An efficient low-cost xylem sap isolation method for bacterial wilt assays in tomato

Abstract

PREMISE: A portable, simple, yet efficient method was developed for the rapid extraction of xylem sap from the stems and petioles of tomato plants for diagnostic and quantification assays of the xylem-colonizing wilt bacterium Ralstonia solanacearum. METHODS AND RESULTS: Xylem saps were extracted from tomato stem sections using negative pressure generated from handheld needleless syringes. The samples were collected from plants grown under different soil moisture levels at four days after inoculation with the pathogen. Pipette tips were modified to serve as adapters for the stem sections. The quantification of the bacterial load in the extracted sap was performed by plating sap dilutions in Kelman's triphenyltetrazolium chloride (TTC) medium. Pathogen identity was further confirmed by performing a PCR using R. solanacearum-specific primers. CONCLUSIONS: Due to its simplicity, portability, and thoroughness of extraction from predetermined tissue sizes, the method can potentially facilitate high-throughput onsite sampling from a large number of samples in a short time, which cannot be achieved with other available techniques.