Nitrogen Fertilization Has an Inhibitory Effect on Colonization of Sugarcane by Acetobacter diazotrophicus

Sugarcane, a vegetatively propagated plant, is been shown to be colonized endophytically by A. diazotrophicus. Attempts to isolate A. diazotrophicus have suggested that N-fertilization in sugarcane inhibits the population numbers of this bacterium colonizing the plant (Fuentes-Ramirez et al, 1993). Using microscopy techniques, this bacterium has been located inside root cortex cells, inside root xylem vessels of plantlets, and inside intercellular spaces of the stem of adult sugarcane plants (James et al, 1994; Dong et al, 1994). The xylem has been proposed to be used by this bacterium as a route for spreading to different plant regions (James et al, 1994). Nevertheless, in mature stems, the bacterium was observed solely in apoplastic intercellular spaces (Dong et al, 1994). An A. diazotrophicus strain constitutivelly expressing gusA was constructed in attempt to define its colonization pattern in sugarcane (UAP5541/pRGS561; see Caballero- Mellado et al., this volume). A. diazotrophicus numbers were determined in inoculated plants grown under two different N-fertilization doses. Sugarcane stems (var. ZMEX5532) were inoculated with the strain UAP5541/pRGS561, prior to sprouting. The plants were grown for 10 and 30 days (d.). A. diazotrophicus cells were quantified and the identity of the isolates was checked by antibiotic resistance and by GUS activity (GUSa). Segments from plants of the same age and from 3, 5, and 7 month old plants were tested for GUSa and by optical microscopy and by scanning electron microscopy. GUSa in plant sections was observed only in plants grown with low N fertilization and when the incubation of the GUS assay (ca. 12 h, 30°C) was done in the absence of the bacterial growth inhibitor NaN3 (0.02%). Likewise, GUSa was not detected in 10 d.