Primers specific for Xylella fastidiosa based on RAPD differential fragments

• Author(s): Ferreira, Henrique; Goncalves, Edmilson; Neto, Julio; Rosato, Y.;
• Abstract: A 1.0 kb fragment of X. fastidiosa, isolated from plants showing CVC (citrus variegated chlorosis), was selected for primer construction based on differential amplification products from a RAPD reaction. The fragment was sequenced at its flanking termini and a pair of primers, named JB1 and JB2, was synthesized. These primers were tested in a polymerase chain reaction (PCR) using DNA from X. fastidiosa isolated from citrus (12 strains), coffee (4 strains), grapevine, oleander and plum. A unique amplification product of apprx1.0 kb was obtained from all X. fastidiosa strains at annealing temperatures of 64 degreeC, but at 68 degreeC only CVC and coffee-related strains were amplified. Other bacterial species such as Agrobacterium, Burkholderia, Erwinia, Pseudomonas and Xanthomonas and citrus pathogens (Xanthomonas axonopodis pv. citri) and coffee (Burkholderia andropogonis, Pseudomonas cichorii, P. syringae pv. garcae) were also tested and no amplification product was observed. The primers showed a high sensitivity to detect a low number of cells (2 to 20) and they were able to detect X. fastidiosa in extracts prepared from symptomatic and healthy/inoculated citrus and coffee plants.
• Publication Date: Feb 2000
• Journal: Summa Phytopathologica