Polyporales Brown Rot Species Fomitopsis pinicola: Enzyme Activity Profiles, Oxalic Acid Production, and Fe3+-Reducing Metabolite Secretion

Abstract

Basidiomycota fungi in the order Polyporales are specified to decomposition of dead wood and woody debris and thereby are crucial players in the degradation of organic matter and cycling of carbon in the forest ecosystems. Polyporales wood-decaying species comprise both white rot and brown rot fungi, based on their mode of wood decay. While the white rot fungi are able to attack and decompose all the lignocellulose biopolymers, the brown rot species mainly cause the destruction of wood polysaccharides, with minor modification of the lignin units. The biochemical mechanism of brown rot decay of wood is still unclear and has been proposed to include a combination of nonenzymatic oxidation reactions and carbohydrate-active enzymes. Therefore, a linking approach is needed to dissect the fungal brown rot processes. We studied the brown rot Polyporales species Fomitopsis pinicola by following mycelial growth and enzyme activity patterns and generating metabolites together with Fenton-promoting Fe³⁺-reducing activity for 3 months in submerged cultures supplemented with spruce wood. Enzyme activities to degrade hemicellulose, cellulose, proteins, and chitin were produced by three Finnish isolates of F. pinicola Substantial secretion of oxalic acid and a decrease in pH were notable. Aromatic compounds and metabolites were observed to accumulate in the fungal cultures, with some metabolites having Fe³⁺-reducing activity. Thus, F. pinicola demonstrates a pattern of strong mycelial growth leading to the active production of carbohydrate- and protein-active enzymes, together with the promotion of Fenton biochemistry. Our findings point to fungal species-level "fine-tuning" and variations in the biochemical reactions leading to the brown rot type of wood decay.IMPORTANCEFomitopsis pinicola is a common fungal species in boreal and temperate forests in the Northern Hemisphere encountered as a wood-colonizing saprotroph and tree pathogen, causing a severe brown rot type of wood degradation. However, its lignocellulose-decomposing mechanisms have remained undiscovered. Our approach was to explore both the enzymatic activities and nonenzymatic Fenton reaction-promoting activities (Fe³⁺ reduction and metabolite production) by cultivating three isolates of F. pinicola in wood-supplemented cultures. Our findings on the simultaneous production of versatile enzyme activities, including those of endoglucanase, xylanase, β-glucosidase, chitinase, and acid peptidase, together with generation of low pH, accumulation of oxalic acid, and Fe³⁺-reducing metabolites, increase the variations of fungal brown rot decay mechanisms. Furthermore, these findings will aid us in revealing the wood decay proteomic, transcriptomic, and metabolic activities of this ecologically important forest fungal species.

Keywords: Agaricomycetes; Fenton reaction; Polyporales; basidiomycetes; biodegradation; brown rot; fungal enzymes; lignocellulose; oxalic acid; wood decay fungi.

FIG 1

Extracellular enzyme activities of Fomitopsis pinicola against hemicellulose and cellulose. Endo-β-1,4-xylanase (a), endo-β-1,4-glucanase (b), and β-glucosidase (c) activities were detected from wood-LNAS cultures during 10 weeks of cultivation. F. pinicola isolates FBCC1181 (dotted line, closed square), FBCC1243 (long dash-dot, closed triangle), and FBCC1468 (continuous line, closed circle) are shown. Bars indicate the standard error (SE) (n = 5, biological replicates). In some cases, the error bar is not visible due to small deviation values.

FIG 2

Extracellular acidic protease activity of F. pinicola isolates on wood-LNAS (a) and ME (b) media during 10 weeks of cultivation. Isolates shown are FBCC1181 (dotted line, closed square), FBCC1243 (long dash-dot, closed triangle), and FBCC1468 (continuous line, closed circle). Peptidase activity is represented as the activity generated by the amount of pepsin equivalent in milligrams per liter of culture filtrate. Bars indicate SE (n = 5, biological replicates). In some cases, the error bar is not visible due to small deviation values.

FIG 3

Extracellular chitinase activity of F. pinicola isolates on wood-LNAS (a) and ME (b) media during 10 weeks of cultivation. Isolates shown are FBCC1181 (dotted line, closed square), FBCC1243 (long dash-dot, closed triangle), and FBCC1468 (continuous line, closed circle). Chitinase activity is represented as the amount of 4-methylumbelliferone (MU) released per liter of culture filtrate. Bars indicate SE (n = 5, biological replicates). In some cases, the error bar is not visible due to small deviation values.

FIG 4

Ergosterol content of the F. pinicola isolates on wood-LNAS (a) and ME (b) media during 12 weeks of cultivation. Ergosterol content is estimated per gram (dry weight) of fungus-colonized wood (wood-LNAS) and dry weight fungal mycelia (ME). Bars indicate the SE (n = 3, biological replicates). In some cases, the error bar is not visible due to small deviation values. Fp 1468, F. pinicola FBCC1468; Fp 1243, F. pinicola FBCC1243; Fp 1181, F. pinicola FBCC1181.

FIG 5

Accumulation of secreted oxalic acid and decrease in culture filtrate pH during cultivations of the three F. pinicola isolates on wood-LNAS (a) and ME (b) media over the 10-week cultivation period. Oxalic acid concentrations are represented as columns, and pH values are indicated as line graphs. The key identifies oxalic acid production (OX) and pH for both media. Bars indicate SE (n = 5, biological replicates). In some cases, the error bar is not visible due to small deviation values.

FIG 6

Fe²⁺ generation by the culture filtrate (CF) and ethyl acetate-extracted culture filtrate (ET) obtained from wood-LNAS (a) and ME (b) media. Activities for time points of 2, 8, and 12 weeks of cultivation are presented for all three F. pinicola isolates and are normalized against Fe²⁺ generation detected in the uninoculated sterile medium (control samples derived on day 0 of cultivations). Fe²⁺ generated is represented per liter of culture filtrate for both CF and ET. Bars indicate SE (n = 3, biological replicates). In some cases, the error bar is not visible due to small deviation values.

FIG 7

Aromatic compound profiles in cultures of the F. pinicola isolate FBCC1468 cultivated on wood-LNAS and ME media. HPLC chromatograms of ethyl acetate-extracted culture filtrates were derived at time points of 0, initial medium-dissolved compound profile of wood-LNAS substrate at the beginning of cultivation (a); after 12 weeks of cultivation on wood-LNAS (b); 0, initial medium-dissolved compound profile of ME substrate at the beginning of cultivation (c); and after 12 weeks of cultivation on ME medium (d). Aromatic compound peaks predicted to be generated as either secretion products or by bioconversion of medium-existing aromatic and organic molecules are indicated with their retention times. For peaks with Fe³⁺-reducing activities, see Table 1.