Isolation of Fungal Cellobiohydrolase I Genes from Sporocarps and Forest Soils by PCR

Abstract

ABSTRACT Cellulose is the major component of plant biomass, and microbial cellulose utilization is a key step in the decomposition of plant detritus. Despite this, little is known about the diversity of cellulolytic microbial communities in soil. Fungi are well known for their cellulolytic activity and mediate key functions during the decomposition of plant detritus in terrestrial ecosystems. We developed new oligonucleotide primers for fungal exocellulase genes (cellobiohydrolase, cbhI ) and used these to isolate distinct cbhI homologues from four species of litter-decomposing basidiomycete fungi ( Clitocybe nuda , Clitocybe gibba , Clitopilus prunulus , and Chlorophyllum molybdites ) and two species of ascomycete fungi ( Xylaria polymorpha and Sarcoscypha occidentalis ). Evidence for cbhI gene families was found in three of the four basidiomycete species. Additionally, we isolated and cloned cbhI genes from the forest floor and mineral soil of two upland forests in northern lower Michigan, one dominated by oak ( Quercus velutina , Q. alba ) and the other dominated by sugar maple ( Acer saccharum ) and American basswood ( Tilia americana ). Phylogenetic analysis demonstrated that cellobiohydrolase genes recovered from the floor of both forests tended to cluster with Xylaria or in one of two unidentified groups, whereas cellobiohydrolase genes recovered from soil tended to cluster with Trichoderma , Alternaria , Eurotiales, and basidiomycete sequences. The ability to amplify a key fungal gene involved in plant litter decomposition has the potential to unlock the identity and dynamics of the cellulolytic fungal community in situ.