Presence of endophytic bacteria in Vitis vinifera leaves as detected by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy (CLSM) was applied to detect and localize bacterial colonies in leaf tissues of Vitis vinifera. Leaves were cleared to minimize the autofluorescence of plant fragments. The use of fluorescently labeled bacterial probe EUB338 on discolored grapevine leaf disks allowed the estimation of the spatial distribution of different bacterial colonies. In particular, bacterial colonies were found in veins, cells, hairs, intercellular spaces, and in cut edges of leaf disks of both non-Acremonium byssoides-colonized and A. byssoides-colonized leaves of five different cultivars. Furthermore, CLSM confirmed that bacteria were located in different layers of the leaf tissue. At the same time, one cleared disk of each foliar sample was crushed and plated on Plate Count Agar to isolate cultivable endophytic bacteria. Some of these microorganisms were presumptively identified, by partial 16S rRNA gene sequencing, as Burkholderia phytofirmans, thus suggesting their potential role as plant growth promoters and sources of resistance against pathogenic agents, such as fungi.