The use of GUS-reporter gene to study the effect of Azospirillum-Rhizobium coinoculation on nodulation of white clover

Abstract

The gusA-marked Azospirillum lipoferum T1371, constructed by inserting transposon mTn5SSgusA20 from Escherichia coli S17-1 λ-pir into the genome of Azospirillum lipoferum 137, was used to evaluate its effect on nodulation of white clover with and without Rhizobium inoculation. When inoculated alone, Azospirillum colonized the tap roots, secondary roots and root hairs. The combined inoculation of white clover with Rhizobium leguminosarum bv trifolii and A. lipoferum enhanced the number of nodules by 2–3 times from 5 to 20 days after inoculation (DAI). The combined inoculation also enhanced acetylene reduction activity by 2.3–2.7 times at 20 DAI. Moreover, Azospirillum was observed colonizing the tap root, root hairs and sites near or on the nodules. These results suggest that the formation of additional infection sites by A. lipoferum, with a combined inoculation, may be the mechanism that will enhance nodulation and nitrogen fixation of white clover.