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Horseradish (Armoracia rusticana) is a perennial plant, belonging to the Brassicaceae family, whose roots are widely used as a cooking spice and flavouring agent across the world and has recently acquired considerable scientific interest due to the richness of phytochemicals. Aim of the study was to explore water relations in plants grown in pots and subject to severe stress conditions and gas exchange, plant growth, and agronomic characteristics of two horseradish accessions grown in open field under rainfed Mediterranean conditions. Total and osmotic leaf water potential were significantly reduced by water stress to a similar extent at predawn and middle day. The carbon isotope composition (δ13C) (on average –34.2 and –28.9‰ in leaves and roots, respectively) resulted statistically different among water stress treatments in leaves collected at middle day. In open field, during the vegetative development, plants regulate the foliage expansion depending on rainfall distribution and temperature. When the dry period occurs, plants get rid of several mature leaves that dry completely, and might develop new leaves in the next period, when the air-cools and rainfall increases. During the dry period, horseradish optimised gas exchange during the day by increasing the net CO2 assimilation rate during the early morning (up to 31 μmol CO2 m–2s–1, on average), when the sunlight intensity and the air temperature conditions are more favourable. The species, which rely on an annual rainfall of about 680 mm, produced about 11.5 and 24.3 t/ha of roots from one- and two-year-old plantations, respectively. Regardless of accessions, close relationships were found between the number of leaves and root weight (R2=0.88 and P≤0.01) and diameter (R2=0.82 and P≤0.01). The results advise the importance to know the physiology and agronomic characteristics of horseradish to purposefully apply agronomic practices and strategies to maximise benefits for production and root quality.