Antimicrobials in Phytophthora isolation media and the growth of Phytophthora species

Abstract

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.

A comparison of methods for the detection of Phytophthora infestans on potatoes in Mauritius

“…Equally, using "healthy" leaves or tubers for recovery of P. infestans could present further risks of contamination by other microorganisms. Indeed, the contamination of plates has been attributed to the growth of background microflora on the host tissue (Tumwine et al, 2000) or delayed sub-culturing of hyphal tips onto media (Sarker et al, 2020). On the other hand, the direct transfer of mycelial growth from infected leaves onto media was successful in this study, as demonstrated by the onset of growth after 7 days of incubation.…”

“…In fact, no growth or occasional outgrowth by background contaminants was observed on incubated plates. The contamination could be attributed to the absence of antibiotics in the media, possibly favouring the growth of bacterial contaminants, as reported in the study done by Sarker et al (2020). Equally, using "healthy" leaves or tubers for recovery of P. infestans could present further risks of contamination by other microorganisms.…”

“…Moreover, compared to PSA, CSA and FPDA-1, other media (CPA, CPDA, FPDA-2, OMA and WA) did not promote the growth of the pathogen since microscopic examination of the plates revealed the presence of sporangia, which were initially transferred from the infected leaves, without sporangiophores. The absence of sporangiophores could be attributed to the lack of one or more nutrients needed for the development of P. infestans (Kumbar, 2017;Sarker et al, 2020). For instance, Kumbar (2017) noted that the absence of sucrose in CPA inhibited the sporangial germination of P. infestans.…”

Calcium chelate is as effective as phosphite in controlling Phytophthora root rot in glasshouse trials

“…The roots and shoots were dried at 37°C for 7 days and then at 60°C for 24 h. Representative samples were weighed on consecutive days, and when no difference in weight was obtained, all the dry weights were recorded. P. cinnamomi was recovered from the fresh roots of inoculated plants by plating them onto NARH, a Phytophthora selective medium (Sarker et al, 2020). The trial was conducted between February and July 2017, with mean maximum and minimum temperatures of 26.6 and 15.6°C, respectively.…”

Towards Eradication of Phytophthora cinnamomi Using a Fallow Approach in a Mediterranean Climate

“…Inoculum was grown for 3 months before use. Inoculum viability was assessed by plating 10 plugs/flask onto NARH, a Phytophthora selective agar [31]. Inoculum was only used from flasks that were not contaminated and had 100% recovery of P. cinnamomi.…”