Microscopic elucidation of abundant endophytic bacteria colonizing the cell wall–plasma membrane peri-space in the shoot-tip tissue of banana

Abstract

This study was aimed at generating microscopic evidence of intra-tissue colonization in banana in support of the previous findings on widespread association of endophytic bacteria with the shoot tips of field- grown plants and micropropagated cultures, and to understand the extent of tissue colonization. Leaf-sheath tissue sections (� 50- 100 mm) from aseptically gathered shoot tips of cv. Grand Naine were treated with Live/ Dead bacterial viability kit components SYTO 9 (S9) and propidium iodide (PI) followed by epifluorescence or confocal laser scanning microscopy (CLSM). The S9, which targets live bacteria, showed abundant green-fluorescing particles along the host cell periphery in CLSM, apparently in between the plasma membrane and the cell wall. These included non-motile and occasional actively motile single bacterial cells seen in different x -y planes and z-stacks over several cell layers, with the fluorescence signal similar to that of pure cultures of banana endophytes. Propidium iodide, which stains dead bacteria, did not detect any, but post-ethanol treatment, both PI and 4 ' ,6-diamidino-2- phenylindole detected abundant bacteria. Propidium iodide showed clear nuclear staining, as did S9 to some extent, and the fluorophores appeared to detect bacteria at the exclusion of DNA-containing plant organelles as gathered from bright-field and phase-contrast microscopy. The S9- PI staining did not work satisfactorily with for- malin- or paraformaldehyde-fixed tissue. The extensive bacterial colonization in fresh tissue was further confirmed with the suckers of different cultivars, and was supported by transmission electron microscopy. This study thus pro- vides clear microscopic evidence of the extensive endophytic bacterial inhabitation in the confined cell wall- plasma membrane peri-space in shoot tissue of banana with the organisms sharing an integral association with the host. The abundant tissue colonization suggests a possible involvement of endophytes in the biology of the host besides recognizing cell wall - plasma membrane peri-space as a major niche for plant-associated bacteria.