New isolation methods and polymerase chain reaction strain discrimination techniques for natural products screening programs

Abstract

A new isolation method of fungi was established by using the Percoll density gradient centrifugation. We found that the difference in the specific gravity allowed us to separate mixtures of fungal spores of Aspergillus, Fusarium, Listeromyces, Penicillium, and Trichoderma. When this technique was applied to soil samples, a wider variety of fungi were isolated compared with those obtained from an ordinary serial dilution method. For screening purposes, it is necessary to eliminate redundant strains from the strains initially isolated. For the efficient discrimination of strains, the random amplified polymorphic DNA (RAPD) method was applied. The establishment of a standard method for RAPD enabled us to minimize the deviation of Rf values between gels to less than 0.03. Conversion of the Rf values to binary matrices provided a matrix data that could be analyzed statistically. Based on the RAPD, it was possible to select genetically distinct fungal strains within a species. Key words: fingerprinting, isolation method, microbial screening, PCR, Percoll density gradient, RAPD.