In recent years, foliar inoculation has gained acceptance among the available methods to deliver plant beneficial microorganisms to crops under field conditions. Colonization efficiency by such microorganisms 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 visualise 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 PGPR strain.

中文翻译：

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巴西固螺菌 Az39 在大豆叶片中的定位及存活

近年来，在田间条件下将植物有益微生物输送到作物的可用方法中，叶面接种已获得认可。此类微生物的定殖效率很大程度上取决于它们在叶子上的存活能力。在这项工作中，我们评估了 Azospirillum brasilense Az39 (Az39) 在切除的大豆叶片中的存活和定位。Az39 的红色荧光转化变体的扫描电子显微镜和共聚焦激光扫描显微镜用于确定细菌定位，而最可能的数量和平板计数方法用于细菌定量。显微镜观察表明，处理后 24 小时叶片表面 Az39 细胞的数量减少，而内叶表皮的中脉和细胞 - 细胞连接成为人口稠密的区域。杀菌后 6 小时证实了木质部血管内 Az39 的存在。Az39 种群在 24 小时内没有显着减少。我们可以观察大豆叶片表面和内部组织中的 Az39 细胞，并通过培养方法恢复它们。这些结果证明了 Az39 在叶子上和叶子内的存活能力，并表明这种众所周知的 PGPR 菌株具有以前未被注意到的内生潜力。