Molecular and conventional detection and identification of Cladosporium tenuissimum on two-needle pine rust aeciospores

An integrated approach, based on the analysis of both molecular and morphol ogical characters, has led to the unambiguous detection and identification of the rust hyperparasite Cladosporium tenuissimum from aeciospores of the two-needle pine rust fungi Cronartium flaccidum and Peridermium pini. Clado sporium tenuissimum was first detected from contaminated field-collected ru st spores using the polymerase chain reaction (PCR) method. The similar-siz ed amplified DNA of the parasite was then separated from rust DNA using ele ctrophoretic migration, reamplified separately with the nested PCR, and seq uenced. Sequence comparison in the data banks enabled the hyperparasite to be recognised as a species of Cladosporium. Molecular detection was followe d by conventional identification, obtained by plating rust spores on potato dextrose agar, a selective medium for rusts, since they are unable to grow on such a common substrate, and isolating the hyperparasite in pure cultur e. It was subsequently identified as C. tenuissimum. Traditional identifica tion would not have been possible without guidance from the molecular data, which focused attention on the mycoparasite. Macro- and micro-scopic featu res of colonies are also given to help with future identification on spore sources from other geographical areas and, if this should occur future iden tification on other rusts.