SELECTION OF DRUG-TOLERANT STRAINS OF PYTHIUM-SYLVATICUM USING SUBLETHAL ENRICHMENT

Sublethal enrichment was used to generate mutants of Pythium sylvaticu m tolerant to kanamycin and tetracycline. Kanamycin tolerance was read ily generated, and mutants had growth rates similar to wild-type isola tes at antibiotic concentrations lethal to wild-type isolates. Based o n crosses between wild-type and mutant isolates, two types of inherita nce of tolerance were identified. A high level of kanamycin tolerance was inherited in progeny only when the maternal parent was drug tolera nt and was correlated with the inheritance of maternal mitochondrial D NA. A lower level of tolerance was observed in some progeny when the p aternal parent was tolerant to the antibiotic and, based on the lack o f inheritance of paternal mitochondrial DNA, was presumably nuclear-en coded. Selection of mutants tolerant to tetracycline took longer to ge nerate than kanamycin-tolerant mutants. Based on crosses between toler ant and wildtype parents, tolerance to tetracycline was nuclear-encode d. Tolerance to both antibiotics was stable, with cultures retaining t olerance following repeated transfers on nonamended medium and after s torage for 7 years.