Detection of endophytic bacteria in leaves of Vitis vinifera by using fluorescence in situ hybridization

Abstract

Previous investigation on five cultivars of healthy Sicilian grapevine allowed the isolation of endophytic bacteria belonging to Bacillus genus from different organs (bud, leaf, stalk and shoot). The aim of this work was to use fluorescence in situ hybridization (FISH) experiments in healthy and damaged leaf tissues of Vitis vinifera to visualize and localize bacteria associated with plant materials. The leaves were cleared to minimize the autofluorescence of the plant fragments. The use of fluorescently labelled bacterial probe Eub338 in FISH experiments on discoloured grapevine leaf disks allowed the estimation of the spatial distribution of different bacterial colonies. At the same time, one cleared disk each foliar sample was minced on Plate Count Agar to detect cultivable endophytic bacteria. The combined microscopic approach showed a differential location of microbial colonies within the leaf tissues examined. Particularly, the bacterial colonies were found in the veins, cells, hairs, intercellular spaces and more in the cut edges of leaf disk. Several bacterial species were isolated from leaf tissue. The high presence of microbial colonies in leaf tissue suggests a potential use of these endophytic bacteria as plant growth promoting and sources of resistance against pathogenic agents, such as fungi. Further studies are required to understand the role of bacterial endophytes in plant-microbe inter- actions. Besides, the DGGE (Denaturing Gradient Gel Electrophoresis) analysis from foliar disks will be carried out to detect uncultivable endophytic bacterial populations.