2.3. ARA measurements
Dinitrogen fixation was estimated by measuring
the Acetylene Reduction Activity (ARA) using an
in situ method [20]. The pot was tightly sealed and
acetylene was injected into the soil around the root
system; the acetylene volume amounted to 10% of
total porosity of the mixture in the pot. After a
7-min incubation, samples were removed to determine
ethylene concentration by gas chromatography
(Delsi Model DI 200, Paris, France). The
measurements were carried out at reproductive
stages [21] R2 (full bloom), R5 (start seed), R6
(full green seed) and 10 days after R6. The results
are expressed in mmol C2H4 h−1 plant−1
. The
integrated ARA (iARA) was calculated according
to Patterson and LaRue [22] through the period
R2–R6+10 d and is expressed in mmol C2H4
plant−1