Protocol for histological analysis of the interaction Vanilla planifolia - Fusarium oxysporum f. sp. vanillae

Abstract

Fusarium oxysporum f. sp. vanillae (Fov) is the main phytosanitary problem of Vanilla planifolia, invading the root and causing the rotting of up to 80% of the plants. The histological process is used to study the morphological characteristics of the tissues of interest and published a protocol to check Fov-V. planifolia interaction that was not reproducible under our conditions, so the study aimed to adapt a procedure that allows differential analysis of the mechanism of Fov invasion in V. planifolia by confocal microscopy. The bioassays consisted of analyzing Fov-infected roots. The experimental design consisted of three replicates evaluated at 12, 24, 36, 48, 60 and 72 hours post-inoculation. Roots without inoculum were used as controls. The samples were fixed in paraformaldehyde and 4% sucrose (PBS). Subsequently, the tissues were dehydrated by a combination of alcohols at different concentrations. The cuts were made with a microtome. The sections were observed under a Leica confocal microscope with a 63X objective with ap. No. 1.4, plus immersion oil; excitation lines: 405, 488, 532. The FAA + kerosene method was the procedure that allowed complete tissue sections. Keywords: Fusariosis, Vanilla, interaction.