Antimicrobials in Phytophthora isolation media and the growth of Phytophthora species

Many recently described Phytophthora species detected using high‐throughput sequencing have never been isolated into culture. NARH is a commonly used isolation medium containing cornmeal agar with nystatin 22.72 ppm, ampicillin 100 ppm, rifampicin 10 ppm, and hymexazol 50 ppm. We investigated whether the antimicrobial compounds in this medium selectively inhibit growth of some Phytophthora species. Growth of 10 Phytophthora species from 10 Phytophthora clades was tested in NARH medium with antimicrobials in a range of concentrations, singly and in combination: nystatin 12.5–100 ppm, ampicillin 62.5–500 ppm, rifampicin 5–40 ppm, hymexazol 12.5–100 ppm and in addition, chloramphenicol 5–40 ppm. Two combinations of antimicrobials that supported good growth of the greatest number of species were selected: (a) nystatin 12.5 ppm, ampicillin 250 ppm, rifampicin 10 ppm, and hymexazol 12.5 ppm, and (b) these compounds with the addition of chloramphenicol 5 ppm. Subsequently the growth of 47 Phytophthora species from all clades was tested in media with these two combinations and standard NARH, both in aseptic culture and in plates with leaf baits infected from soils known to be infested with Phytophthora. Although growth of some Phytophthora species was better with the new combinations of antimicrobials in aseptic culture, suppression of competing microorganisms was best in standard NARH. Sensitivity to the NARH antimicrobials is not the reason for the difficulty or failure of isolating many species detected using high‐throughput sequencing. NARH is a robust and appropriate medium for isolation of Phytophthora species from all clades. The failure of baiting techniques to isolate many of the Phytophthora species, shown to be present in a sample using molecular techniques, is unlikely to be due to sensitivity to the antimicrobials in the selective isolation medium.