DETECTION OF PHANEROCHAETE-CHRYSOSPORIUM IN SOIL BY PCR AND RESTRICTION ENZYME ANALYSIS

A nonradioactive method to detect Phanerochaete chrysosporium grown in a soil matrix was developed. This method involved DNA extraction, PCR amplification, and restriction enzyme analysis. Amplification of lign inase H8 DNA from pure cultures of P. chrysosporium was not as sensiti ve as amplification of the internal transcribed spacer (ITS) of the hi ghly repetitive nuclear ribosomal DNA, Amplified ITS DNA was digested with restriction enzymes for analysis. The restriction enzyme pattern of PCR-amplified ITS DNA of P. chrysosporium was unique compared with those of unrelated fungi. Two strains of Phanerochaete chrysosporium a nd two strains of Phanerochaete sordida were indistinguishable by rest riction enzyme analysis, while a third strain of P. chrysosporium had an unique pattern. These results were confirmed by sequence informatio n and indicate that species designations of Phanerochaete spp. should be reexamined. The restriction enzyme pattern of DNA extracted and PCR amplified from P. chrysosporium grown in soil was identical to that f rom P. chrysosporium grown in pure culture. The ITS sequence was detec ted in 14 ng of the 100 mu g of total DNA extracted from 1 g of soil.