Combined XRF techniques as a valuable tool for the study and early detection of plant viruses

Viruses can cause modifications of mineral elements homeostasis in plants, both at the cell and tissue level. Leaves are particularly sensitive to viruses and other biotic stresses, showing increase or decrease of certain elements’ concentrations or uneven distributions. Such phenomena can be ascribed to the pathogen’s activity and/or to the plant physiological response to the pathogen itself. In this field of research, X-ray fluorescence (XRF) techniques can be very useful analytical tools since they allow both to quantify many important elements in plants’ tissues and map their distribution. In particular, micro-XRF (μXRF), either performed at a synchrotron facility or with laboratory instruments, allows to easily and non-destructively visualize elemental distributions in leaves and identify elements correlations. Such information, together with the visual observation of plant’s symptoms as well as molecular and physiological data, may provide additional clues for the early identification of viruses and study their mode of action. In this study, leaves of flowering ash (Fraxinus ornus L.) have been sampled for three consecutive years in the city of Hamburg (Germany). Some of the trees showed the typical symptoms of ash shoestring-associated virus (ASaV), i.e. leaf deformation, shoestrings and spotting. Both healthy and ASaV infected (ASaV+, verified by RT-PCR) samples were analyzed by μXRF, using both laboratory and synchrotron (SR) X-ray sources. Important differences between infected and healthy leaves were observed: ASaV+ samples showed inhomogeneous element distribution and regions of the lamina with severe depletions of P, S, and Ca. Differently, K appeared more concentrated. In addition, SR μXRF allowed to assess variations also in distribution of some micronutrients: Mn was more intense in the lamina of the infected leaf while Zn decreased. In healthy samples, Ca was always more abundant than K which, on its turn, was less abundant in ASaV+ samples. Based on this evidence, 139 leaves (healthy n=69; ASaV+ n=70) from various trees and different years were analyzed with a portable XRF (pXRF) instrument, and K and Ca concentrations were quantified. In general, the K/Ca ratio was significantly higher in infected samples than in healthy samples. Such correlation between the ASaV infection and the K/Ca ratio allowed to correctly classify most of the infected samples. Therefore, this parameter could be used, together with visual evaluation of symptoms, for a rapid, non-destructive and cheap indirect pathogen detection. In addition, pXRF could be potentially used directly in the field. These results demonstrate the potential of combined XRF techniques to study plants viruses (or more generally plant pathogens) and develop fast diagnostic methods potentially applicable directly in the field.