The plant microbiome includes pathogenic, symbiotic, epiphytic and endophytic bacteria. Endophytes colonize plants internally, often in the intercellular region, without causing any adverse effects. Endophytic bacteria are becoming increasingly recognized in crop production on account of their potential utility as agents in plant growth promotion, stress alleviation and phytoremediation.
Banana (Musa sp.) forms a major fruit and food crop worldwide. Since commerical banana no longer contain viable seeds, micropropagation is of vital importance. The true stem in this herbaceous plant is the underground corm, while leaf sheaths constitute the pseudostem. The deep-seated shoot tip, protected from the exterior by numerous leaf bases, forms the starting material for micropropagation in banana, a practice now commonly adopted for the rapid clonal multiplication of elite types. Studies employing tissue-cultured and field-grown bananas have indicated widespread association of bacterial endophytes predominantly in a non-culturable form in micropropagated stocks.
A recent paper in AoB PLANTS demonstrates the application of Live/Dead bacterial viability testing with confocal imaging on fresh tissue sections as a simple and efficient tool for documenting native endophytic bacteria, and also showed extensive bacterial colonization in the peri-space between the cell wall and the plasma membrane in the growing shoot-tip region of banana. The microorganisms, present in innumerable numbers, share a deep and integral association with the host, but show apparently mutualistic benefits with no obvious adverse or pathogenic effect on the host. The observations reported here open the path to further in-depth investigations on the plant–endophyte association and interactions.
See the data in video form here
Thomas, P., & Reddy, K. M. (2013) Microscopic elucidation of abundant endophytic bacteria colonizing the cell wall–plasma membrane peri-space in the shoot-tip tissue of banana. AoB Plants 5: plt011 doi: 10.1093/aobpla/plt011
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 µm) 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 formalin- 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 provides 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.