Imaging methods to evaluate rootstock colonization by a GTD related pathogen and its control in grapevine nursery process

The application of imaging methods, already applied to study plant-microbe interactions, could represent a crucial tool to understand the effect of experimental treatments on Grapevine Trunk Disease (GTD) related pathogens and on the tissues they colonized. Trials were carried out to assess the efficacy of treatments based on 2 copper salts and an innovative coadjutant applied to control the wood colonization by a GTD related pathogen on propagating material. The distribution and the effect of the treatments in rootstock that had been inoculated with a Phaeoacremonium minimum or with the wild-type strain, compared to non-inoculated control rootstocks were studied. Treatments were applied during the hydration water process of rootstocks (Vitis Berlandieri x Vitis riparia cv. K5BB) and scions (Vitis vinifera L, cv. Chardonnay). After callusing, grafted vines were grown under greenhouse conditions. Grafted vines were inoculated with an agar plug containing the pathogen by drilling a hole in the rootstock. Plants were then harvested fifteen weeks post-inoculation and woody tissues close to the inoculation site were observed using a Confocal Laser Scanning Microscopy (CSLM) and an Environmental Electron Scanning Microscope (ESEM). Results were presented according to (i) the P. minimum strain, wild-type strain and P. minimum, (ii) the analyzed tissues in the inoculation site, i.e. cavity, pith border and xylem plus parenchyma, (iii) and the applied treatment. The imaging methods revealed distinctly the fungal presence and colonization within and around the inoculation site in the rootstocks, treated with experimental substances and untreated. By processing the images with Image J software 1.47v it was possible to estimate the area of P. minimum colonization confirming the large quantity of pathogen present in the untreated but inoculated control rootstocks and in uninoculated control rootstocks treated by the pure delivery system. Treatments based on copper limited the pathogen colonization to different extents. The correlation between the observed P. minimum at the CLSM and any morphological response induced by the treatments was verified by observing the noninoculated plant material by the ESEM.