The polymerase chain reaction (PCR) is widely used in modern biology and medicine. However, PCR artifacts can complicate the interpretation of PCR-based results. The internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster is the consensus fungal barcode marker and suspected PCR artifacts have been reported in many studies, especially for the analyses of environmental fungal samples. At present, the patterns of PCR artifacts in the whole fungal ITS region (ITS1+5.8S+ITS2) are not known. In this study, we analyzed the error rates of PCR at three template complexity levels using the divergent copies of ITS from the mushroom Agaricus subrufescens. Our results showed that PCR using the Phusion® High-Fidelity DNA Polymerase has a per nucleotide error rate of about 4 × 10–6 per replication. Among the detected mutations, transitions were much more frequent than transversions, insertions, and deletions. When divergent alleles were mixed as templates in the same reaction, a significant proportion (∼30%) of recombinant molecules were detected. The in vitro mixed-template results were comparable to those obtained from using the genomic DNA of the original mushroom specimen as template. Our results indicate that caution should be in place when interpreting ITS sequences from individual fungal specimens, especially those containing divergent ITS copies. Similar results could also happen to PCR-based analyses of other multicopy DNA fragments as well as single-copy DNA sequences with divergent alleles in diploid organisms.

Biogas projects are rapidly expanding in China, but there is insufficient cropland to degrade these biogas residues. Mushroom cultivation has been used to degrade various agricultural wastes. In this study, to screen the feasibility of utilizing biogas residues as potential substrates for the cultivation of Pleurotus ostreatus, seven different concentrations (0 to 60%) of biogas residue mixed with cottonseed hull, wheat bran, and lime were used to cultivate P. ostreatus. The mycelial growth rate, mycelial colonization time, yield, biological efficiency, chemical compositions, and content of four heavy metals, Cd (cadmium), Pb (lead), Hg (mercury), and As (arsenic), were analyzed. The results showed that 10 to 30% of biogas residue mixed in the substrates induced the growth of P. ostreatus mycelia faster than the others. A lower percentage (10 to 20%) of biogas residue added to the substrates is beneficial to the production and nutrient components of P. ostreatus, and the fruiting bodies produced on biogas residue-containing substrates conform to the safety standards for edible mushrooms. Although the total harvest is not significantly increased when biogas residue is added, the utilization of cheap biogas residues can conceivably reduce the practical cost and benefit the environment.