also called Gibberella zeae) causes Gibberella stalk rot in maize (Zea mays) and results in lodging and yield loss (Jackson et al., 2009; Santiago et al., 2007). F. graminearumcan also cause seedling blight and ear rot of maize, and Fusarium head blight of wheat and barley (Jackson et al., 2009). In the field, ascospores of F. graminearumoverwinter on infected crop residues such as maize stalks and wheat straw, and may infect other plants through wounds (e.g., caused by hail or pests), or may enter through young roots, and start a new infection cycle (Jackson et al., 2009). Although the entering routes and disease development time course may differ, the final symptom for maize Gibberella stalk rot is in the stalk of adult maize plant, which directly leads to lodging and yield loss.


Figure 1. Anatomy of maize stalk. Uninfected internodes of maize stalk at V11 stage shown as reference for understanding F. graminearumprogression. V: vascular bundle. P: parenchyma cells. Green bar = 5 cm; White bars = 100 μm.

Compared to the inoculation method for assessing wheat head blight development (Pritsch et al., 2000 and 2001; Proctor et al., 1995), the inoculation method for maize stalk rot is less well established. To assess plant resistance or fungal virulence regarding maize stalk rot, two major types of inoculation methods have been reported. One is inoculation of young roots (Yang et al., 2010), which mimics the route of one type of natural infection, but has great variance in the time before stalk symptoms develop among individual maize plants, which makes follow-up microscopic observations difficult. The other is inoculation of mature stalk by wounding, usually at the internode immediately below the tassel (Zhou et al., 2010; Zheng et al., 2012) or at the internode immediately above aerial root node (Reid et al., 1996; Santiago et al., 2007) and then assessing lesions after 14 days. Recently we reported a method of wounding inoculation of maize stalks at lower internodes in combination with fluorescent protein-expressing fungi, which provided more synchronic symptom development for microscopic tracking of the infection process at the cellular scale (Zhang et al., 2016).