Genetic relationships among isolates of Phoma ligulicola from pyrethrum and chrysanthemum based on ITS sequences and its detection by PCR

Variation within the internal transcribed spacer (ITS1, 5.8S gene and ITS2) region of the rDNA (ITS) was used to characterise the phylogenetic relationships among Phoma ligulicola isolates infecting pyrethrum crops in Tasmania, P. ligulicola isolates from the USA, Germany and mainland Australia, and other closely related fungal species. This study reports the first characterisation of the ITS region of P. ligulicola. Sequence homology within P. ligulicola isolates varied between 99.3 and 100%. For 9 of the 11 isolates from Tasmania, Australia, the nucleotide sequences in this region were identical, whereas the sequences for the remaining two isolates differed only by two nucleotides in the ITS1 region. Isolates from Australia and the USA failed to metabolise NaOH on malt-extract agar and were characterised as P. ligulicola var. inoxydablis. The two isolates from ray blight disease of chrysanthemum in Germany (DSMZ 63133 and DSMZ 62547) were classified as P. ligulicola var. ligulicola. Phylogenetic analyses suggested that the ITS sequences of P. ligulicola isolates were more similar to other Phoma species than selected representatives of the Mycosphaerella genus. Didymella bryoniae had the greatest interspecific homology with P. ligulicola of the fungi used in this study. This information was used to design specific primers within the ITS regions for the detection of P. ligulicola.