Further observations on the interaction between sugar cane and Gluconacetobacter diazotrophicus under laboratory and greenhouse conditions

Sugar cane (Saccharum spp.) variety SP 70‐1143 was inoculated with Gluconacetobacter diazotrophicus strain PAL5 (ATCC 49037) in two experiments. In experiment 1 the bacteria were inoculated into a modified, low sucrose MS medium within which micropropagated plantlets were rooted. After 10 d there was extensive anatomical evidence of endophytic colonization by G. diazotrophicus, particularly in lower stems, where high numbers of bacteria were visible within some of the xylem vessels. The identity of the bacteria was confirmed by immunogold labelling with an antibody raised against G. diazotrophicus. On the lower stems there were breaks caused by the separation of the plantlets into individuals, and at these ‘wounds’ bacteria were seen colonizing the xylem and intercellular spaces. Bacteria were also occasionally seen entering leaves via damaged stomata, and subsequently colonizing sub‐stomatal cavities and intercellular spaces. A localized host defence response in the form of fibrillar material surrounding the bacteria was associated with both the stem and leaf invasion. In experiment 2, stems of 5‐week‐old greenhouse‐grown plants were inoculated by injection with a suspension of G. diazotrophicus containing 108 bacteria ml−1. No hypersensitive response (HR) was observed, and no symptoms were visible on the leaves and stems for the duration of the experiment (7 d). Close to the point of inoculation, G. diazotrophicus cells were observed within the protoxylem and the xylem parenchyma, where they were surrounded by fibrillar material that stained light‐green with toluidine blue. In leaf samples taken up to 4 cm from the inoculation points, G. diazotrophicus cells were mainly found within the metaxylem, where they were surrounded by a light green‐staining material. The bacteria were growing in relatively low numbers adjacent to the xylem cell walls, and they were separated from the host‐derived material by electron‐transparent ‘haloes’ that contained material that reacted with the G. diazotrophicus antibody.