Retrieving the Scopoletin Fluorescence Excitation Band Allows the Non-invasive Investigation of the Plant-Pathogen Early Events in Tobacco Leaves.

In this study, we developed and applied a new spectroscopic fluorescence method for the detection of the early events in the interaction between tobacco ( L.) plants and pathogenic bacteria. The leaf disks were infiltrated with a bacterial suspension in sterile physiological solution (SPS), or with SPS alone as control. The virulent pv. strain ATCC 11528, its non-pathogenic mutant, and the avirulent pv. strain DC3000 were used. At different post-infiltration time-points, the fluorescence spectra on leaf disks were acquired by a fiber bundle-spectrofluorimeter. The excitation spectra of the leaf blue emission at 460 nm, which is mainly due to the accumulation of coumarins following a bacterial infiltration, were processed by using a two-bands Gaussian fitting that enabled us to isolate the scopoletin (SCT) contribution. The pH-dependent fluorescence of SCT and scopolin (SCL), as determined by data and their intracellular localization, as determined by confocal microscopy, suggested the use of the longer wavelength excitation band at 385 nm of 460 nm emission (F) to follow the metabolic evolution of SCT during the plant-bacteria interaction. It was found to be directly correlated ( = 0.84) to the leaf SCT content, but not to that of SCL, determined by HPLC analysis. The technique applied to the time-course monitoring of the bacteria-plant interaction clearly showed that the amount and the timing of SCT accumulation, estimated by F, was correlated with the resistance to the pathogen. As expected, this host defense response was delayed after pv. ATCC 11528 infiltration, in comparison to pv. DC3000. Furthermore, no significant increase of F (SCT) was observed when using the non-pathogenic mutant of pv. ATCC 11528, which lacks a functional Type Three Secretion System (TTSS). Our study showed the reliability of the developed fluorimetric method for a rapid and non-invasive monitoring of bacteria-induced first events related to the metabolite-based defense response in tobacco leaves. This technique could allow a fast selection of pathogen-resistant cultivars, as well as the on-site early diagnosis of tobacco plant diseases by using suitable fluorescence sensors.