Genetic characterization of Phytophthora nicotianae by the analysis of polymorphic regions of the mitochondrial DNA

A new method based on the analysis of mitochondrial intergenic regions characterized byintraspecific variation in DNA sequences was developed and applied to the study of theplant pathogen Phytophthora nicotianae. Two regions flanked by genes trnY and rns andtrnW and cox2 were identified by comparing the whole mitochondrial genomes of Phytophthorainfestans, Phytophthora ramorum, and Phytophthora sojae and amplified using primersdesigned from the flanking conserved genes. These regions were sequenced from 51isolates of P. nicotianae of both A1 and A2 mating type recovered from different hostsand geographic regions. Amplicon length varied from 429 bp to 443 bp (trnY/rns) and322 bp to 373 bp (trnW/cox2) with intraspecific variation due to single nucleotide polymorphismsand indels. Seventeen, seven and 20 different haplotypes were detected by individuallyanalyzing regions trnY-rns, trnW-cox2 and the combined data set of sequences fromboth regions, respectively. Phylogenetic analysis inferred with three different methodsenabled the grouping of isolates in five clades, each containing different mitochondrialhaplotypes and revealed diversity in the mitochondrial genome of P. nicotianae. The majorityof isolates from citrus grouped in a single clade indicating either movement of isolateson planting stock or an association of particular isolates with this host. Phylogeneticgroups were not correlated with the radial growth rate of the isolates or the rapidity ofapple flesh colonization. The method developed in the present study represents an innovativemolecular tool for the characterization of natural populations of P. nicotianae andshould be easily expanded to other species of Phytophthora as well as other plant pathogens.It can be used to track specific haplotypes and, thanks to its high genetic resolution, it couldbe standardized and applied in a DNA barcoding like strategy for the precise identificationof sub-specific taxa. Compared to alternative molecular methods, a major advantage is thatresults are unbiased (a list of nucleotides) and highly reproducible, thus enabling thecomparison of data from different laboratories and time periods. Furthermore, the methodcould be further enhanced by the identification of additional variable mitochondrial and/ornuclear genomic regions.