Lignocellulose-converting enzyme activity profiles correlate with molecular systematics and phylogeny grouping in the incoherent genus Phlebia (Polyporales, Basidiomycota)

Abstract

Background: The fungal genus Phlebia consists of a number of species that are significant in wood decay. Biotechnological potential of a few species for enzyme production and degradation of lignin and pollutants has been previously studied, when most of the species of this genus are unknown. Therefore, we carried out a wider study on biochemistry and systematics of Phlebia species.

Methods: Isolates belonging to the genus Phlebia were subjected to four-gene sequence analysis in order to clarify their phylogenetic placement at species level and evolutionary relationships of the genus among phlebioid Polyporales. rRNA-encoding (5.8S, partial LSU) and two protein-encoding gene (gapdh, rpb2) sequences were adopted for the evolutionary analysis, and ITS sequences (ITS1+5.8S+ITS2) were aligned for in-depth species-level phylogeny. The 49 fungal isolates were cultivated on semi-solid milled spruce wood medium for 21 days in order to follow their production of extracellular lignocellulose-converting oxidoreductases and carbohydrate active enzymes.

Results: Four-gene phylogenetic analysis confirmed the polyphyletic nature of the genus Phlebia. Ten species-level subgroups were formed, and their lignocellulose-converting enzyme activity profiles coincided with the phylogenetic grouping. The highest enzyme activities for lignin modification (manganese peroxidase activity) were obtained for Phlebia radiata group, which supports our previous studies on the enzymology and gene expression of this species on lignocellulosic substrates.

Conclusions: Our study implies that there is a species-level connection of molecular systematics (genotype) to the efficiency in production of both lignocellulose-converting carbohydrate active enzymes and oxidoreductases (enzyme phenotype) on spruce wood. Thus, we may propose a similar phylogrouping approach for prediction of lignocellulose-converting enzyme phenotypes in new fungal species or genetically and biochemically less-studied isolates of the wood-decay Polyporales.

Fig. 1

Maximum likelihood trees of the phlebioid clade and Phlebia clade of Polyporales based on ITS1-5.8S-ITS2 sequences. (a) Maximum likelihood tree illustrating the separation of four clades (Phlebia, Byssomerulius, Phlebiopsis and Phanerochaete) in the phlebioid clade. For the tree, 481 ITS sequences were aligned and phylogenetical analysis was performed using RAxML v. 7.2.8. and 100x bootstrapping. (b) Maximum likelihood analysis of ITS1-5.8S-ITS2 sequences from the Phlebia clade. Fungi of this study (shaded in green, ITS accession numbers are presented in Table 1) are compared with related taxons with sequences retrieved from NCBI (http://www.ncbi.nlm.nih.gov/) database. Bootstrap values (100 replications) higher than 50 % are indicated for the nodes. Quotation marks represent uncertain identification or provisional names suggested [20]. An ITS sequence of Byssomerulius corium was used as an outgroup. Scale bar represents 0.01 nucleotide substitutions per position

Fig. 2

Maximum likelihood phylogeny and exon-intron structure of partial gapdh nucleotide sequences of the Phlebia isolates. (a) Maximum likelihood phylogeny of the Phlebia isolates showing the phylogroups formed. 5.8S, partial LSU, and partial sequences from two protein-encoding genes (gapdh, rpb2) were concatenated for an alignment, and the phylogenetic analysis was performed using RAxML v. 7.2.8. and 100x bootstrapping. Sequences of related Agaricomycetes species (taxons without FBCC-identifier) were retrieved from JGI MycoCosm database [76] and NCBI (http://www.ncbi.nlm.nih.gov/). Species names are followed by isolate culture collection identifiers. The sequences from species Heterobasidion irregulare (Russulales, Basidiomycota) were used as an outgroup. Bootstrap values higher than 50 % are indicated for the nodes. Scale bar represents 0.01 nucleotide substitutions per position. (b) Exon-intron structure of partial gapdh nucleotide sequences from the Phlebia phylogroups studied. Black and white areas indicate exons and introns, respectively

Fig. 3

Fitted values of enzyme activities of each phylogenetic group. Fitted values (mean predicted value) of (a) laccase (b) MnP (c) CBH (d) β-glucosidase and (e) endoglucanase activities of each phylogenetic group during 21 days of cultivation in semi-solid milled spruce cultures

Fig. 4

Enzyme activities and pH values of culture liquids of Phlebia isolates. Extracellular (a) laccase (b) MnP (c) CBH (d) β-glucosidase and (e) endoglucanase activities after 14 days of cultivation in semi-solid milled spruce cultures of Phlebia isolates. (f) pH values of the semi-solid milled spruce culture liquids after 21 days of cultivation. Initial pH of the culture medium was 4.5. Error bars represent standard deviation of the mean activity value or pH value from two parallel cultivations. The isolates were numbered as listed in Table 1

Fig. 5

Hierarchical clustering of the Phlebia isolates. Hierarchical clustering presentation of lignocellulose-converting enzyme activities from fungal cultures on milled spruce wood on day 14. The normalized values taking into account the hyphal growth rates were used for calculations. The isolates were numbered as listed in Table 1