SPECIFIC DETECTION OF PHYTOPHTHORA-NICOTIANAE USING THE POLYMERASE CHAIN-REACTION AND PRIMERS BASED ON THE DNA-SEQUENCE OF ITS ELICITIN GENE PARA1

Six primers based on the sequence of the flanking and coding regions o f the elicitin gene ParA1 of Phytophthora nicotianae were tested for s pecific detection of the fungus by the polymerase chain reaction (PCR) . One combination, IL7/IL8, with IL7 in a flanking region and IL8 in a coding region of the gene, gave an intense 378 bp signal with a diver se collection of isolates of P. nicotianae, that included some from bl ack shank disease of tobacco and others from a variety of hosts. The s equence of the amplification product obtained with an isolate that pro duces elicitin and one that does not, was homologous with the known se quence of the ParA1 gene. The same primer combination gave no signal w ith sixteen other Phytophthora species tested except for two isolates P. palmivora with which it gave a weak 800 bp signal. It gave no signa l with DNA from healthy tobacco and tomato plants but P. nicotianae wa s detected in inoculated tobacco and tomato plants. Small numbers of z oospores (> 100) trapped onto a nitrocellulose membrane after filtrati on from suspension were also detected after two successive rounds of P CR.