Nitrogen-fixing capacity
The nitrogen-fixing capacity of nodules was determined in vivo by measuring the reduction of acetylene to ethylene, as previously described as acetylene-reducing activity (Hardy et al. , 1968). Nodulated roots were harvested and incubated at 30 °C for 1 h in rubber-capped tubes containing a 10% acetylene atmosphere. Two biological replicates have been performed with five technical replicates. Ethylene concentrations were determined by gas chromatography (Agilent GC 6890N, Agilent Technologies, Santa Clara, CA, USA) equipped with a GS-Alumina (30 m × 0.534 mm) separating capillary column.