Abstract:
The genus Ramularia includes numerous phytopathogenic species, several of which are economically
important. Ramularia eucalypti is currently the only species of this genus known to infect Eucalyptus by causing
severe leaf-spotting symptoms on this host. However, several isolates identified as R. eucalypti based on morphology
and on nrDNA sequence data of the ITS region have recently been isolated from other plant hosts, from
environmental samples and also from human clinical specimens. Identification of closely related species based
on morphology is often difficult and the ITS region has previously been shown to be unreliable for species level
identification in several genera. In this study we aimed to resolve this species-complex by applying a polyphasic
approach involving morphology, multi-gene phylogeny and matrix assisted laser desorption ionization time of
flight mass spectrometry (MALDI-TOF MS). Six partial genes (ITS, ACT, TEF1-α, HIS3, GAPDH and RPB2) were
amplified and sequenced for a total of 44 isolates representing R. eucalypti s.lat. and closely related species.
A multi-gene Bayesian phylogenetic analysis and parsimony analysis were performed, and both the resulting trees
showed significant support for separation of seven species in R. eucalypti s.lat., including two previously described
(R. eucalypti and R. miae), four novel species here described (R. haroldporteri, R. glennii, R. mali and R. plurivora)
and one undescribed Ramularia species (sterile). Additionally, Mycosphaerella nyssicola is newly combined in
Ramularia as R. nyssicola. Main mass spectra (MSPs) of several R. eucalypti s.lat. strains were generated using
MALDI-TOF MS and were compared through a Principal Component Analysis (PCA) dendogram. The PCA
dendrogram supported three clades containing R. plurivora, R. glenni /R. mali and R. eucalypti /R. miae. Although
the dendrogram separation of species differed from the phylogenetic analysis, the clinically relevant strains were
successfully identified by MALDI-TOF MS.
