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 Gluconac etobacter diazotrophicus strain PAL5 (ATCC 49037) in two experiments. In ex periment 1 the bacteria were inoculated into a modified, low sucrose MS med ium within which micropropagated plantlets were rooted. After 10 d there wa s extensive anatomical evidence of endophytic colonization by G. diazotroph icus, particularly in lower stems, where high numbers of bacteria were visi ble within some of the xylem vessels. The identity of the bacteria was conf irmed by immunogold labelling with an antibody raised against G. diazotroph icus. On the lower stems there were breaks caused by the separation of the plantlets into individuals, and at these 'wounds' bacteria were seen coloni zing the xylem and intercellular spaces. Bacteria were also occasionally se en entering leaves via damaged stomata, and subsequently colonizing sub-sto matal cavities and intercellular spaces. A localized host defence response in the form of fibrillar material surrounding the bacteria was associated w ith both the stem and leaf invasion, In experiment 2, stems of 5-week-old g reenhouse-grown plants were inoculated by injection with a suspension of G. diazotrophicus containing 10(8) bacteria ml(-1). No hypersensitive respons e (HR) was observed, and no symptoms were visible on the leaves and stems f or 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 l ight-green with toluidine blue. In leaf samples taken up to 4 cm from the i noculation points, G. diazotrophicus cells were mainly found within the met axylem, 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-t ransparent 'haloes' that contained material that reacted with the G, diazot rophicus antibody.