The vector regulation hypothesis: dynamic competition between pathogen and vector behaviors constrains <i>Xylella fastidiosa</i> biofilm development in sharpshooter foreguts

<i>Xylella fastidiosa</i> (<i>Xf</i>) bacteria form biofilm on the cuticular surfaces of the functional foregut (precibarium and cibarium) of its vectors, xylem fluid-ingesting sharpshooter leafhoppers and spittlebugs. While much is known about <i>Xf</i> biofilm development and maturation <i>in vitro</i>, little is known about these processes in vectors. Real-time (RT)-PCR was used to quantify <i>Xf</i> genomes daily in the functional foreguts of blue-green sharpshooters, <i>Graphocephala atropunctata,</i> over 7 days of exposure to infected grapevines. Scanning electron microscopy (SEM) was used to examine <i>Xf</i> biofilm formation at 4 and 7 days of that time course. PCR showed populations building and reducing over a 4-day cycle. SEM revealed that foreguts at 4 days showed variability in quantity and location of bacterial attachment. Only early-stage biofilm formation occurred in low-turbulence areas of the cibarium, while high-turbulence areas of the cibarium and precibarium had rare but older, more developed macro-colonies. Biofilm was almost absent at 7 days but left behind adhesive material and remnants of prior colonization. Evidence supports the hypothesis that bacterial colonization was repeatedly interrupted and constrained by the vector. Behaviors such as egestion and enzymatic salivation likely can loosen and eject <i>Xf</i> biofilm, perhaps when profuse biofilm interferes with ingestion. Thus, vector acquisition of <i>Xf</i> is a dynamic and stochastic process of interactions between bacteria and insects. We further hypothesize for future testing that the insect can regulate this interaction. A deep understanding of <i>Xf</i> acquisition will aid the ongoing development of grapevine resistance to vector transmission of xylellae diseases.IMPORTANCE<i>Xylella fastidiosa</i> (<i>Xf</i>) is one of the most destructive invasive plant pathogens in the world, able to hijack new vectors when it invades a region; yet the temporal interplay of bacterial colonization and insect behavior is unknown. This paper describes important findings about the process of <i>Xf</i> biofilm formation and maturation in a vector, contrasting similarities and differences with such formation <i>in vitro</i>. Results support the hypothesis that the behavior of the vector constrains and may regulate <i>Xf</i> biofilm formation, in dynamic competition with the bacterium. The data from this paper partly explain why <i>Xf</i> is so successful at invasion. Because the bacterium can be acquired and inoculated very quickly, it can move readily from old to new vectors and host plants in all-new environments. Our findings are relevant to biosecurity decisions because they demonstrate the importance of identifying potential vector species in the <i>Xylella</i> invasion front.