Background

Following the observation that transcripts of the rice gene OsJAC1 accumulated after pathogen attack or treatment with chemical resistance inducers, transgenic rice plants with constitutive expression or knockout of this gene were investigated in response to inoculation with fungal pathogens. To cover a broad pathogen spectrum, economically important representatives of ascomycete fungi (Magnaporthe oryzae, Blumeria graminis f. sp. hordei), basidiomycete fungi (Rhizoctonia solani) and oomycetes (Pythium graminicola) were chosen. Using protocols for standardized and even inoculation, an enhanced disease resistance phenotype was established for the transgenic plants constitutively expressing OsJAC1 while the respective knockout plants showed enhanced susceptibility (Weidenbach et al., 2016). A detailed bio-protocol for M. oryzae inoculation on rice is already available (Akagi et al., 2015), therefore we focus here on the inoculation protocols for R. solani, P. graminicola and Bgh.

As causal agent of rice sheath blight R. solani is one of the two most important rice diseases (Lee and Rush, 1983). The fungus overwinters as sclerotia or mycelium in the soil and infects rice sheaths by cuticular penetration or through stomata resulting in lesions, necrosis and leaf death (Ou, 1985). Of different methods available for R. solani inoculation, in the present study a time- and space-saving detached leaf assay is described, that was slightly modified from a protocol provided by Monika Höfte (Ghent University, personal communication).

P. graminicola is a causal agent of seedling damping-off and root rot resulting in stunting and yield loss (Hendrix and Campbell, 1973). In this study P. graminicola was inoculated on rice roots growing on agar plates using a protocol adapted from Van Buyten and Höfte (2013).

Fungi of the B. graminis species invade epidermal cells of their host plants with specialized feeding structures called haustoria. All other parts of the fungal mycelium are developed on the leaf surface thereby causing typical powdery mildew disease symptoms. The disease is of permanent importance in cereal agriculture (Dean et al., 2012). Rice plants do not have any powdery mildew pathogens. However, rice can be inoculated with Bgh which allows the investigation of nonhost resistance mechanisms (e.g., Abbruscato et al., 2012; Weidenbach et al., 2016). For an evenly distributed inoculation density, rice leaves have to be fixed and inoculated in a settling tower, as described for barley (Weidenbach et al., 2014).