A simple extraction procedure for efficient routine detection of pathogenic bacteria in plant material by polymerase chain reaction.
A simple and rapid method for extracting DNA from plants based on the use of an extraction buffer and precipitation with isopropanol was assayed to see its usefulness in detecting pathogenic bacteria in plant material. The method was compared with a phenol-chloroform standard procedure obtaining higher sensitivity levels of detection. The protocol developed was efficient for detecting a Gram-positive bacterium, Clavibacter michiganensis subsp. sepedonicus and several Gram-negative pathogenic bacteria (Ralstonia solanacearum, Erwinia amylovora, Xanthomonas axonopodis pv. citri) with a sensitivity of 10(2)-10(3) cfu/ml in spiked samples. It was also efficient to specifically identify such bacteria in naturally infected plant material. This procedure is proposed as a routine tool for detection of plant pathogenic bacteria, as well as in environmental microbiology and biotechnology studies.
Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones, Agrarias (IVIA), Apartado Oficial, Moncada, Valencia, Spain.
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D019840:2-Propanol; D002725:Chloroform; D004783:Environmental Microbiology; D019652:Extraction and Processing Industry / Q000379:methods; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000302:isolation & purification*; D006094:Gram-Positive Bacteria / Q000235:genetics / Q000302:isolation & purification*; D019800:Phenol; D010944:Plants / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*
D019800:Phenol; D002725:Chloroform; D019840:2-Propanol