Definitive elucidation of the inoculation mechanism of Xylella fastidiosa by sharpshooter leafhoppers

Xylella fastidiosa is a vector-borne xylem-limited bacterium of agricultural and economic importance, yet it remains to be demonstrated how this pathogen is inoculated into plants by insect vectors. Recent DC-EPG (Electrical Penetration Graph) research with the meadow spittlebug, Philaenus spumarius tentatively correlated bacterial inoculation with a non-stereotypical "spiking waveform" termed Xe; this behavior occurred within the first minutes from initial stylets contact with a xylem vessel. Here, using the Graphocephala atropunctata-grapevine-X. fastidiosa pathosystem, we conclusively demonstrate that the DC-EPG waveform Xe represents the vector probing behavior correlated with X. fastidiosa inoculation into a host plant, and successful systemic persistent pathogen infection. Our work supports the previous hypothesis that Xe represents an egestion behavior, with a mixture of xylem sap-bacterial cells propelled by the collapse of the cibarial diaphragm while the precibarial valve is fluttering, potentially to remove obstructions from the precibarium and its chemosensilla. The outward flow delivering the mixture sap-bacterial cells into the plant is permitted by the valve that flutters instead of sealing the food canal. The inoculation behavior Xe is more frequent in vectors harboring X. fastidiosa cells within the foregut, independent of the bacterial population size in insects.