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Plant response to their environment stresses is a complex mechanism involving secondary metabolites. Stilbene phytoalexins, namely resveratrol, pterostilbene, piceids and viniferins play a key role in grapevine (Vitis vinifera) leaf defense. Despite their well-established qualities, conventional analyses such as HPLC-DAD or LC-MS lose valuable information on metabolite localization during the extraction process. To overcome this issue, a correlative analysis combining mass spectroscopy imaging (MSI) and fluorescence imaging was developed to localize in situ stilbenes on the same stressed grapevine leaves. High-resolution images of the stilbene fluorescence provided by macroscopy were supplemented by specific distributions and structural information concerning resveratrol, pterostilbene, and piceids obtained by MSI. The two imaging techniques led to consistent and complementary data on the stilbene spatial distribution for the two stresses addressed: UV-C irradiation and infection by Plasmopara viticola. Results emphasize that grapevine leaves react differently depending on the stress. A rather uniform synthesis of stilbenes is induced after UV-C irradiation, whereas a more localized synthesis of stilbenes in stomata guard cells and cell walls is induced by P. viticola infection. Finally, this combined imaging approach could be extended to map phytoalexins of various plant tissues with resolution approaching the cellular level.

Flavonols and hydroxycinnamic acids are known to contribute to plant resistance against pathogens, but there are few reports on the implication of flavonols in the resistance of grapevine against Plasmopara viticola, and none on the involvement of hydroxycinnamic acids. In order to analyze the effect of flavonols on P. viticola infection, variable amounts of flavonols were induced by different light conditions in otherwise phenologically identical leaves. Differences in content of leaf hydroxycinnamic acids were induced at the same time. A non-invasive monitoring of flavonols and hydroxycinnamic acids was performed with Dualex leaf-clip optical sensors. Whatever the light condition, there were no significant changes in flavonol or in hydroxycinnamic acid contents for control and inoculated leaves during the development of P. viticola until 6 days after inoculation. The violet-blue autofluorescence of stilbenes, the main phytoalexins of grapevine that accumulate in inoculated leaves, was used as an indicator of infection by P. viticola. The implication of leaf constitutive flavonols and hydroxycinnamic acids in the defence of Vitis vinifera against P. viticola could be investigated in vivo thanks to this indicator. The increase in stilbene violet-blue autofluorescence started earlier for leaves with low flavonol content than for leaves with higher content, suggesting that constitutive flavonols are able to slow down the infection by P. viticola. On the contrary, constitutive hydroxycinnamic acids did not seem to play a role in defence against P. viticola. The non-destructive nature of the methods used alleviates the major problem of destructive experiments: the large variability in leaf phenolic contents.

A 15-day survey of autofluorescence has been conducted upon infection by downy mildew [Plasmopara viticola (Berk. & M.A. Curtis) Berl. & de Toni] of leaves of a susceptible grapevine genotype. Different autofluorescence signals were followed from the cellular to the whole-leaf level by using four types of devices for fluorosensing: a macroscope, a spectrofluorimeter, a portable field optical sensor (the Multiplex 3), and a field fluorescence sensor prototype with 335 nm excitation. It was shown for the first time, by the three different techniques and at three different scales, that the stilbene-dependent violet-blue autofluorescence (VBF) had a transitory behaviour, increasing to a maximum 6 days post-inoculation (DPI) and then decreasing to a constant lower level, nevertheless significantly higher than in the control leaf. This behaviour could be sensed from both sides of the leaf. On the abaxial side, VBF could discriminate the presence of infection from 1 DPI, and on the adaxial side from 3 DPI. There was a constant increase in blue-excited green fluorescence starting from 8 DPI, concomitant with a decrease in leaf chlorophyll content sensed by one reflectance and two fluorescence indices available on the Multiplex 3 sensor. These results show that a pre-symptomatic and symptomatic sensing of downy mildew is possible by autofluorescence-based sensors, and this is potentially applicable in the field.