Background: Soybean iron deficiency chlorosis (IDC) is an important nutrient stress frequently found in high pH and/or soils high in calcium carbonates. To advance the understanding of IDC resistance in soybean, a rapid (21-day) controlled-environment assay was developed to investigate the effects of nodulation, pH, and calcium carbonate levels on soybean iron deficiency traits. This system was tested on four genotypes known to exhibit differences in iron efficiency, including two standard IDC check cultivars and a pair of near-isogenic lines exhibiting variation at an IDC resistance quantitative trait locus. Visual score, chlorophyll content, plant height, root dry mass, and shoot dry mass were measured to quantify iron stress.

Results: Calcium carbonate levels and nodulation were found to have the greatest effects on IDC severity. Increasing calcium carbonate levels worsened IDC symptoms, while nodulation reduced symptoms in all genotypes. Higher pH levels increased iron deficiency symptoms in check genotypes 'Corsoy 79' and 'Dawson', but did not induce iron deficiency symptoms in near-isogenic lines. A significant interaction was observed between genotype, nodulation, and calcium carbonate level, indicating that a specific treatment level could discern IDC symptoms between genotypes differing in resistance to IDC.

Conclusions: IDC symptoms were successfully induced in the Check Genotypes Experiment as well as the NIL Experiment, indicating the success of using this assay for inducing IDC in controlled environments. However, our results suggest that treatment levels that best differentiate genotypes for their IDC resistance may need to be determined for each experiment because of the unique way in which different genotypes display symptoms and respond to iron deficiency conditions. Core ideas: An assay was developed to independently assess factors leading to IDC in soybean. Calcium carbonates have a greater influence on IDC symptoms than pH. Nodulation improved resistance to IDC in highly resistant and low resistant soybean genotypes. The developed assay is highly sensitive and can detect slight differences in IDC resistance.