Localization and survival of Azospirillum brasilense Az39 in soybean leaves

• Published in: Letters in applied microbiology Vol. 72; no. 5; pp. 626 - 633
• Main Authors: Puente, M.L., Maroniche, G.A., Panepucci, M., Sabio y García, J., García, J.E., Criado, M.V., Molina, R., Cassán, F.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.05.2021
• Subjects: Azospirillum brasilense; Azospirillum brasilense - growth & development; Cell culture; Cell junctions; Colonization; Confocal microscopy; Endophytes; Epidermis; Fluorescence; foliar inoculation; Glycine max - microbiology; Inoculation; leaf epidermis; leaf localization; Leaves; Localization; Microscopy; Microscopy, Confocal; Microscopy, Electron, Scanning; Most probable number; most probable number technique; PGPR; Plant growth; plant growth-promoting rhizobacteria; Plant Leaves - microbiology; Plant Roots - microbiology; plate count; Scanning electron microscopy; Scanning microscopy; soybean; Soybeans; Survival; Xylem; soybean; Azospirillum brasilense; foliar inoculation; leaf localization; PGPR
• ISSN: 0266-8254; 1472-765X; 1472-765X
• DOI: 10.1111/lam.13444

In recent years, foliar inoculation has gained acceptance among the available methods to deliver plant beneficial micro‐organisms to crops under field conditions. Colonization efficiency by such micro‐organisms largely depends on their ability to survive when applied on the leaves. In this work, we evaluated the survival and localization of Azospirillum brasilense Az39 (Az39) in excised soybean leaves. Scanning electron microscopy and confocal laser scanning microscopy of a red fluorescent‐transformed variant of Az39 were used to determine bacterial localization, while the most probable number and plate count methods were applied for bacterial quantification. Microscopic observations indicated a decrease in the number of Az39 cells on the leaf surface at 24 h after treatment, whereas midribs and cell–cell junctions of the inner leaf epidermis became highly populated zones. The presence of Az39 inside xylem vessels was corroborated at 6 h after bacterization. Az39 population did not significantly decrease throughout 24 h. We could visualize Az39 cells on the surface and in internal tissues of soybean leaves and recover them through culture methodologies. These results evidence the survival capacity of Az39 on and inside leaves and suggest a previously unnoticed endophytic potential for this well‐known plant growth‐promoting rhizobacteria strain. Significance and impact of the study: The plant growth‐promoting rhizobacterium Azospirillum brasilense Az39 is widely used to formulate inoculants for nonlegume crops in Argentina. Today, seed treatment is the preferred methodology to deliver Azospirillum under field conditions. However, recent experiments demonstrated that foliar application might be an alternative delivery method. The combination of microscopy techniques, DsRed‐tagging and standard counting procedures allowed us to confirm the survival of A. brasilense Az39 cells on and inside soybean leaves for at least 24 h. The potential of this strain to become a foliar inoculant is demonstrated.