DNA-based method for rapid identification of the pine pathogen, Phytophthora pinifolia.
Phytophthora pinifolia causes a needle and shoot disease in Pinus radiata, referred to as 'Daño Foliar del Pino'. This newly discovered disease requires intensive research efforts that necessitate the processing of large numbers of samples for which accurate identification, often by people not experienced in Phytophthora taxonomy, is required. The aim of this study was, therefore, to develop species-specific primers for P. pinifolia that amplify the internal transcribed spacer region of the ribosomal operon and the nuclear Ypt1 gene, respectively. The primers were tested over several Phytophthora spp., as well as fungi isolated from P. radiata. In all cases, only P. pinifolia was amplified. In addition to the species-specific primers, a PCR-restriction fragment length polymorphism protocol using available Phytophthora genus-specific primers was also used to generate a species-specific profile for P. pinifolia. This provided a characteristic profile that allows the identification of P. pinifolia, and it could also discriminate between 27 different species of Phytophthora. Both techniques reported in this study make it possible to identify large numbers of P. pinifolia cultures accurately and efficiently, which will be important for both quarantine work and biological research on this important new pathogen.
Durán
Alvaro
A
Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa. alvaro.duran@fabi.up.ac.za
Slippers
Bernard
B
Gryzenhout
Marieka
M
Ahumada
Rodrigo
R
Drenth
Andre
A
Wingfield
Brenda D
BD
Wingfield
Michael J
MJ
D020418:Algal Proteins / Q000235:genetics; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D048028:DNA, Algal / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D008969:Molecular Sequence Data; D010838:Phytophthora / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D028223:Pinus / Q000382:microbiology*; D010935:Plant Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D020418:Algal Proteins; D017931:DNA Primers; D048028:DNA, Algal; D021903:DNA, Ribosomal Spacer