A new in vitro monitoring system reveals a specific influence of Arabidopsis nitrogen nutrition on its susceptibility to Alternaria brassicicola at the seedling stage

Abstract

Background: Seedling growth is an early phase of plant development highly susceptible to environmental factors such as soil nitrogen (N) availability or presence of seed-borne pathogens. Whereas N plays a central role in plant-pathogen interactions, its role has never been studied during this early phase for the interaction between Arabidopsis thaliana and Alternaria brassicicola, a seed-transmitted necrotrophic fungus. The aim of the present work was to develop an in vitro monitoring system allowing to study the impact of the fungus on A. thaliana seedling growth, while modulating N nutrition.

Results: The developed system consists of square plates placed vertically and filled with nutrient agar medium allowing modulation of N conditions. Seeds are inoculated after sowing by depositing a droplet of conidial suspension. A specific semi-automated image analysis pipeline based on the Ilastik software was developed to quantify the impact of the fungus on seedling aerial development, calculating an index accounting for every aspect of fungal impact, namely seedling death, necrosis and developmental delay. The system also permits to monitor root elongation. The interest of the system was then confirmed by characterising how N media composition [0.1 and 5 mM of nitrate (NO₃^-), 5 mM of ammonium (NH₄⁺)] affects the impact of the fungus on three A. thaliana ecotypes. Seedling development was strongly and negatively affected by the fungus. However, seedlings grown with 5 mM NO₃^- were less susceptible than those grown with NH₄⁺ or 0.1 mM NO₃^-, which differed from what was observed with adult plants (rosette stage).

Conclusions: The developed monitoring system allows accurate determination of seedling growth characteristics (both on aerial and root parts) and symptoms. Altogether, this system could be used to study the impact of plant nutrition on susceptibility of various genotypes to fungi at the seedling stage.

Keywords: Ammonium; Image analysis; Necrosis; Necrotrophic fungus; Nitrate; Root; Seedling; Symptom.