Molecular assays to detect the presence and viability of Phytophthora ramorum and Grosmannia clavigera.

Wood and wood products can harbor microorganisms that can raise phytosanitary concerns in countries importing or exporting these products. To evaluate the efficacy of wood treatment on the survival of microorganisms of phytosanitary concern the method of choice is to grow microbes in petri dishes for subsequent identification. However, some plant pathogens are difficult or impossible to grow in axenic cultures. A molecular methodology capable of detecting living fungi and fungus-like organisms in situ can provide a solution. RNA represents the transcription of genes and can become rapidly unstable after cell death, providing a proxy measure of viability. We designed and used RNA-based molecular diagnostic assays targeting genes essential to vital processes and assessed their presence in wood colonized by fungi and oomycetes through reverse transcription and real-time polymerase chain reaction (PCR). A stability analysis was conducted by comparing the ratio of mRNA to gDNA over time following heat treatment of mycelial cultures of the Oomycete Phytophthora ramorum and the fungus Grosmannia clavigera. The real-time PCR results indicated that the DNA remained stable over a period of 10 days post treatment in heat-treated samples, whereas mRNA could not be detected after 24 hours for P. ramorum or 96 hours for G. clavigera. Therefore, this method provides a reliable way to evaluate the viability of these pathogens and offers a potential way to assess the effectiveness of existing and emerging wood treatments. This can have important phytosanitary impacts on assessing both timber and non-timber forest products of commercial value in international wood trade.

Phylogeny and taxonomy of species in the Grosmannia serpens complex.

Grosmannia serpens was first described from pine in Italy in 1936 and it has been recorded subsequently from many countries in both the northern and southern hemispheres. The fungus is vectored primarily by root-infesting bark beetles and has been reported to contribute to pine-root diseases in Italy and South Africa. The objective of this study was to consider the identity of a global collection of isolates not previously available and using DNA sequence-based comparisons not previously applied to most of these isolates. Phylogenetic analyses of the ITS2-LSU, actin, beta-tubulin, calmodulin and translation elongation factor-1 alpha sequences revealed that these morphologically similar isolates represent a complex of five cryptic species. Grosmannia serpens sensu stricto thus is redefined and comprises only isolates from Italy including the ex-type isolate. The ex-type isolate of Verticicladiella alacris was shown to be distinct from G. serpens, and a new holomorphic species, G. alacris, is described. The teleomorph state of G. alacris was obtained through mating studies in the laboratory, confirming that this species is heterothallic. Most of the available isolates, including those from South Africa, USA, France, Portugal and some from Spain, represent G. alacris. The remaining three taxa, known only in their anamorph states, are described as the new species Leptographium gibbsii for isolates from the UK, L. yamaokae for isolates from Japan and L. castellanum for isolates from Spain and the Dominican Republic.

Multigene phylogeny of filamentous ambrosia fungi associated with ambrosia and bark beetles.

Most 'ambrosia' fungi are members of a heterogeneous group of ophiostomatoids that includes the anamorph genera Ambrosiella, Raffaelea and Dryadomyces. The taxonomy of these fungi based on morphological features has been complicated by these features being poorly descriptive and having evolved convergently. In this work we report maximum parsimony and Bayesian phylogenetic analysis of a multigene dataset (nSSU rDNA, nLSU rDNA and beta-tubulin gene) from sixty-seven taxa that include members of genera Ambrosiella, Raffaelea and Dryadomyces and a diverse set of ophiostomatoid relatives. We discuss the phylogenetic status of genus Ambrosiella and its relationships with representatives of Ophiostomatales teleomorph and anamorph genera. Our analysis shows that ten of the thirteen species that had been assigned to the genus Ambrosiella are related to the teleomorph genera Grosmannia or Ophiostoma, within the Ophiostomatales. The multigene analysis and expanded taxon samplings provide a higher resolution for the species phylogeny and clarify detailed relationships between Ambrosiella associates of ambrosia and bark beetles and the closely related species of genera Raffaelea and Dryadomyces. We discuss difficulties in using the morphology of conidiophores and the mode of conidiogenesis to re-define the phylogenetic classification of Ambrosiella species. Finally, we report a correlation between the molecular classification of Ophiostomatales-related species of Ambrosiella and Raffaelea and their ecological niches.