Molecular identification and phylogenic analysis by sequencing the rDNA of copper-tolerant soft-rot Phialophora spp.

• Phylogenetic analysis of Phialophora spp. showed incompatibility between species and strains.
• PCR and sequencing showed Phialophora malorum, Phialophora mutabilis, Phialophora sp. A. to differentiate into 3 clades.
• Differences in species/strains were confirmed by nucleotide composition and compatibility tests.
• Screening assays on copper media showed Phialophora copper tolerance to be species/strain-related.
• P. malorum 211-C-15-1 was most Cu-tolerant strain growing at 6.4% CuSO4 on MEA and 3.2% liquid media.

The genus Phialophora includes some of the most copper-tolerant and frequently isolated fungi causing soft rot of copper-treated wood in service. The aim of this work was to conduct a phylogenic analysis of different Phialophora species and strains isolated from treated wood, evaluate strain compatibility, and examine their copper-tolerance variability under liquid- and solid-culture conditions. Phylogenic analysis of Phialophora malorum (four strains), Phialophora mutabilis (two strains), Lecythophora mutabilis (one strain), and Phialophora sp. A. (three strains) originating from different parts of the world showed clustering into three major clades. Phialophora sp. A. refers to a number of fungal isolates identified as Phialophora spp. previously isolated by our laboratory from preservative-treated stakes and poles in Sweden. P. mutabilis strains fell into a single cluster together with Phialophora lignicola and Phialophora hoffmanii; P. malorum strains clustered in their own group (93% bootstrap value) and Phialophora sp. A. clustered together with Phialophora botulispora. Compatibility studies with the different Phialophora species/strains showed discriminative behavior. All Phialophora species showed mutual inhibition (incompatibility), as did strains of P. malorum and P. mutabilis. Phialophora sp. A. strains showed mutual intermingling but incompatibility with P. botulispora. Species/strain variability regarding copper tolerance was evaluated using in-vitro liquid and solid media containing different CuSO4 concentrations. P. malorum 211-C-15-1 showed the highest copper-tolerance growing on 6.4% CuSO4 supplemented agar and 3.2% CuSO4 in liquid cultures, followed by P. malorum ATCC 66716 (3.2% w/v CuSO4-agar). P. mutabilis strains and L. mutabilis showed growth on 0.64% CuSO4 incorporated in liquid and agar cultures and Phialophora sp. A. (25M3) at 0.32% w/v CuSO4 on agar. The results emphasize that copper tolerance varies greatly within and between species of Phialophora.