Evaluation of procedures for reliable PCR detection of Ralstonia solanacearum in common natural substrates.
Several procedures were compared for reliable PCR detection of Ralstonia solanacearum in common substrates (plant, seed, water and soil). In order to prevent the inhibition of PCR by substances contained in crude extracts, numerous DNA extraction procedures as well as additives to buffers or PCR mixtures were checked. Our results showed that the efficiency of these methods or compounds depended greatly upon the nature of the sample. Consequently, preparation of samples prior to PCR depended upon sample origin. Simple methods such as a combined PVPP/BSA treatment or the association of filtration and centrifugation for detecting the bacterium in plant or water samples were very powerful. DNA capture also efficiently overcame PCR inhibition problems and ensured the detection of R. solanacearum in environmental samples. However, the commercial DNA extraction QIAamp kit appeared to be the most effective tool to guarantee the accurate PCR detection of the pathogen whatever the origin of the sample; this was particularly true for soil samples where the commonly used methods for the detection of R. solanacearum were inefficient. This study demonstrates that using an appropriate procedure, PCR is a useful and powerful tool for detecting low levels of R. solanacearum populations in their natural habitats.
Poussier
Stéphane
S
Pôle de Protection des Plantes, CIRAD, 7, chemin de l'IRAT, 97455 St Pierre, La Réunion, France.
Chéron
Jean-Jacques
JJ
Couteau
Annie
A
Luisetti
Jacques
J
D020562:Betaproteobacteria / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D004783:Environmental Microbiology; D010935:Plant Diseases / Q000382:microbiology; D010944:Plants / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012639:Seeds / Q000382:microbiology; D012988:Soil Microbiology; D014871:Water Microbiology
D004269:DNA, Bacterial