PCR detection of Xylella fastidiosa in plant tissue
A PCR protocol for the specific detection of Xylella fastidiosa (EPPO A1 quarantine pest) in plant tissue has been developed. By using oligonucleotide specific primers, it was possible to amplify a fragment of genomic DNA in 33 strains of X. fastidiosa, from grapevine, citrus, plum, oak, golden rod (Solidago ssp.), Platanus occidentalis and also from Brazilian strains isolated from citrus trees presenting symptoms of citrus variegated chlorosis. The PCR method consistently detected X. fastidiosa in extracts from three naturally infected grapevine tissue sources and citrus rootstocks, as well as from artificially contaminated samples. Plant extracts were obtained by maceration of grape petioles and by vacuum extraction of citrus stems. However, as DNA amplification was inhibited in the presence of plant extract, it was necessary to add acid-washed polyvinylpyrrolidone and sodium ascorbate to the extracts. Detection by PCR is 100-fold more sensitive than by ELISA, and the detection limits are respectively, 1 x 10ý cfu/ml for PCR and 2 x 104 cfu/ml for ELISA. In addition, restriction endonuclease digestion of the PCR products allowed for the differentiation of two pathotypes. The authors felt that this result correlates other studies which have shown the existence of at least two pathotypes corresponding to Pierce's disease group and the group causing other diseases (often referred to as the phony peach group). However, further studies are needed to find new primers that will allow for finer differentiation within and between the pathotypes of X. fastidiosa. The authors concluded that the PCR method for detecting X. fastidiosa in plant tissue is a useful tool for research and diagnostic programmes, and noted that this protocol may also be adapted for detection of the pathogen in insect vectors.
Sources
Minsavage, G.V.; Thompson, C.M.; Hopkins, D.L.; Leite, R.M.V.B.C.; Stall, R.E. (1994) Development of a polymerase chain reaction protocol for detection of Xylella fastidiosa in plant tissue.
Phytopathology, 84 (5), 456-461.