A combination of baiting and PCR techniques for the detection of Phytophthora quercina and P. citricola in soil samples from oak stands

Abstract

A description is given of the use of a combination of polymerase chain reaction (PCR) and baiting techniques for the specific detection of Phytophthora quercina and Phytophthora citricola from soil around declining oak trees. The soil was flooded with water and subjected to a specific baiting procedure using Quercus robur leaflets as baits. Single round or nested PCR, respectively, with species-specific primers allowed the detection of P. quercina and P. citricola in infected oak leaflets used as baits and in the water from the same bait tests. PCR detection of both fungi was also possible after soil samples had been thoroughly mixed with water and the floating organic debris had been collected. Phytophthora quercina and P. citricola could be readily detected in almost every case in the water from these tests by PCR but less frequently in the organic debris. The identities of P. quercina and P. citricola were confirmed by restriction digests of the corresponding PCR amplicons. The presence of both fungi was also confirmed in parallel in soil samples tested by baiting with oak leaflets. Nested PCR with the primers used allowed the detection of as few as five zoospores of P. citricola and 300 zoospores of P. quercina in a volume of 100 μl. The methods presented here allow detection and identification of species of Phytophthora in soil without the need or direct extraction of soil samples, and without specific knowledge of the morphological characteristics of the genus.